afm biomed conference...2016/11/04 · 14 april 2016 18:00 bus from biblioteca almeida garrett to...

AFM BioMedConferencePorto | Portugal

11-15 APRIL2016

http://afmbiomed.org

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welcomeOn behalf of the AFM BioMed Conference organizing committee, I am honored and delighted to welcome you to the VII Conference on AFM for Life Sciences and Nanomedicine, in Porto.Building up on the success of the previous editions, this year’s event will bring closer specialists from cell biology and medical science to materials science and physics, to discuss the latest technological advances and applications of AFM in the field of BIOIMAGING, MECHANOBIOLOGY, HEALTH AND DISEASE, and NANOMEDICINE.The Conference is hosted by i3S/INEB (Instituto de Investigação e Inovação em Saúde/Instituto de Engenharia Biomédica) at the Auditorium of Biblioteca Municipal Almeida Garrett, and is preceded by a workshop on AFM training.

Guest Speakers and Invited Speakers lectures, as well as oral and poster presentations will take place, and participants will be encouraged to bring questions that may contribute to the advancement of knowledge in the field of AFM for Life Sciences.Our scientific program is rich and varied with 12 invited talks from internationally renowned speakers, 46 oral presentations, and 35 poster presentations (129 participants all together). This year’s program also features vast geographical diversity with presentations from 18 different countries across 4 continents!The meeting will also host a full day of free pre-Conference workshops by Bruker Nano. Their staff will be available throughout the meeting to highlight the features of their equipment.Prizes for best oral and poster presentation will be awarded. New to this year’s program is the AFM image competition. As part of the Conference, we invited the AFM users to select their best AFM images to enter a competition for the best image. Selected images will be showcased during the meeting.

As Conference chair, I know the success of the Conference depends ultimately on the many people who have worked with us in planning and organizing both the technical program and supporting local arrangements. In particular, I would like to thank the Conference’s organizing committee as well as i3S, Rita Matos, Anabela Nunes, and Natália Lima. Lastly, I would like to thank you for attending the Conference. Your attendance helps ensuring the meeting’s success.We hope that you will seize this opportunity to join us at this exciting meeting and visit Porto, a world heritage city!

Susana R. Sousa, Conference Chair

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venueAuditorium of Biblioteca Almeida GarrettJardins do Palácio de CristalRua de D. Manuel II, 4050-239 Porto

Almeida Garrett Library, in Portuguese Biblioteca Almeida Garrett, is a library in Porto, Portugal. It is located at the gardens of Palácio de Cristal. The library was inaugurated on 2001 and it is named after the writer Almeida Garrett (1799–1854).

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social program12 APRIL 2016

20:00 Bruker’s Party EveningÁrvore RestaurantRua Azevedo de Albuquerque, nº 1 | 4050-076 Porto, PortugalWebsite: http://www.arvorecoop.pt/

The Árvore (“Tree”) - artistic cooperative CRL, a cultural cooperative recognized by the state of Portugal as a private organism of public utility. It was founded in 1963 by artists, writers, architects and intellectuals interested in create new conditions for the cultural production and diffusion in a free and independent way. The Árvore aims the production, dissemination and commercialization of artistic and editorial work and the training of its’ partners and general public in the visual arts, art studies and others creation and knowledge areas.Placed since it’s creation in the historical center, Passeio das Virtudes, the Árvore has a privileged place and view of Douro River.

Directions from Biblioteca Almeida Garret to Árvore Restaurant

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14 APRIL 2016

18:00 Bus from Biblioteca Almeida Garrett to Taylor’s Cellars18:30 Visit to Taylor’s Port Wine Cellars19:30 Dinner at Taylor’s Cellars22:00 (approximately) Bus to Porto

Taylor’s CellarsRua do Choupelo 250, 4400-088 Vila Nova de Gaia, Portugal Website: http://www.taylor.pt/en/

Taylor’s Port wine cellars in the heart of the historic area of Vila Nova de Gaia, across the river from the old city centre of Oporto, house most of the company’s extensive reserves of wood aged Port as well its Vintage Port cellars. The cellars are long cool, dark warehouses, their thick granite walls and high ceilings helping to keep out the heat and maintain an even temperature throughout the year. This ensures that the wines age slowly, gradually acquiring the sublime complex flavours and aromas of maturity.

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informationsREGISTRATION DESKThe Registration desk will open at 13:00 on 11 April and at 8:30 on 12-15 April.

NAME BADGESFor identification and security purposes, participants must wear their name badges when in the venue. The use of the badge is mandatory for the access to the coffee breaks and lunches.

PRESENTATIONS INSTRUCTIONSSpeakers presenting in the morning should hand in their presentations in the auditoriums until 9:00 of the presentation day. Speakers presenting in the afternoon sessions, should hand in their presentations during lunch break. A data show and personal computer will be at the presenters’ disposal. Technicians will be available to make sure that you have successfully submitted your presentation. You will be requested to provide your presentation in a USB key.

POSTER PRESENTATIONSPosters should have 1.20m high and 0.90m wide, and will be presented on the designated poster area, in the Auditorium. Conference staff will be present to provide assistance. Authors should remain next to their poster during their poster sessions.

All posters must be placed before Poster Session I and remain in place until the end of Poster Session IV. The posters presentation will be on:- Poster Session I - HEALTH AND DISEASE - 12 April 2016- Poster Session II - NANOMEDICINE - 13 April 2016- Poster Session III – MECHANOBIOLOGY - 14 April 2016- Poster Session IV – BIOIMAGING - 15 April 2016

program

Enabling scientists to make new discoveries

Visit www.bruker.com/PeakForce_Tapping, email [email protected]

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How AFM Should Be

Innovation with Integrity Atomic Force Microscopy

Bruker Workshop & Training Day 11th April 2016 AFM BioMed Conference Site, Porto

Contact Us: [email protected]

AGENDA OVERVIEW (detailed programme in next pages) 13h30 Welcome 13h45 Introduction 14h High-resolution imaging of biological sites using PeakForce QNM Alexander Dulebo, Bruker Nano Surfaces 14h20 Understanding cells mechanical properties using Atomic Force Microscopy Samuel Lesko, Bruker Nano Surfaces 14h40 Q&A 15h-15h15 Coffee Break 15h15-16h45 Hands-On Sessions 1 session to be selected between the 3 choices available on pages 4,5 & 6 16h45-17h Break and group switch 17h-18h30 Hands-On Sessions 1 session to be selected between the 3 choices available on pages 4,5 & 6 18h30-19h Additional demonstrations if needed 19h End



13h30 Welcome 13h45 Introduction 14h High-resolution imaging of biological sites using PeakForce QNM Alexander Dulebo, Bruker Nano Surfaces Understanding chemical, mechanical and structural properties of small molecules and complexes in their native state remains important for many research groups. To realize this task, be-sides high spatial resolution, to which AFM is known for, high force sensitivity is required. Various even gentle AFM modes however are not capable to maintain precise force control when working with soft samples like biopolymers or living cells in vitro. In this lecture we will present what is so unique in Bruker’s proprietary PeakForce Tapping mode that enables to publish more than thousand publications in high impact journals by various researches within last 5 years, with the majority of them coming from life science. We will show how PeakForce Tapping technology combined with chemically functionalized probes made possible to localize single biologi-cal and chemical sites on native proteins and on living cells at high resolution.



14h20 Understanding cells mechanical properties using Atomic Force Microscopy Samuel Lesko, Bruker Nano Surfaces In the last years, Atomic Force Microscopy has become a pow-erful tool not only to study the surface morphology but also the nanomechanics of all kind of samples. Together with the recent evidence that the deformation characteristics of cells represent a potential biomarker of cells’ disease status, makes it a suitable instrument to diagnose cells state. In this lecture we will present recent technological advances for accurate and fast mechanical mapping using Bruker technology. We will demonstrate how we probe samples elastic properties by us-ing multi-frequency sinusoidal PeakForce Tapping mode and linear FASTForce Volume mode. 14h40 Q&A

15h-15h15 Coffee Break

15h15-16h45 Hands-On Sessions #1 (see next page)

16h45-17h Break and group switch

17h-18h30 Hands-On Sessions #2 (see next page)

18h30-19h Additional Demonstrations

19h End

Hands-On Sessions #1 “Dynamics of DNA Self Assembly using Dimension FastScan Bio” During this workshop, we will use the Dimension FastScan Bio AFM to visualize DNA and DNA self-assembled structures in liquid with high spatial and temporal resolution. DNA self-assembled structures (also known as scaffolded DNA origami) are small nanoarchitectures with a large variety of geometries, topologies, and periodicities. Modified by functional groups, those DNA nanostructures could serve as scaffolds to control the positioning of other molecules, and study such processes like protein-protein interactions or build artificial multicomponent nanomachines. FastScan Bio Nanotrack software will be used to continuously track this mobile sample with the possibility to compensate for any intrinsic or extrinsic movements. We will investigate real-time dynamics and structural changes of these self-assembled DNA structures using on-board MovieMaker software.

DNA origami imaged at 12Hz scan rate in liquid


Hands-On Sessions #2 “Mechanobiology of Cells and Biomaterials using Bioscope Resolve” During this workshop, we will use the BioScope Resolve AFM to visualize and map mechanical properties of cells and soft materi-als in high resolution. We will demonstrate how latest PeakForce Tapping innovations, including the new nanomechanics package and dedicated life science probes, could significantly expand mechanobiology applications of the AFM. We will present how novel MIROView optical-AFM integration workflow enables seamless navigation and automated scanning using different scanning modes over various locations. The workshop will in-clude a real-time demonstration of the functional synchroniza-tion of light microscopy techniques with AFM in order to conduct time correlated optical and AFM imaging.

Height Sneddon Deformation


Hands-On Sessions #3 “Topography screening with higher frequencies PeakForce Tapping using MultiMode” During this workshop, we will use the MultiMode to visualize DNA in air. We will demonstrate how the latest PeakForce Tap-ping innovations and software improvements boost speed and throughput, accelerating statistical studies. We will present new multi site imaging as well as PeakForce Tap-ping at 8kHz in air and at 4 kHz in liquid. The workshop will in-clude a real-time demonstration of force curve & FASTForce Vol-ume recording and post-processing of acquired data.

Plasmid DNA strand immobilized on mica using low (5 mM) NiCl2 concentra-tion. Major groove, minor groove, and the right-handedness of the helix are

clearly visible along the entire strand. Courtesy of Pyne et al, "Single-Molecule Reconstruction of Oligonucleotide Secondary Structure by Atomic

Force Microscopy," Small, 2014. doi: 10.10002/smll.2012400265 . LINK


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Registration / Poster InstallationWelcome

The Dubrovnik Procedure: An european approach to standartize cell elasticity measurementsHerman Schillers | Institute of Physiology II, University Münster, Germany

Analysis of the Effect of LRP-1 Silencing on the Invasive Potential of Cancer Cells by Nanomechanical Probing and Adhesion Force Measurements using Atomic Force Microscopy (01)Anthony Le Cigne | LRN EA4682– Laboratoire de Recherche en Nanosciences, Université de Reims Champagne Ardenne, France

Single Molecule Force Spectroscopy Applied to Heparin-Induced Thrombocytopenia (02)Thi-Huong Nguyen | Institut für Immunologie und Transfusionsmedizin/ 2ZIK HIKE - Center for Innovation Competence, Humoral Immune Reactions in Cardiovascular Diseases, Universitätsmedizin Greifswald, Germany

The tumor microenvironment modulates the strength of cell-cell contacts in human melanoma (03)Verena Hofschröer | Institute of Physiology II, University of Münster, Germany

Coffee break

Stiffer Intercalated Discs and Thicker Extracellular Matrix Maintains Contractile Function of the Aged Heart (04) Adam Engler | Department of Bioengineering and Biomedical Sciences Program, University of California / Sanford Consortium for Regenerative Medicine, USA

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12 APRIL 2016

Topic: Health and Disease

SELECTED ORAL COMMUNICATIONS

Anticancer Drugs Increase Disorder And Reduces Complexity In The Macrophage Membrane (05)Arkady Bitler | Physics Department and Institute of Nanotechnology, Bar Ilan University / Department of Chemical Research Support, Faculty of Chemistry, Weizmann Institute of Science, Israel

A new therapeutical approach against Pseudomonas aeruginosa: the LecA lectin/galactoclusters affinity studied by Single Molecule Force Spectroscopy (06) Magali Phaner-Goutorbe | INL- Institut des Nanotechnologies de Lyon (UMR CNRS 5270), Ecole Centrale de Lyon, Université de Lyon, France


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Lunch and Poster Session I

Are Cancer Cells different? Insights from visco-elastic creep response Manfred Radmacher | Professor (Full) Universität Bremen, Bremen · Institute of Biophysics, Germany

How endothelial cells can feel hydrostatic pressure (07)Valeria Prystopiuk | Institute of Physiology II, University of Münster, Germany

Measuring viscoelastic parameters of cells directly from atomic force microscopy force-distance curves (08)Yuri M. Efremov | Birck Nanotechnology Center / School of Mechanical Engineering, Purdue University, USA

Stress-strain curve of a single antibody in liquid (09)Alma P. Perrino | Instituto de Ciencia de Materiales de Madrid, Spain

Probing mechanical properties of the extracellular matrix with AFM (10)Daniel Navajas | Institute for Bioengineering of Catalonia (IBEC)/ School of Medicine, University of Barcelona/ CIBER of Respiratory Diseases, Spain

Coffee break

Biomechanical analysis of breast cancer cells to study P-cadherin/SFK mechanotransduction signalling (11)Filomena A. Carvalho | Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Portugal

Morphology and Nanomechanical Properties of Murine Isolated Liver Sinusoidal Endothelial Cells (12)Bartlomiej Zapotoczny | Centre for Nanometer-Scale Science and Advanced Materials, NANOSAM, Faculty of Physics, Astronomy, and Applied Computer Science, Jagiellonian University, Poland

Quantitating membrane bleb stiffness using AFM force spectroscopy and an optical sideview setup (13)Clemens M. Franz | Center for Functional Nanostructures, Karlsruhe Institut of Technology, Germany

Welcome Dinner by Bruker at Árvore Restaurant

Topic: Mechanobiology



12:30

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Realising the impact of SPM in pharmaceutical science and industryPhil Williams | Laboratory of Biophysics and Surface Analysis, School of Pharmacy, University of Nottingham, UK

AFM study of diamond particles internalization by monitoring MCF7 cell membrane stiffness changes (14)Marta Martin-Fernandez | Laboratoire Charles Coulomb (L2C), UMR 5221 CNRS-Université de Montpellier, France

Crowding redefines Endonuclease Recognition in Highly Dense DNA Nanoreactors (15)Abimbola Feyisara Adedeji | Dept. of Physics, University of Trieste/ Dept. of Medical and Biological Sciences, University of Udine, Italy

Fluorescence Tracking of Genome Release during Mechanical Unpacking of Single Viruses (16) Pedro J. de Pablo | Departamento de Física de la Materia Condensada, Universidad Autónoma de Madrid, Spain

Coffee break

High-Speed Atomic Force Microscopy: The dawn of dynamic structural biochemistrySimon Scheuring | INSERM / Aix-Marseille Université, France

Structural insights into translation repression via AFM imaging of mRNA/protein complexes (17)Loic Hamon | Laboratoire Structure-Activité des Biomolécules Normales et Pathologiques, INSERM U1204 and Université d’Evry, Université Paris-Saclay, France

Proteins sense different grain orientations in hydroxyapatite during adsorption (18)Thomas Faidt | Department of Experimental Physics, Saarland University, Germany

Group Photo

Lunch and Poster Session II

09:00

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13 APRIL 2016



Topic: Nanomedicine

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Atomic force microscopy as a tool to evaluate the risk for cardiovascular diseases in patientsNuno C. Santos | Faculdade de Medicina da Universidade de Lisboa, iMM Lisboa, Portugal

Probing mechanical properties of young and senescent human fibroblasts: a critical comparison of Force Volume and Peak Force QNM (19)Simone Bovio | Cellular Microbiology and Physics of Infection Group, Center of Infection and Immunity of Lille - Institut Pasteur, CNRS UMR8204, INSERM U1019, regional Hosp. Center of Lille, University of Lille, France

Detection of endothelial dysfunction in ex vivo vessels from ApoE/LDLR-/- mice: A multimodal approach (20)Martina Maase | Institute of Physiology II, University of Münster, Germany

Early Impairment Of Lung Mechanics In A Murine Model Of Marfan Syndrome (21)Juan Jose Uriarte | Unitat Biofísica i Bioenginyeria, Universitat de Barcelona / Institute for Bioengineering of Catalonia / CIBER de Enfermedades Respiratorias, Spain

Coffee break

Biomolecular mechanisms in Alzheimer’s disease: investigation by high speed Atomic Force Microscopy and NanoInfrared spectroscopy of the interaction between toxic amyloid peptides and model membranes (22)Michael Molinari | LRN EA4682– Laboratoire de Recherche en Nanosciences, Université de Reims Champagne Ardenne, France

Single molecule force spectroscopy study for curli-mediate bacterial adhesion (23)Yoojin Oh | Institute of Biophysics, Johannes Kepler University Linz, Austria

The impact of hyperglycemia on adhesion between endothelial and cancer cells (24)Katarzyna Malek-Zietek | Centre for Nanometer-Scale Science and Advanced Materials, NANOSAM, Faculty of Physics, Astronomy and Applied Computer Science, Jagiellonian University, Krakow, Poland

end

Topic: Health and Disease



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Elasticity as a cellular response to changing properties of surrounding microenvironmentMalgorzata Lekka | Institute of Nuclear Physics, Polish Academy of Sciences, Poland

A Microfluidic Chip To Measure The Effect Of Oxygen On Cell Mechanics Probed With AFM (25)Ignasi Jorba | Unitat de Biofísica i Bioenginyeria, Universitat de Barcelona / Institute for Bioengineering of Catalonia, Spain

Mapping the viscoelastic creep behavior of cancer cells by atomic force microscopy (26)Nicolas Schierbaum | IAP - Institute of Applied Physics, University of Tübingen, Germany

Towards higher-throughput platforms for cell mechanobiology (27)Sanjay Kumar | Department of Bioengineering, Department of Chemical & Biomolecular Engineering, University of California, USA

Coffee break

Quantitative measurements of the viscosity of biomimetic membranes using the AFM circular mode (28)Sebastian Jaramillo Isaza | Roberval CNRS, UMR 7337 - Sorbonne University, Université de technologie de Compiègne, France

High-Resolution Studies of Cell Structure and Mechanics by Atomic Force Microscopy (29)Alexander Dulebo | Bruker Nano Surfaces, USA

Lunch and Poster Session III

Real-time visualisation of membrane pore formation by bacterial toxinsBart Hoogenboom | London Centre for Nanotechnology, University College London, UK

Simultaneous advanced microscopies for live cell signaling dynamics investigations (30)Pieter A. A. De Beule | International Iberian Nanotechnology Laboratory, Portugal

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14:30

14 APRIL 2016



Topic: Mechanobiology

Topic: Bioimaging


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Force-controlled Patch Clamp and Scanning Ion Conductance Microscopy with the FluidFM (31)Livie Dorwling-Carter | LBB – Laboratory of Biosensors and Bioelectronics, Institute for Biomedical Engineering, ETH Zürich, Switzerland

Revealing contact formation characteristics of bacteria (32)Karin Jacobs | Experimental Physics, Saarland University, Germany

Coffee break

Sphingomyelin-rich domains in bilayer models of the milk fat globule membrane: temperature governs structural and mechanical heterogeneity (33)Fanny Guyomarc’h | INRA / AGROCAMPUS OUEST UMR 1253, Science and Technology of Milk and Egg, France

Nanoscale Infrared Spectroscopy and functional imaging of proteins (34)Andrzej Kulik | Physics of Living Matter, EPFL, Switzerland

Real-time interactions of pharmaceutical compounds with lipid rafts: from nanoscale lipid organization to immunosensing (35)Joaquim T. Marques | Centro de Química e Bioquímica. Faculdade de Ciências, Universidade de Lisboa, Portugal

Departure from the Biblioteca Almeida Garrett to the social programVisit to Taylor’s Port Wine CellarsDinner at Taylor’s Port Wine Cellars

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17:3018:0020:00


15 APRIL 2016

Recognition force microscopy and high-speed AFMPeter Hinterdorfer | Johannes Kepler University, Institute for Biophysics, Austria

The Architecture of Neutrophil Extracellular Traps revealed by AFM Imaging and Force Spectroscopy (36)Ricardo H. Pires | ZIK-HIKE, University of Greifswald, University Medicine Greifswald, Germany / DZHK (German Centre for Cardiovascular Research), Germany

Correlated atomic force microscopy and single molecule localization microscopy (37)Pascal D. Odermatt | Laboratory for Bio- and Nano-Instrumentation, Institute of Bioengineering, School of Engineering, EPFL, Switzerland

Resolving the division process in Mycobacterium Smegmatis; from milliseconds to days (38)Georg E. Fantner | Interfaculty Institute for Bioengineering – Laboratory for Bio and Nanoinstrumentation, École Polytechnique Fédéral de Lausanne, Switzerland

09:00

09:40 SELECTED ORAL COMMUNICATIONS

Topic: Bioimaging

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Coffee break

Local hydration properties of single bacterial cells and spores by lift-mode Electrostatic Force Microscopy (39)Marc Van Der Hofstadt | IBEC – Institut de Bioenginyeria de Catalunya, Spain

The organization of membrane proteins in rod outer segments revealed by AFM imaging and SMFS (40)Sourav Maity | International School for Advanced Studies (SISSA-ISAS), Italy

ESCRT-III spiral springs: polymerization and depolymerization observed by High-Speed AFM (41)Lorena Redondo-Morata | 1U1006 INSERM, Aix-Marseille Université, France

Fluctuating Lipid Nanodomains Near Critical Transitions (42)Simon D. Connell | School of Physics and Astronomy, University of Leeds, United Kingdom

Lunch and Poster Session IV

Nanotechnology at the service of nerve regenerationAna Paula Pêgo | NanoBiomaterials for Targeted Therapies i3S/INEB, Portugal

On chip biodetection, analysis and nanometrology of blood microparticles (43)Celine Elie-Caille | FEMTO-ST Institute, UFC, CNRS, ENSMM, UTBM, UBFC, France

The conformational equilibria of the prion protein characterized by AFM-based single-molecule force spectroscopy (44)Giampaolo Zuccheri | Department of Pharmacy and Biotechnologies, University of Bologna/ S3 Center, Institute of Nanoscience of the Italian CNR, Italy

Cardiomyocyte syncytium combined with Atomic force microscopy, advanced setup of universal biosensor for phenotype screening (45)Martin Pesl | International Clinical Research Center, St. Anne’s University Hospital / Department of Biology, Faculty of Medicine, Masaryk University, Czech Republic Coffee breakAwards and Closing Session

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Topic: Nanomedicine


invitedspeakers

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Institute of Physiology II, University Münster, Germany

Hermann Schillers is Assistant Professor in the Department of Physiology II at the University of Münster. He studied Chemistry and earned his PhD with a thesis about blood coagulation factors. His postdoctoral career began 1997 in the lab of Hans Oberleithner where he started to work with AFM on biological samples. Dr. Schillers was awarded the Habilitation at the medical faculty 2009. His clinical part is the diagnosis of cystic fibrosis with transepithelial nasal potential difference measurements. He filed a patent for blood based CF diagnostics and was leading project coordinator of a clinical multicenter trial. His research activity is focused on the interplay of physiological and biomechanical properties of cells in health and diseases.

BIOGRAPHY

Hermann Schillers

The Dubrovnik Procedure: An european approach to standartize cellelasticity measurements

Cell’s elasticity is an integrative parameter summarizing the biophysicaloutcome of many known and unknown cellular processes. This includesintracellular signalling, cytoskeletal activity, changes of cell volume andmorphology and many others. Not only intracellular processes defines acell`s elasticity but also environmental factors like their biochemical andbiophysical surrounding. Cell mechanics represents a comprehensive variableof life. A cell in its standard conditions shows variabilities ofbiochemical and biophysical processes resulting in a certain range ofcell’s elasticity. Changes of the standard conditions, endogenously orexogenously induced, are frequently paralleled by changes of cellelasticity. Therefore cell elasticity could serve as parameter tocharacterize different states of a cell.

H SchillersInstitute of Physiology II, University of Münster, Münster, Germany; [email protected]

ABSTRACT

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Atomic force microscopy (AFM) combines high spatial resolution with veryhigh force sensitivity and allows investigating mechanical properties ofliving cells under physiological conditions. However, elastic modulireported in the literature showed a large variability, sometimes by anorder of magnitude (or even more) for the same cell type assessed indifferent labs. Clearly, a prerequisite for the use of cell elasticity todescribe the actual cell status is a standardized procedure that allowsobtaining comparable values of a cell independent from the instrument, fromthe lab and operator. The approach described here uses four steps todevelop such a standardized protocol: (1) measuring same samples (gels andcells) in ten different European labs according to their own protocols (2)central data processing and identification of error sources (3) developmentof a procedure that eliminates these errors (4) validation of the procedurein ten different European labs. Step 1 revealed variations of the elasticmoduli of +/- 50%. Main sources of error were identified as the accuracy ofdetermining the deflection sensitivity and cantilevers spring constant. Weimplemented a novel method named Dubrovnik procedure which needs thevibrometer determined spring constant to calculate the exact deflectionsensitivity. Application of the Dubrovnik procedure reduced variability inelastic moduli of PAA gels down to 1% and increased accuracy of living cellelasticity measurement by factor 2. Biologically derived variations ofelasticity could not be reduced due to the nature of living cells buttechnically and methodologically derived variations could be minimized bythe Dubrovnik procedure.Together, the high reproducibility of elasticity measurements induced bythe Dubrovnik procedure allows to use this mechanical property of livingcells as a parameter to characterize their status in health and disease.

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Manfred Radmacher

Professor Manfred Radmacher studied physics at the Technical University of Munich and was awarded a PhD in Biophysics there. Then he spent two years as a post-doc in Santa Barbara (Ca), before returning to the Ludwig Maximilian University in Munich. After habilitation in Munich, he spent two years as professor at the University of Göttingen, before he was promoted to a full professorship at the University of Bremen. His research focuses on mechanical properties of cells investigated by Atomic Force Microscopy. He has published over 70 research papers and 10 book chapters.

BIOGRAPHY

Institut für Biophysik, Universität Bremen, Germany

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Are Cancer Cells different? Insights from visco-elastic creep response

We used atomic force microscopy (AFM) technique to measure the viscoelastic creep response of cancer and normal cells on different stiffness polyacrylamide gels. After applying a force step in contact we recorded the creep relaxation of cells in order to measure the viscous and elastic responses independently. This allows measuring separately the viscous and elastic properties of cells, which is not possible with conventional force curves. With the help of an extended version of the Hertz contact model, we could quantify for the first time the true elastic modulus and the dynamic viscosity of cells on substrates with different stiffness. We have cultured cancer and normal cells on standard plastic Petri dishes and on stiff (~50kPa) and soft (~5 kPa) polyacrylamide gels. Whereas normal cell adapt their stiffness according to the stiffness of the underlying support (from 1.2 to 2.7 kPa) exhibit cancer cells a elastic modulus of around 1.4. kPa regardless of the support’s stiffness. This difference in sensing and reacting to the substrate stiffness may point to differences in the way cancer and normal cells interact with the neighboring tissue. One possible explanation may be the ability of cancer cells to form metastasis, during which they invade the surrounding tissue and move from the primary tumor site to different locations. Similarly, they may try to indent soft gels, applying additional forces and increasing their stiffness, compared to normal cells.

Carmela Rianna, Achu Yango & Manfred RadmacherInstitut für Biophysik, Universität Bremen

ABSTRACT

29

I graduated with a Bachelors of Pharmacy degree from the University of Nottingham 1988 and qualified as a Pharmacist in 1989, after which I returned to the University to undertake a PhD on “Computational Studies in Scanning Probe Microscopy” under the supervision of Martyn Davies, David Jackson and Saul Tendler. I undertook postdoctoral research in the School developing the biological applications of SPM, in particular AFM, with one focus on the analysis and compensation for tip-induced artifacts. I was appointed to a lectureship in the School in 1996. In 2000, I was awarded a five-year Advanced Research Fellowship from the Engineering and Physical Sciences Research Council during which time I was able to develop research links with Evan Evans working at the University of British Columbia in Vancouver developing the theoretical understanding of force-induced dissociation and protein unfolding experiments. I was promoted to Associate Professor and Reader in Theoretical Biophysics in 2003.I am a member of the Laboratory of Biophysics and Surface Analysis, a research division with the School. The LBSA has an international leading reputation and track record in scanning probe microscopy and surface chemical analysis of pharmaceuticals, polymers and biomaterials. The LBSA remain the only grouping to receive the GlaxoSmithKline International Achievement Award, given for “Internationally recognized work on drug delivery and new techniques for surface and interface analysis”. In 2004, at the age of 37, I was one of the youngest pharmacists ever to be made a Fellow of the Royal Pharmaceutical Society of Great Britain, made in recognition of “distinction in science related to pharmacy”. I was promoted to a personal chair in 2008 as Professor of Biophysics. I have been involved with the formation of a number of University spin-out companies and I am the co-founder of a successful spin-out from the LBSA, Molecular Profiles Ltd (www.molprofiles.com) (now Juniper Pharmaceuticals) which was awarded the Queens Award for Enterprise in the category of Innovation in 2007 and 2011.I have published over 170 research publications (including papers in Nature, Physical Review Letters, Journals of American Chemical Society, Journal of Molecular Biology, and the Biophysical Journal) and 23 invited papers and book chapters since 1990. I am currently the Head of the Laboratory of Biophysics and Surface Analysis, and Director of Research for the School of Pharmacy at Nottingham; a top-ten QS world-ranked school.

BIOGRAPHY

PhilWilliams

Laboratory of Biophysics and Surface Analysis, School of Pharmacy, The University of Nottingham, U.K.

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Realising the impact of SPM in pharmaceutical science and industry

The Laboratory of Biophysics and Surface Analysis (LBSA), one of six research divisions within the School of Pharmacy at the University of Nottingham, first ventured into the exciting world of biological scanning probe microscopy in the late 1980s. In this time SPM has seen unique application as an investigative tool to further understanding of and help create new pharmaceutical formulations. In this talk I will review some of the areas in which SPM has made a real impact in pharmaceutical science and industry, and some areas in which the LBSA has made a contribution to the work:Seven out of the top eight top-selling medicines of 2014 were biological in origin (so-called biopharmaceuticals or biologics). Successful formulation of such biopharmaceuticals has created new challenges to the pharmaceutical industry since the physical and chemical properties of the biological molecule (protein, peptide, RNA, DNA) differ from those of small ‘classical drug’ molecules. I will highlight our development of single molecule force experiments as a promising method to screen for excipients that may stabilize protein structure in formulation and storage. I will also highlight the extension of this work as a development of a method for fragment-based lead discovery, and the use of force measurements to aid in biomaterials discovery.

Phil WilliamsLaboratory of Biophysics and Surface Analysis, School of Pharmacy, University of Nottingham, UK

ABSTRACT

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Simon Scheuring is the founder and head of the “Bio-AFM-Lab” laboratory U1006 INSERM / Université Aix-Marseille. He is a trained biologist from the Biozentrum at the University of Basel, Switzerland (1992-1996). During his PhD in the laboratory of Andreas Engel (1997-2001), he learned electron microscopy and atomic force microscopy, and got interested in membrane proteins. During this period he worked on the structure determination of aquaporins in collaboration with Peter Agre. During his postdoc at the Institut Curie in Paris, France, in the laboratory of Jean-Louis Rigaud (2001-2004), he learned membrane physical chemistry and developed atomic force microscopy for the study of native membranes. As a permanent researcher (2004-2007) and junior research director (2007-2012) he set up his lab at the Institut Curie in Paris, France. He has been awarded an INSERM Avenir grant (2005) and the “Médaille de la Ville de Paris” (2007). He then moved to Marseille (2012) where he built a larger laboratory named “Bio-AFM-Lab” administrated by INSERM and Université Aix-Marseille, where he got promoted senior research director (2012). In 2013 he received the ‘Grand Prix Robert Debré à orientation fondamentale’. His objective is to head a dynamic young research team with members from different scientific fields ranging from biology, physics and engineering to develop and use the atomic force microscopy to provide novel insights into the processes taking place in biological membranes. Simon Scheuring obtained 22 grants, published about 95 papers (61 scientific articles, 24 reviews, 10 didactic writings), and is co-inventor of 2 AFM-based patents. In his career he was invited to about 70 international conferences, about 50 seminars at renowned institutions and was organizer of several international conferences. He has trained (or still trains) 7 PhD students and 16 postdocs. Simon Scheuring has been awarded an European Research Council (ERC) grant (2012).

BIOGRAPHY

SimonScheuring

INSERM / Aix-Marseille Université, France

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High-Speed Atomic Force Microscopy: The dawn of dynamic structural biochemistry

The advent of high-speed atomic force microscopy (HS-AFM(1)) has opened a novel research field for the dynamic analysis of single bio-molecules: Molecular motor dynamics(2,3) membrane protein diffusion(4) and assembly(5) could be directly visualised. Further developments for buffer exchange(6) and temperature control(7) during HS-AFM operation are breakthroughs towards the performance of dynamic structural biochemistry using HS-AFM.

[1] Ando et al., Chem Rev. 2014 Mar 26;114(6):3120-88.[2] Kodera et al., Nature. 2010 Nov 4;468(7320):72-6.[3] Uchihashi et al., Science. 2011 Aug 5;333(6043):755-8.[4] Casuso et al., Nat Nanotechnol. 2012 Aug;7(8):525-9.[5] Chiaruttini et al., Cell. 2015 Nov 5;163(4):866-79.[6] Miyagi et al., Nat Nanotechnol. 2016[7] Takahashi et al., in preparation

Simon ScheuringINSERM / Aix-Marseille Université, France

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Nuno C. Santos was born in Lisbon, Portugal, in 1972. He graduated in Biochemistry from the Faculty of Science, University of Lisbon, in 1995, and received his PhD in Theoretical and Experimental Biochemistry in 1999 from the same University, although all the experimental work was conducted at Instituto Superior Técnico (Technical University of Lisbon) and University of California (Santa Barbara). Currently, he is Associate Professor with Habilitation of the Faculty of Medicine, University of Lisbon, and Head of the Biomembranes Unit of the Institute of Molecular Medicine (IMM). Among other distinctions, his research work was awarded with the Gulbenkian Prize for young researchers (2001), Dr. José Luis Champalimaud Prize – Basic Research (2004) and Dr. José Luis Champalimaud Prize – Applied Research and Technology (2005). He is (co)author of 98 articles in per-reviewed international journals, which received more than 2500 citations (h-index 25, publishing since 1996), presenting an ISI - Web of Knowledge impact factor sum of more than 450. In addition to these publications, he (co)authored 10 articles in Portuguese scientific journals, 12 book chapters (mostly edited outside Portugal, in English) and 2 patents. Among different National and European research projects, he was the coordinator of a consortium funded by the 7th Framework Programme of the European Union (FP7), including 10 different research groups from Europe and Brazil.

BIOGRAPHY

NunoC. Santos

Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Portugal

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Atomic force microscopy as a tool to evaluate the risk for cardiovascular diseases in patients

The availability of biomarkers to evaluate the risk of cardiovascular diseases is limited. High fibrinogen levels have been identified as a relevant cardiovascular risk factor, but the biological mechanisms remain unclear. Increased aggregation of erythrocytes (red blood cells) has been linked to high plasma fibrinogen concentration. Here, we show using atomic force microscopy (AFM) that the interaction between fibrinogen and erythrocytes is modified in chronic heart failure patients. Ischemic patients showed increased fibrinogen-erythrocyte binding forces compared to non-ischemic patients. Cell stiffness in both patient groups was also altered. A 12-month follow-up shows that patients with higher fibrinogen-erythrocyte binding forces initially were subsequently hospitalized more frequently. Our results show that AFM can be a promising tool for pinpointing patients with increased risk for cardiovascular diseases.

[1] Guedes et al. (2016) Nature Nanotechnology, in press.

Ana Filipa Guedes1, Filomena A. Carvalho1, Inês Malho1, Nuno Lousada2, Luís Sargento2, Nuno C. Santos1

1Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Portugal; 2Heart Failure Unit, Cardiology Department, Hospital Pulido Valente, Centro Hospitalar Lisboa Norte, Lisbon, Portugal

ABSTRACT

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Assoc. Prof. Małgorzata Lekka was appointed at the Institute of Nuclear Physics, Polish Academy of Science (IFJ PAN) in 1993, directly after graduation from the at the Faculty of Mathematics and Physics at the Jagiellonian University defencing her MSc thesis entitled “Preparation of biological targets for PIXE methods on example of post-partum placenta and blood”. Afterwards, she carried out work on biological applications of scanning force microscopy in the frame of PhD Studies at the Henryk Niewodniczański Institute of Nuclear Physics in Kraków. In 1998, she obtain the degree of doctor of physical sciences in the field of biophysics.Her activity is mainly focused on the characterisation of differences between normal and cancerous cells/tissues by means of atomic force microscopy (AFM). The main aim of these studies is to establish the AFM technique as a method that allows the direct detection of altered cancer cells at the single molecules level. In the period 2004–2006 she was a fellow within a NATO Collaborative Linkage Grant (”Molecular interaction in normal and cancerous cells”). Currently, she is a head of Laboratory of Biophysical Microstructures at the IFJ PAN. She has published more than 85 refereed publications and book chapters.

BIOGRAPHY

MalgorzataLekka

Department of Biophysical Microstructures, Institute of Nuclear Physics, Polish Academy of Sciences, Poland

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Elasticity as a cellular response to changing properties of surrounding microenvironment

(ECM), includes various processes, such as differentiation, migration, proliferation, cell-cell and/or cell-ECM adhesion [1]. Much of the effort has been put to understand the mechanisms of signal transduction at the molecular lever but, within last decade, the focus was placed to understand how cells respond to mechanical changes occurring within the environment surrounding single cells. There is much evidence showing that on hydrogels substrates, mimicking viscoelastic properties of ECM, cellular response of normal and stem cells is stiffness-depended. In most cases, on highly rigid hydrogels, cells spread extensively, form prominent stress fibres and mature focal adhesions. As hydrogel stiffness decreases, cells appear to be round [2]. These morphological changes are accompanied by alterations in the elastic properties of single living cells. Understanding the relation between ECM mechanics and cellular response is particularly important in the context of cancer development, which is typically associated with alterations in rigidity due to local accumulation of a dense, crosslinked proteins network [3]. With the development of new techniques that enable to probe elastic properties of single cells, it is now possible to identify and detect a single, mechanically altered cell [4]. Using this approach, it has been shown that cancerous cells are mostly more deformable (i.e. they are softer) [5] but they change their elastic properties due to interactions with surrounding environment, namely, with neighbouring cells and extracellular matrix proteins.

REFERENCES[1] Paszek et al. Cancer Cell 2005 8:241.[2] Tee et al. Biophys. J. 100: L25–27 (2011).[3] McGrail et al. J. Cell Sci. 127 2621-2626 (2014).[4] Guck et al. Integr. Biol. 2010 2:575.[5] Lekka et al. Micron 43(12) (2012): 1259-1266.[6] The work has been financed by the NCN project no DEC-2014/15/B/ST4/04737.

M Lekka1, J Pabijan1, B Orzechowska1, J Bobrowska1, P Bobrowski2, J Danilkiewicz1, Sz. Prauzner-Bechcicki1

1IFJ PAN – Institute of Nuclear Physics, Polish Academy of Sciences, Kraków, Poland: 2IMMS PAS - Institute of Metallurgy and Materials Science, Polish Academy of Sciences, Kraków, Poland; the corresponding author: [email protected]

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Dr Bart Hoogenboom is a Reader (UK eq. Associate Professor) at the London Centre for Nanotechnology and at the Department of Physics and Astronomy, University College London. After training in solid state physics in the Netherlands and Switzerland, he developed AFM instrumentation for atomic-resolution imaging of solid-liquid interfaces and for submolecular-resolution imaging of membrane proteins in aqueous solution.Since 2007, he has lead a research group in London, where he uses AFM in combination with other techniques to understand physical mechanisms of biomolecular processes. His recent research highlights include the first visualisation of the DNA double helix in solution, the nanomechanical characterisation of the transport barrier in nuclear pore complexes, and the elucidation of mechanisms of membrane perforation by antimicrobial peptides and by bacterial toxins.

BIOGRAPHY

Bart Hoogenboom

London Centre for Nanotechnology, University College London, United Kingdom

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Real-time visualisation of membrane pore formation by bacterial toxins

Pore forming proteins are widely used weapons of attack and defence in all domains of life. In the course of their action, they are released as monomers, bind to the target membrane, and assemble into oligomeric transmembrane pores that can have diameters up to several 10s of nm. Our understanding of these processes has been enhanced by atomic force microscopy studies of membrane pore formation, complementing studies by other methods and by electron microscopy in particular. Here I will illustrate this with our recent advances in imaging pore formation by bacterial cholesterol dependent cytolysins and in elucidating their pathways of assembly on and into the membrane.

REFERENCES[1] Leung et al., eLife 3, e04247 (2014). doi:10.7554/eLife.04247[2] Lukoyanova et al., J. Cell Sci. (in press)[3] Hodel et al., Curr. Opin. Struct. Biol. (in press)

Bart HoogenboomLondon Centre for Nanotechnology, University College London, United Kingdom

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Peter Hinterdorfer has 20 years of experience in atomic force microscopy of biomolecules, single-molecule force spectroscopy and recognition imaging. His publication list includes 160 original publications, 36 reviews, and 25 book chapters. His particular research interests cover structural elucidation, molecular recognition, and transport in the following fields: antibody/antigen interactions, bacterial surface layers, cells of the immune system, nuclear envelope membranes, virus/membrane interactions, transmembrane transporters, and structured sensor templating, molecular structures, membranes, cells and proteins. His group was the first to study single-molecular interaction forces between antibodies and antigens. He has invented the technique of combined topography and molecular recognition imaging and molecular structures, and done pioneering work in high speed AFM on antibodies.

BIOGRAPHY

PeterHinterdorfer

Johannes Kepler University Linz, Institute of Biophysics, Austria

40

Recognition force microscopy and high speed AFM

Binding of antibodies to their cognate antigens is fundamental for adaptive immunity. Molecular engineering of antibodies for therapeutic and diagnostic purposes emerges to be one of the major technologies in combating many human diseases. Despite its importance, a detailed description of the nanomechanical process of antibody-antigen binding and dissociation on the molecular level is lacking. Here, we utilize single molecule force spectroscopy and high-speed atomic force microscopy to examine the dynamics of antibody recognition and uncover a new principle. Contradicting the current textbook view, antibodies do not remain stationary on surfaces of regularly spaced epitopes; they rather exhibit “bipedal” random walking. As monovalent Fabs do not move, steric strain is identified as the origin of short-lived bivalent binding. Randomly walking antibodies gather in transient clusters that might serve as docking sites for the complement system and/or phagocytes. Our findings will inspire the rational design of antibodies and multivalent receptors to exploit/inhibit steric strain-induced dynamic effects.

Peter HinterdorferInstitute for Biophysics, Johannes Kepler University Linz, Gruberstr. 40, A-4020 Linz, Austria

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Ana Pêgo got her Ph.D. in Polymer Chemistry and Biomaterials from the University of Twente, the Netherlands, in 2002. In 2003 she became a researcher at INEB where she is a Principal Investigator since 2012. She is the Coordinator of the nBTT - nanoBiomaterials for Targeted Therapies Group and leader of the nanoBiomaterials for Neurosciences team. By using nanomedicine strategies the Biomaterials for Neurosciences team aims at providing in situ and in a targeted manner the required signals to promote nervous tissue regeneration.The research on new biomaterials for application in neurosciences includes the development of new polymers for the design of alternative vectors to viruses for efficient gene/siRNA delivery and preparation of nerve grafts for spinal cord injury treatment. Societal and ethical issues that concern Regenerative Medicine and NanoMedicine are also a topic in which Ana Pêgo is involved. She has been appointed the Scientific Director of the Bioimaging Centre for Biomaterials and Regenerative Therapies of INEB and she is an Invited Auxiliary Professor at the Faculty of Engineering of the University of Porto (FEUP) and Affiliated Professor at Instituto de Ciências Biomédicas Abel Salazar (ICBAS) of the University of Porto.

BIOGRAPHY

Ana PaulaPêgo

i3S - INEB, Portugal

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Nanotechnology at the service of nerve regeneration

The impact of a physical injury or disease on the nervous system can have devastating consequences to the individual and most frequently significant personal, societal and economical repercussions. Nervous system problems are common and encompass a large spectrum of traumatic injuries, diseases or iatrogenic lesions (e.g. after radio- or chemotherapy). Among these, neurodegenerative diseases like Alzheimer and Parkinson are becoming significantly prevalent in our aging population. The poor regenerative capacity, particularly in the case of the central nervous system (CNS), cannot be attributed to an intrinsic inability of neurons to sprout and re-grow after injury, as these axons are able to regenerate in the presence of a permissive growth environment. One of the challenges facing the neurosciences field is the development of effective therapies that can spur the regenerative capacity of the nervous system based on the advances achieved in basic research.The application of biomaterials in neurosciences has given so far a substantial contribute to the development of implantable and interfaceable devices dedicated to the restoration of a number of neural functions. But the use of biomaterials in the context of nervous system regenerative medicine is still in its infancy. We have been dedicated to using nano-enabled solutions to the design of new therapeutic approaches towards the enhancement of the process of nerve regeneration.Two main strategies are being followed and will be discussed:- the development of materials designed at the nanoscale for the preparation of grafts to be used in nerve defect correction;- the design of biomaterial-based nanoparticles for targeted nucleic acid delivery to neurons.In particular, the use of Atomic Force Microscopy for the understanding and characterization of the processes of neuron-material interaction with important outcomes for the design and optimization of material design towards attaining neuron specificity and, ultimately, nerve regeneration, will be presented.

AP Pêgo1,2,3

1i3S – Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Portugal; 2INEB - Instituto de Engenharia Biomédica, Universidade do Porto, Portugal; 3FEUP – Faculdade de Engenharia da Universidade do Porto & ICBAS – Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto, Portugal

ABSTRACT

oral com-munica-tions

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oral communica-tions’ listAnalysis of the Effect of LRP-1 Silencing on the Invasive Potential of Cancer Cells by Nanomechanical Probing and Adhesion Force Measurements using Atomic Force MicroscopyLe Cigne A., Chièze L., Beaussart A., El-Kirat-Chatel S., Dufrêne Y.F., Devy J., and Molinari M.

01

Single Molecule Force Spectroscopy Applied to Heparin-Induced ThrombocytopeniaTH Nguyen, M Decea, A Greinacher

02

The tumor microenvironment modulates the strength of cell-cell contacts in human melanomaV Hofschröer, FT Ludwig, K Koch, C Stock and A Schwab

03

Stiffer Intercalated Discs and Thicker Extracellular Matrix Maintains Contractile Function of the Aged Heart

Gaurav Kaushik, Alice Spenlehauer, Ayla O. Sessions, Adriana S. Trujillo, Alexander Fuhrmann, Sanford I. Bernstein, Anthony Cammarato, Adam J. Engler

04

Anticancer Drugs Increase Disorder And Reduces Complexity In The Macrophage MembraneA. Bitler, R. Dover and Y. Shai

05

A new therapeutical approach against Pseudomonas aeruginosa: the LecA lectin/galactoclusters affinity studied by Single Molecule Force SpectroscopyF. Zuttion, D. Sicard, C. Ligeour, Y. Chevolot, F. Morvan, S. Vidal, J. J. Vasseur, M. Phaner-Goutorbe

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How endothelial cells can feel hydrostatic pressure Valeria Prystopiuk, Hans Oberleithner, Johannes Fels

07Measuring viscoelastic parameters of cells directly from atomic force microscopy force-distance curves Yu.M Efremov, A Raman

08

Stress-strain curve of a single antibody in liquidAP Perrino, R Garcia

09Probing mechanical properties of the extracellular matrix with AFM

Daniel Navajas

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Biomechanical analysis of breast cancer cells to study P-cadherin/SFK mechanotransduction signallingFA Carvalho, J Monteiro, AS Ribeiro, AF Guedes, J Paredes, NC Santos

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Morphology and Nanomechanical Properties of Murine Isolated Liver Sinusoidal Endothelial Cells B. Zapotoczny, K. Szafranska, K. Owczarczyk, E. Maslak, S.K. Chlopicki, M. Szymonski

12

Quantitating membrane bleb stiffness using AFM force spectroscopy and an optical sideview setup

C Gonnermann, J Kashef, CM Franz

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AFM study of diamond particles internalization by monitoring MCF7 cell membrane stiffness changesM. Martin, M. Gulka, B. Varga, T. Cloitre, P. Cígler, M. Nesládek, C. Gergely

14

Crowding redefines Endonuclease Recognition in Highly Dense DNA NanoreactorsAbimbola Adedeji, Dianne Choi, Vincent Inverso, Shiv K. Redhu, Marco Vidonis, Luca Crevatin, Allen W. Nicholson, Giacinto Scoles, Matteo Castronovo

15

Fluorescence Tracking of Genome Release during Mechanical Unpacking of Single Viruses P. J. de Pablo, A. Ortega-Esteban, K. Bodensiek, C. San Martín, M. Suomalainen, U. Greber, I. Schaap

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Structural insights into translation repression via AFM imaging of mRNA/protein complexes D.A. Kretov, S. Abrakhi, D. Pastre, L.Hamon

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Proteins sense different grain orientations in hydroxyapatite during adsorption T Faidt, J Schmauch, MJ Deckarm, S Grandthyll, F Müller, K Jacobs

18

Probing mechanical properties of young and senescent human fibroblasts: a critical comparison of Force Volume and Peak Force QNMS. Bovio, V. Dupres, N. Martin, C. Abbadie, F. Lafont

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Detection of endothelial dysfunction in ex vivo vessels from ApoE/LDLR-/- mice: A multimodal approach20

Early Impairment of Lung Mechanics in a Murine Model of Marfan SyndromeJuan J. Uriarte, Thayna Meirelles, Darya Gorbenko del Blanco, Paula N. Nonaka, Noelia Campillo, Elisabet Sarri, Gustavo Egea, Ramon Farré, Daniel Navajas

21

Biomolecular mechanisms in Alzheimer’s disease: investigation by high speed Atomic Force Microscopy and nanoInfrared spectroscopy of the interaction between toxic amyloid peptides and model membranes

M Ewald, S Henry, N. Bercu, C. Culin, S Lecomte, M. Molinari

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Martina Maase, Anna Rygula, Magdalena Sternak, Agnieszka Serwadczak, Marta Pacia, Malgorzata Baranska, Stefan Chlopicki, Kristina Kusche-Vihrog

Single molecule force spectroscopy study for curli-mediate bacterial adhesion YJ Oh, M Hubauer-Brenner, HJ Gruber, Y Cui, L Traxler, S Park, P Hinterdorfer

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A Microfluidic Chip to Measure the Effect of Oxygen on Cell Mechanics Probed with AFMIgnasi Jorba, Noelia Campillo, Ramon Farré, Daniel Navajas

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Mapping the viscoelastic creep behavior of cancer cells by atomic force microscopyN Schierbaum, J Rheinlaender, FM Hecht, WH Goldmann, B Fabry, TE Schäffer

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The impact of hyperglycemia on adhesion between endothelial and cancer cellsKatarzyna Malek-Zietek, Marta Targosz-Korecka, and Marek Szymonski

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Towards higher-throughput platforms for cell mechanobiologyS Kumar

27Quantitative measurements of the viscosity of biomimetic membranes using the AFM circular modeS Jaramillo Isaza, R N Baiti, K El-Kirat, O Noël, P.-E Mazeran

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High-Resolution Studies of Cell Structure and Mechanics by Atomic Force MicroscopyAL Slade, B Pittenger, I Medalsy, S Hu, JE Shaw, H Schillers, M Radmacher

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Simultaneous advanced microscopies for live cell signaling dynamics investigations30

Force-controlled Patch Clamp and Scanning Ion Conductance Microscopy with the FluidFM L. Dorwling-Carter, D. Ossola, J. Vörös, H. Abriel, T. Zambelli

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Revealing contact formation characteristics of bacteria K.Jacobs, N. Thewes, C. Spengler, F. Nolle

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Adelaide Miranda, Marco Martins, Pieter A. A. De Beule

Sphingomyelin-rich domains in bilayer models of the milk fat globule membrane: temperature governs structural and mechanical heterogeneity

F. Guyomarc’h, A.V.R. Murthy, C. Lopez

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Nanoscale Infrared Spectroscopy and functional imaging of proteins AJ Kulik, FS Ruggeri, WI Gruszecki, G. Dietler

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Real-time interactions of pharmaceutical compounds with lipid rafts: from nanoscale lipid organization to immunosensingJT Marquês, I Almeida, TO Paiva, RFM de Almeida, AS Viana

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The Architecture of Neutrophil Extracellular Traps revealed by AFM Imaging and Force SpectroscopyRicardo H. Pires, Stephan B. Felix, and Mihaela Delcea

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Correlated atomic force microscopy and single molecule localization microscopyPascal D. Odermatt, Arun Shivanandan, Hendrik Deschout, Radek Jankele, Adrian P. Nievergelt, Lely Feletti, Michael W. Davidson, Aleksandra Radenovic and Georg E. Fantner

37

Resolving the division process in Mycobacterium Smegmatis; from milliseconds to days Georg E. Fantner, Pascal D. Odermatt, Haig-Alexander Eskandarian, Joëlle Ven, Mélanie Hannebelle, John McKinney

38

Local hydration properties of single bacterial cells and spores by lift-mode Electrostatic Force Microscopy39

The organization of membrane proteins in rod outer segments revealed by AFM imaging and SMFSSourav Maity, Monica Mazzolini, Vincent Torre

40

ESCRT-III spiral springs: polymerization and depolymerization observed by High-Speed AFM

L. Redondo-Morata, N. Chiaruttini, A. Roux, S. Scheuring

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Marc Van Der Hofstadt, R. Fabregas, M. C. Biagi, L. Fumagalli, A. Juárez, G. Gomila

Fluctuating Lipid Nanodomains Near Critical Transitions George R. Heath, Stephen D. Evans, Simon D. Connell

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On chip biodetection, analysis and nanometrology of blood microparticles S. Obeid, C. Decourbey, A. Ceroi, G. Mourey, P. Ducoroy, G. Belliot, P. Saas, W. Boireau, C. Elie-Caille

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The conformational equilibria of the prion protein characterized by AFM-based single-molecule force spectroscopyG Zuccheri, A Raspadori, V Vignali, G Legname, B Samorì

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Cardiomyocyte syncytium combined with Atomic force microscopy, advanced setup of universal biosensor for phenotype screeningMartin Pesl, Jan Pribyl, Sarka Jelinkova, Ivana Acimovic, Anton Salykin, Petr Dvorak, Vladimir Rotrekl, Petr Skladal

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Analysis of the Effect of LRP-1 Silencing on the Invasive Potential of Cancer Cells by Nanomechanical Probing and Adhesion Force Measurements using Atomic Force Microscopy

Low-density lipoprotein receptor-related protein 1 (LRP-1) [1] can internalize proteases involved in cancer progression and is thus considered a promising therapeutic target. However, it has been demonstrated that LRP-1 is also able to regulate the endocytosis of membrane-anchored proteins. Thus, strategies that target LRP-1 to modulate proteolysis could also affect adhesion and cytoskeleton dynamics [2]. Here, we investigated the effect of LRP-1 silencing on parameters reflecting cancer cells’ invasiveness by atomic force microscopy (AFM). The results show that LRP-1 silencing induces changes in the cells’ adhesion behavior, particularly the dynamics of cell attachment. Clear alterations in morphology, such as more pronounced stress fibers and increased spreading, leading to increased area and circularity, were also observed. The determination of the cells’ mechanical properties by AFM showed that these differences are correlated with an increase in Young’s modulus. Moreover, the measurements show an overall decrease in cell motility and modifications of directional persistence. An overall increase in the adhesion force between the LRP-1-silenced cells and a gelatin-coated bead was also observed. Ultimately, our AFM-based force spectroscopy data, recorded using an antibody directed against the β1 integrin subunit, provide evidence that LRP-1 silencing modifies the rupture force distribution. Together, our results show that techniques traditionally used for the investigation of cancer cells can be coupled with AFM to gain access to complementary phenotypic parameters that can help discriminate between specific phenotypes associated with different degrees of invasiveness.

[1] J. Herz, U. Hamann, S. Rogne, O. Myklebost, H. Gausepohl and K. K. Stanley, EMBO J., 1988, 7, 4119–4127. [2] S. Dedieu, B. Langlois, J. Devy, B. Sid, P. Henriet, H. Sartelet, G. Bellon, H. Emonard and L. Martiny, Mol. Cell. Biol., 2008, 28, 2980–2995.

Le Cigne A.1, Chièze L.1, Beaussart A.2, El-Kirat-Chatel S.2, Dufrêne Y.F.2, Devy J.3, and Molinari M.1

1LRN EA4682– Laboratoire de Recherche en Nanosciences, Université de Reims Champagne Ardenne, France; 2Institute of Life Sciences, Université Catholique de Louvain, Louvain-la-neuve, Belgique; 3Laboratoire Matrice Extracellulaire et Dynamique Cellulaire, Université de Reims Champagne-Ardenne, France; [email protected]

ORAL COMMUNICATION

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Single Molecule Force Spectroscopy Applied to Heparin-Induced Thrombocytopenia

Heparin (the most important antithrombotic drug in hospitals) binds to the endogenous tetrameric protein platelet factor 4 (PF4), form PF4/Heparin (PF4/H) complexes, which allow binding of anti-PF4/H antibodies (anti-PF4/H-ABS). Misdirection of anti-PF4/H-ABS to PF4/H complexes bridges platelets and causes the adverse drug reaction: heparin-induced thrombocytopenia (HIT). The extreme manifestation of the anti-PF4/H immune response is life-threatening autoimmune HIT. However, little is known about the characteristics of these interactions. We present here an overview of our recently achievements of HIT complication by single molecule force spectroscopy (SMFS) with the support of other biophysical techniques, e.g., Isothermal Titration Calorimetry, CD spectroscopy, Scanning electron microscopy, Scanning laser microscopy, and ELISA. The investigated interactions include: heparin drugs with PF4;1,2 patients heparin-dependent anti-PF4/H-ABS and pathologic anti-PF4/H-autoantibodies with PF4, and PF4/H complexes;3 and single platelets with other single platelets exposing different degree of activation.4 For heparin drugs, the boundary between antigenic and non-antigenic heparin drugs was determined between 8- and 12-mers. We introduced a model for long Heparin/PF4 interaction, that is, it is not only due to charge differences as generally assumed, but also due to additional interaction between two PF4s, which are brought closely to each other by long heparin, induced more stable complexes than other ligand-receptor interactions. For heparin-dependent anti-PF4/H-ABS, their interaction with PF4/H complexes is weaker than pathologic anti-PF4/H autoantibodies. Like heparins, anti-PF4/H autoantibodies induced a conformational change in PF4, allowing binding of heparin-dependent anti-PF4/H-ABS. These results provide a new mechanism of human antibody-mediated autoimmunity: a small subset of autoantibodies changes the conformation of an endogenous protein, thereby catalyzing binding of physiologic antibodies, recruiting them into an autoimmune process. For measurements of single platelet interactions, we could avoid rapid surface-activation of platelets and manipulate the degree of platelet activation by surface modification. We found that the rupture force increased proportionally to the degree of platelet activation, but reduced with blockade of specific receptors. Our results revealed that SMFS in combination with other biophysical techniques allowed characterizing the unknown interactions/mechanisms in HIT. It can be also

applied to characterize other biological complications.

TH Nguyen1,2*, M Decea1,2, and A Greinacher1 1Institut für Immunologie und Transfusionsmedizin, Universitätsmedizin Greifswald, 17475 Greifswald, Germany; 2ZIK HIKE - Center for Innovation Competence, Humoral Immune Reactions in Cardiovascular Diseases, University of Greifswald, 17489 Greifswald, Germany.

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1. Nguyen, T.H., Greinacher, A. & Delcea, M. Quantitative description of thermodynamic and kinetic properties of the platelet factor 4/heparin bonds. Nanoscale 7, 10130-10139 (2015).2. Kreimann, M., et al. Binding of anti-platelet factor 4/heparin antibodies depends on the thermodynamics of conformational changes in platelet factor 4. Blood 124, 2442-2449 (2014).3. Nguyen, T.H., Medvedev N., Delcea, M., Greinacher, A. . Antibodies as Catalyzer of Autoimmunity: Qualitative Characterization of Anti-PF4/Polyanion Antibodies with Different Reactivity Patterns Provides Evidence for a Basic Mechanism in Antibody-Mediated Autoimmunity (2016) submitted.4. Nguyen, T.H., Raghavendra, P. , Bui, V-C., Medvedev, N., Delcea, M., Greinacher, A. . Rupture Forces among Human Blood Platelets at different Degrees of Activation (2016) submitted.

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The tumor microenvironment modulates the strength of cell-cell contacts in human melanoma

An early step in the metastatic cascade is the detachment of cells from the primary tumor. The strength of cell-cell adhesion might therefore predict tumor malignancy since low interactive adhesion forces might promote cell detachment, migration, invasion and metastasis. Tumor cell migration and invasion require a well-balanced cell-matrix adhesion. In this context, local pH-regulation, mediated by an ion transporter located in the plasma membrane, the Na+/H+ exchanger NHE1, is critical for melanoma cell migration and cell-matrix adhesion. We hypothesize that the presence and/or function of NHE1, represented by pH changes at the cell surface due to H+ export, can also modulate cell-cell-adhesion as an early step in the formation of metastasis.We used human melanoma cells (MV3) transfected with an NHE1 expression vector or NHE1-deficient cells. Absolute adhesive interaction forces between single living melanoma cells were quantified by mechanically separating individual cells using Atomic Force Microscopy (CellHesion® setup JPK). Studying human melanoma cells with different NHE1 expression levels at different extracellular pH (pHe) values revealed the impact of NHE1 on cell-cell adhesion. Studies on the single cell level were supported by multicellular cell aggregation assays (CAA). Molecular biology and immunofluorescence stainings were conducted to unravel a possible contribution of pHe-dependent adhesion molecules. NHE1 overexpression or extracellular acidification both reduced cell-cell adhesion strength, characterized by a reduction of the maximum pulling force necessary to separate two individual cells when compared to control conditions. This is consistent with cell aggregation assays where NHE1-overexpressing cells and cells kept in an acidic environment no longer formed stable tumor spheroids. The cell adhesion molecules ALCAM and MCAM showed NHE1-dependent expression. Cadherins did not contribute to cell-cell adhesion in our model, whereas MV3 cells with a knockdown of β1-integrin did not form tumor spheroids in the CAA. Our results offer a quantitative BioAFM approach that might be predictive for metastasis in that low cell-cell adhesion could lead to increased detachment of cells from the primary lesion. We showed that NHE1 reduces the strength of cell-cell contacts in human melanoma. These effects are contrary to the impact on cell-matrix interactions which are strengthened by protons extruded via NHE1. We conclude that NHE1 promotes metastasis by first facilitating the detachment of cells from the primary tumor, followed by a modulation of cell-matrix interactions to promote cell migration and invasion. Therefore, NHE1 could serve as a target protein to predict melanoma malignancy and to modify melanoma cell aggressiveness.

V Hofschröer1, FT Ludwig1, K Koch1, C Stock2 and A Schwab1

1Institute of Physiology II, University of Münster, Münster, Germany; 2Department of Gastroenterology, Hannover Medical School, Hannover, Germany; [email protected]

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Stiffer Intercalated Discs and Thicker Extracellular Matrix Maintains Contractile Function of the Aged Heart

Cardiac myocytes are capable of functioning for decades despite minimal cell turnover or regeneration, suggesting that molecular alterations help sustain heart function with age. Identification of compensatory remodeling events in the aging heart remains elusive. Vinculin acts a unique regulator of cytoskeletal mechanics during cardiac aging: increased cardiac vinculin expression reinforces the cortical cytoskeleton, as measured by cortical stiffness via atomic force microscopy, enhances myofilament organization, improves cellular contractility, elevates hemodynamic stress tolerance, and leads to prolonged lifespan in fly. Though alterations in cortical mechanics are often associated with impaired function, these findings suggest that cytoskeletal reinforcement may compensate for dysfunction in the aging heart. Moreover they prolong the amount of time that the sarcomeres, i.e. the contractile portion of the cell, are organized and highly crystalline, which extends lifespan by more than 2-fold. Conversely, when the extracellular matrix (ECM) that surrounds heart cells is thinner but without a change in stiffness, it mechanically couples myocytes together better and improves their contractility. This also leads to an ECM protein specific response, i.e. knockdown of LamininA results in greater couple and thus longer lifespan than Pericadin or Viking (both collagen IV-like proteins). Together these data imply a tighter link between stiffness, cell structures, heart function, and overall lifespan than previously appreciated.

Gaurav Kaushik1, Alice Spenlehauer1, Ayla O. Sessions2, Adriana S. Trujillo3, Alexander Fuhrmann1, Sanford I. Bernstein3, Anthony Cammarato4, and Adam J. Engler1,2,5

1Department of Bioengineering and 2Biomedical Sciences Program, University of California, San Diego; La Jolla, CA USA; 3Department of Biology, San Diego State University; San Diego, CA USA; 4Department of Medicine, Johns Hopkins University; Baltimore, MD USA; 5Sanford Consortium for Regenerative Medicine; La Jolla, CA USA;

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Anticancer drugs increase disorder and reduces complexity in the macrophage membrane

Macrophages are part of the immune system and play critical role in the host defense and tissue homeostasis ingesting a dead tissue and fighting invading pathogens. Exploring interactions of anticancer therapies with macrophages as part of innate immunity is important both for biomedical researches and applications and can help to combine various approaches with immunotherapy. In this study we evaluated the influence of anticancer drugs colchicine and taxol on the disorder and complexity of the macrophage membrane from atomic force microscope (AFM) images. Both drugs influence cell cytoskeleton integrity and stability but have opposite effect: colchicine inhibits the microtubule polymerization and taxol is microtubule stabilizing agent. Since the cytoskeleton plays a central role in the mechanical function of macrophage adhesion, motility, and migration, it is important to elucidate the influence of both drugs on the membrane state. Mouse RAW 264.7 macrophage membranes were imaged with AFM operating in Peak Force mode. The disorder of the membrane was characterized by entropy and complexity - by fractal dimension. These parameters were calculated for a set of AFM images of untreated macrophages and macrophages pretreated with anticancer drugs colchicine and taxol, using three different methods. Processing of AFM images and calculations were done with custom MATLAB code. We show that even small concentration of both colchicine and taxol yield entropy increase and therefore produces higher disorder of the macrophage membrane, although these drugs have the opposite effect on macrophage skeleton. Furthermore the membrane complexity is also reduced in both cases showing lower fractal dimension. These results demonstrate at the level of single cell that anticancer drugs colchicine and taxol affect macrophage membrane structures producing more disordered state. Finally we discuss some possible consequences of this more disordered state on the macrophage activity.

A. Bitler 1, 2, R. Dover3 and Y. Shai3

1Physics Department and Institute of Nanotechnology, Bar Ilan University, Ramat Gan, Israel, 52900; 2Department of Chemical Research Support, Faculty of Chemistry, Weizmann Institute of Science, Rehovot, P.O.B. 26, Israel, 76100; 3Department of Biological Chemistry, Faculty of Biochemistry, Weizmann Institute of Science, Rehovot, P.O.B. 26, Israel, 76100; E-mail of corresponding author: [email protected]

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A new therapeutical approach against Pseudomonas aeruginosa: the LecA lectin/galactoclusters affinity studied by Single Molecule Force Spectroscopy

The human opportunistic pathogen Pseudomonas aeruginosa (PA) has been largely studied via AFM1,2. PA is especially responsible of the infection of human epithelial of cystic fibrosis patients, causing respiratory difficulties and in the worst case patient’s death. It has developed antibiotic resistances due to its ability of growing biofilm. To do that PA takes advantage of an arsenal of virulence factors, among them one lectin LecA which specifically recognizes the galactose molecules present on the surface of the host cell. A new therapeutic approach against PA is based on the carbohydrate inhibition of lectins that leads to a decreasing in cytotoxicity and cell survival3.Good candidates for carbohydrate inhibition are the galactoclusters, synthesized molecules which present higher affinity to LecA than the natural carbohydrates. By taking advantage of the higher affinity and the galactocluster effect4, we assume that these molecules will link to the lectin and inhibit the PA adhesion and the formation of the biofilm5. It has been demonstrated that the design of the galactocluster influences the interaction:6,7 designing multivalent galactoconjugates that perfectly fit the topology of binding sites will increase their affinity toward the target, and help in fine-tuning their specificity.8

In this study, AFM has been used to study LecA/galactocluster binding with the aim of characterizing the interaction at the nanoscale. Among 300 synthetized galactoclusters, three of them were selected and their affinity with LecA was measured by single-molecule force spectroscopy. The binding force of single lectin/galactocluster pairs were measured and compared according to the architecture of the galactocluster and its dissociation constant value Kd measured by DNA-directed Immobilization (DDI) microarray.9

1. Camesano, T. A. An investigation of bacterial interaction interaction forces and bacterial adhesion to porous media (Ph.D. Thesis Pennsylvania State University, 2000).2. Beaussart, A. et al. Nanoscale Adhesion Forces of Pseudomonas aeruginosa Type IV Pili. ACS Nano 8, 10723–10733 (2014).

F. Zuttion1, D. Sicard1,2, C. Ligeour3, Y. Chevolot1, F. Morvan3, S. Vidal4, J. J. Vasseur3, and M. Phaner-Goutorbe1 1INL- Institut des Nanotechnologies de Lyon (UMR CNRS 5270), Ecole Centrale de Lyon, Université de Lyon, Ecully, France; 2Current address: Mayo Clinic Research, Rochester, Minnesota, USA; 3IBMM-Institut des Biomolécules Max Mousseron, UMR 5247 CNRS,UM, ENSCM, Université de Montpellier, France; 4ICBMS- Institut de Chimie et Biochimie Moléculaires et Supramoléculaires (UMR CNRS 5246), Laboratoire de Chimie Organique 2-Glycochimie, Université Claude Bernard Lyon 1, Université de Lyon, Villeurbanne, France; ([email protected])

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3. Chemani, C. et al. Role of LecA and LecB lectins in Pseudomonas aeruginosa-induced lung injury and effect of carbohydrate ligands. Infect. Immun. 77, 2065–2075 (2009).4. Lundquist, J. J. & Toone, E. J. The cluster glycoside effect. Chem. Rev. 102, 555–578 (2002).5. Ligeour, C. et al. Mannose-centered aromatic galactoclusters inhibit the biofilm formation of Pseudomonas aeruginosa. Org. Biomol. Chem. 13, 8433–8444 (2015).6. Casoni, F. et al. The influence of the aromatic aglycon of galactoclusters on the binding of LecA: a case study with O-phenyl, S-phenyl, O-benzyl, S-benzyl, O-biphenyl and O-naphthyl aglycons. Org. Biomol. Chem. 12, 9166–9179 (2014).7. Sicard, D. et al. AFM investigation of Pseudomonas aeruginosa lectin LecA (PA-IL) filaments induced by multivalent glycoclusters. Chem. Commun. 47, 9483–9485 (2011).8. Bernardi, A. et al. Multivalent glycoconjugates as anti-pathogenic agents. Chem. Soc. Rev. 42, 4709–4727 (2013).9. Dupin L. et al. Effects of the Surface Densities of glycoclusters on the determination of their IC50 and Kd value determination by using a Microarray, ChemBioChem, 16, 2329-2336 (2015).

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How endothelial cells can feel hydrostatic pressure

Hydrostatic pressure is a mechanical force which influences vascular endothelial cells together with shear and tensile stress. We have shown that hydrostatic pressure affects the endothelial cortical cytoskeleton and cellular response depends on its duration. Acute pressurization (1h 100 mmHg) leads to actin-independent and reversible stiffening. In this project we focused on the understanding of molecular mechanisms of pressure action and attempt to identify possible “pressure sensors” in endothelial cells. Blocking non-muscle myosin II with application of 5µM blebbistatin leads to complete dissipation of pressure-related cortical stiffening: “pressurized” cells have cortical elasticity of 0.52 ± 0.01 kPa, control cells - 0.28 ± 0.007 kPa, combination of blebbistatin with elevated pressure results into cortical elasticity of 0.22 ± 0.02 kPa. To verify the central role of myosin in cortical stiffening, we prepare laser ablation experiments to quantify lateral tension within the cortical acto-myosin web. A pulsed UV laser was used to cut single acto-myosin filaments in the cell cortex and further subsequent analysis of filament retraction velocity can be used as a read-out for lateral forces within the dynamic cortical web. In unpressurized cells (ambient control), acto-myosin filaments exhibit a retraction velocity of 480 ± 15 nm/sec. Subsequent to pressurization, retraction velocity was increased to 632 ± 24 nm/sec. Hence, our data indicate that endothelia active myosin in the acute response to elevated pressure. As myosin activity is mainly increased by Ca2+-dependent myosin light chain phosphorylation, mechanosensitive Ca2+-channels (MSCs) are promising candidates in the search for the actual pressure sensor. To test involvement of MSCs in pressure-sensing we quantified pressure-dependent lateral tension in presence of GsmTx-4, a known inhibitor of mechanosensitive Ca2+ channels. While application of 5 μM GsmTx-4 has no effect at ambient pressure, it reduced the pressure-dependent increase in filament retraction velocity from 632 ± 24 to 525 ± 21 nm/sec. However, GsmTx-4 is known to block also a wide spectrum of voltage-gated Na+ channels. To test whether another family of putative MSCs, i.e. the DEG/ENaC family, may play a role in pressure sensing, we applied the blocker amiloride. Remarkably, upon exposure to 100 mmHg for 1h, amiloride even reduced slightly the retraction velocity. Moreover, atomic force experiments show that application of 0.1 µM benzamil (analog of amiloride) prevents pressure-related cortical stiffening and cortical elasticity stays close to control level- 0.27 ± 0.008 kPa. Taken together, blocking mechanosensitive cation channels prevents the pressure-dependent increase in cortical lateral tension. Therefore, we postulate that ENaC and/or mechanosensitive ion channels (TRP) act as endothelial pressure sensors mediating an activation of myosin acute response to elevated pressure.

Valeria Prystopiuk1, Hans Oberleithner1,Johannes Fels2

1Institute of Physiology II, University of Münster, Germany; 2Institute of Cell Dynamics and Imaging, University of Münster, Germany; Corresponding e-mail: [email protected]

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Measuring viscoelastic parameters of cells directly from atomic force microscopyforce-distance curves

Atomic force microscopy (AFM) has become a powerful tool for the analysis of mechanical properties of polymer samples and biological objects such as cells and tissues. Typically, the mechanical properties of samples are evaluated during nanoindentation experiments, when force versus distance curves (force curves, FCs) are collected. Commonly the Hertz model or its modifications are applied to the approach part of FCs to extract Young’s modulus (E), the elastic parameter used for the characterization of the sample’s mechanical properties. However, such models assume the purely elastic nature of the sample, while in reality most biological samples are viscoelastic. Viscoelasticity of such samples reveals in the hysteresis between approach and retraction part of FCs; the indentation speed dependence of E values extracted from FCs with the Hertz model; the force relaxation at constant indentation depth and creep at constant loading force. Studying viscoelasticity in micro and nano scale is crucial to shedding new light on the understanding of cell biomechanics. For example, measured viscoelastic parameters can provide model-independent information about the metastatic potential of the cell line (Rother et al., 2014).While several methods have been proposed to obtain viscoelastic properties of samples with AFM both in time and frequency domain, they generally require modifications in the AFM apparatus and/or in the data acquisition protocol. Here we propose a new method to extract viscoelastic properties of soft samples like cells and hydrogels directly from FCs. Based on the results of Ting (1966), who succeeded in solving analytically, in an implicit form, the problem of the indentation of a linear viscoelastic half-space by a rigid axisymmetric indenter for any load history, we developed an algorithm for reconstruction of the relaxation function of the sample using force and indentation time histories. Our approach can be applied to FCs without an initial assumption about the form of relaxation function. However, we found that for cells the relaxation function was described well with single power-law behavior, with exponents in the range 0.1–0.4, varying with cell type. With obtained relaxation functions and Ting equations we were able to model FCs and they were in good agreement with the experimental FCs, capturing the hysteresis between approach and retraction. We found that benign cells (NIH 3T3 fibroblasts) had larger exponent values than malignant cells (MDA-MB231 and MCF-7 breast cancer cell lines). Overall, obtained values were very close to such obtained in the previous work in the frequency domain for the same cell types (Rother et al., 2014). Observed here power-law rheological behavior confirms the emerging description of the cell as an active soft glassy material (Kollmannsberger and Fabry, 2011). Proposed here approach can be used for automated processing of FCs. The relaxation function can be extracted directly and any viscoelastic models can be adapted for relaxation description.

Yu.M Efremov1,2, A Raman1,2 1Birck Nanotechnology Center, Purdue University, West Lafayette, Indiana 47907, United States; 2School of Mechanical Engineering, Purdue University, West Lafayette, Indiana 47907, United States; [email protected]

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ReferencesRother, J. et al. (2014). Atomic force microscopy-based microrheology reveals significant differences in the viscoelastic response between malign and benign cell lines. Open Biology, 4(5), 140046. Ting, T.C.T. (1966). The contact stresses between a rigid indenter and a viscoelastic half-space. Journal of Applied Mechanics, 33(4), 845–854. Kollmannsberger, P., & Fabry, B. (2011). Linear and Nonlinear Rheology of Living Cells. Annual Review of Materials Research, 41(1), 75–97.

Stress-strain curve of a single antibody in liquid

Understanding the mechanical functionalities of complex biological systems requires the measurement of the mechanical compliance of their smallest components. Here, we develop a force microscopy method to quantify the softness of a single antibody pentamer by measuring the stress-strain curve with force and deformation resolutions, respectively, of 5 pN and 50 pm [1]. The curve shows three distinctive regions. For ultrasmall compressive forces (5-75 pN), the protein’s central region shows that the strain and stress are proportional (elastic regime). This region has an average Young modulus of 2.5 MPa. For forces between 80 and 220 pN, the stress is roughly proportional to

AP Perrino1, R Garcia1 1Instituto de Ciencia de Materiales de Madrid, Madrid, Spain; Email: [email protected]

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References

[1] Alma P. Perrino and R.Garcia. Nanoscale, 10.1039/C5NR07957H (2016)

the strain with a Young modulus of 9 MPa. Higher forces lead to irreversible deformations (plastic regime). Full elastic recovery could reach deformations amounting 40% of the protein height. The existence of two different elastic regions is explained in terms of the structure of the antibody central region. The stress-strain curve explains the capability of the antibody to sustain multiple collisions without any loss of biological functionality.

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Probing mechanical properties of the extracellular matrix with AFM

Cells sense and actively respond to mechanical features of their microenvironment. Moreover, mechanical cues have been shown to mediate critical cell functions including proliferation, differentiation, gene expression, contraction, and migration. Therefore, a precise definition of the mechanical properties of the extracellular matrix (ECM) is needed to further our understanding of the cell-microenvironment interplay. We use atomic force microscopy (AFM) to study nanomechanical properties of lung and heart ECM. Thin slices (10-50 μm thick) of decellularized lung and heart tissues are probed with a custom-built AFM attached to an inverted optical microscope. The Young’s modulus (E) of the ECM is computed by fitting the tip-ECM contact model to force-indentation curves recorded on the ECM. The complex shear modulus (G*) is measured by placing the tip at an operating indentation of β500 nm and superimposing small amplitude (β75 nm) multifrequency oscillations composed of sine waves (0.1-11.45 Hz). G* is computed in the frequency domain from the complex ratio between oscillatory force and indentation signals. ECM exhibits scale-free rheology with a storage modulus (G’, real part of G*) increasing with frequency as a weak power law. The loss modulus (G’’, imaginary part of G*) displays a parallel frequency dependence at low frequencies, but increases more markedly at higher frequencies. ECM rheology of both lung and heart tissues is very well characterized by a two power law model composed of a weak power law with an exponent of 0.05, accounting for a viscoelastic solid regime dominant at physiological frequencies, and a second power law with an exponent of 3/4, accounting for a viscoelastic liquid regime at high frequencies. Our AFM measurements define intrinsic mechanical properties of the ECM at the length scale in which cells sense and probe their microenvironment. Regional changes in mechanical properties of the ECM could provide differential mechanical cues to regulate the spatial distribution, differentiation and function of lung and heart cells.

Daniel Navajas1 1Institute for Bioengineering of Catalonia (IBEC), Barcelona, Spain; School of Medicine, University of Barcelona, Barcelona, Spain; CIBER of Respiratory Diseases. Madrid, Spain; [email protected]

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Funded in part by the Spanish Ministry of Economy and Competitiveness grant FIS-PI14-00280

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Biomechanical analysis ofbreast cancer cells to studyP-cadherin/SFK mechano-transduction signalling

Cell-cell adhesion molecules, such as E- and P-cadherins, have a crucial role in the maintenance of normal epithelia architecture. Molecular alterations in the mechanical balance mediated by their adhesive forces are known to promote tumorigenesis1. In breast cancer, P-cadherin overexpression activates Src family kinase (SFK) signalling and induces cell invasion by inhibiting the E-cadherin suppressive function and presenting a higher tumorigenic capability in vivo 2,3. As so, we aimed to understand the role of P-cadherin/SFK signaling in the activation of a mechanotransduction pathway associated with breast cancer invasion. For that, two main goals were determined: 1) to characterize the morphological and mechanical properties induced by P-cadherin overexpression in E-cadherin-positive cancer cells; and, 2) to evaluate these properties in P-cadherin-overexpressing cells upon dasatinib treatment, an oral FDA-approved SFK inhibitor.Two E-cadherin-positive breast cancer models have been used: MCF-7/AZ cell line, retrovirally transduced to encode P-cadherin cDNA (MCF.7/AZ.Mock and MCF-7/AZ.P-cad), and BT20 cell line, endogenously expressing high levels of P-cadherin. BT20 were transfected with small interfering RNA (siRNA) against P-cadherin. Atomic force microscopy (AFM) was performed in both cell models, with and without inhibition of SFK with dasatinib (100nM treatment). Cells were scanned and AFM images analysed, yielding maximum height, area and volume values. Differences on cell stiffness and cell-cell adhesion forces were evaluated by AFM-based force spectroscopy.Results reveal that P-cadherin overexpressing cells (MCF-7/AZ.Pcad and BT20) have significant alterations in cell morphology, with decreased cell height, as well as an increase in cell area, when compared with their controls (MCF-7/AZ.Mock and BT20 siPcad). Additionally, they present a significantly lower Young’s modulus, which indicates higher tumor cell elasticity. On cell-cell adhesion data, P-cadherin overexpressing cells presented a significantly higher force necessary for cell detachment, which presented a decrease after P-cadherin silencing in BT20 cells. After treatment with dasatinib, P-cadherin overexpressing cells show a significant increase in cell height and decrease in its area, leading to a more epithelial-like phenotype. The Young’s modulus significantly increases with dasatinib, showing a decrease in breast cancer cell elasticity. Additionally, cell-cell adhesion is highly promoted in P-cadherin overexpressing cells treated with dasatinib. This suggests that the inhibition of SFK make cells more adherent to each other, becoming more difficult to spread. Taken together, our findings demonstrate that overexpression of P-cadherin in breast cancer cells promotes alterations in their biomechanical properties, correlating with the increased migratory and invasive potential of these cells.

FA Carvalho1*, J Monteiro2, AS Ribeiro2, AF Guedes1, J Paredes2,3, NC Santos1

1Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Portugal; 2IPATIMUP, Instituto de Patologia e Imunologia Molecular da Universidade do Porto, and i3S, Portugal; 3Faculdade de Medicina, Universidade do Porto, Portugal; *[email protected]

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1. Albergaria A. et al. (2011) Int J Dev Biol 55: 811-22.2. Ribeiro A.S. et al. (2013) J Pathol 229: 705-18.3. Vieira A.F. et al. (2014) Oncotarget 5: 679-92.

These morphological and mechanical properties may be associated with the activation of the SFK signaling mediated by P-cadherin expression in an E-cadherin wild-type context. Therefore, upon treatment with SFK inhibitors such as dasatinib, the functional effect of P-cadherin is repressed, inhibiting the biomechanical alterations promoted by its overexpression.

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Morphology and Nanomechanical Properties of Murine Isolated Liver Sinusoidal Endothelial Cells

Due to its functions and location, the vascular endothelium, i.e., a tight monolayer of cells covering the inner side of the blood vessels, undergoes permanent squeezing and stretching resulting from the neighbouring environment. Dysfunction of endothelial cells is a result and/or a primary cause of many human diseases. In particular, the dysfunction of liver sinusoidal endothelial cells (LSEC) mediated by alterations in hepatocyte-derived paracrine signals is essential for the development of liver steatosis. For an accurate understanding of the functionalities and mechanical response of the cells to both the native and pathological environments it is important to have good knowledge of the mechanical properties of the endothelium at the cellular level [1,2]. LSECs differ from other classes of the endothelial cells. Their characteristic feature is the presence of discontinuity of basal lamina which appear as non-diaphragmed transcellular pores, so called “fenestrations”. They are gathered in groups of few and form sieve plates [3]. The fenestrations participate in the transfer of substances between the lumen of a blood vessel and an extravascular compartment (space of Disse) [4]. Risk factors for atherosclerosis, such as hypertension, high serum cholesterol levels, diabetes and others, lead to the endothelial dysfunction and could change both the size and the number of fenestrations. Many techniques have been applied previously for investigating LSECs. One of the most often applied is an electron microscopy [5]. It provides great resolution but structures are fixated and often dehydrated before measurement. Such processes can alter the true size of fenestrations. Recent studies show that super-resolution optical microscopy, can also be applied to visualize LSECs [6]. It is shown that single fenestrations can be resolved using labeling of the cytoskeleton. Nevertheless, those experiments were performed on fixated cells too. In our work, we focus on the results obtained using the tip of an atomic force microscope as an imaging and nanoindentation spectroscopic probe. AFM-based techniques can be used in liquid conditions and therefore, they can be applied for investigation of living cells. Moreover, they allow for getting a complex physical information from the sample in a single experiment, e.g., on morphology as well as on mechanical, or other properties. In particular, an AFM probe is often used for spectroscopic indentation force analysis, providing information about nanomechanics of the investigated biological cells [1,2]. We present results of our attempts to visualize living and fixated murine isolated LSEC using atomic force microscopy, with an aim to get appropriate resolution for resolving

B. Zapotoczny1, K. Szafranska1, K. Owczarczyk1, E. Maslak2, S.K. Chlopicki2, M. Szymonski1

1Centre for Nanometer-Scale Science and Advanced Materials, NANOSAM, Faculty of Physics, Astronomy, and Applied Computer Science, Jagiellonian University, Krakow, Poland; 2Jagiellonian Centre for Experimental Therapeutics, JCET, Jagiellonian University, Krakow, Poland;

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sieve plates and single fenestrations. Moreover, we show spectroscopic data allowing to determine the nanomechanical properties of both living and glutaraldehyde fixated LSEC cells. Finally, we report on our investigations of changes in the mechanical properties of the glutaraldehyde fixated LSEC samples with different concentrations of the fixative agent. We discuss those data in light of the recently published model developed for EA.hy926 endothelial cells [8].

FIGURE 1: Overview AFM image of a fixed sinusoidal liver endothelial cell (LSEC) that underwent brief fixation with 2% glutaraldehyde. The measurement was performed under PBS (with Mg2+ and Ca2+). Height of the cell is equal to 2 μm.

References[1] J Fels, P Jeggle, I Liashkovicha, W Peters, H Oberleithner, Cell Tissue Research, 355, 727 (2014).[2] M Szymonski, M Targosz-Korecka, KE Malek-Zietek, Pharmacological Reports: PR, 67, 728 (2015).[3] F Braet, E Wisse, Micron, 43, 1252 (2012).[4] F Braet, E Wisse, Comparative Hepatology, 1, 1 (2002). [5] F Braet, Journal of Electron Microscopy 50, 4 (2001).[6] V Mönkemöller et al., Scientific Reports 5, 16279 (2015).[7] F Braet, C Rotsch, E Wisse, M Radmacher, Applied Physics A. 66, 575 (1998). [8] M Targosz-Korecka, GD Brzezinka, J Danilkiewicz, Z Rajfur, M Szymonski, Cytoskeleton, 72, 3 (2015).

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Quantitating membrane bleb stiffness using AFM force spectroscopy and an optical sideview setup

AFM-based force spectroscopy in combination with optical microscopy is a powerful tool for investigating cell mechanics and adhesion on the single cell level. However, standard setups featuring an AFM mounted on an inverted light microscope only provide a bottom view of cell and AFM cantilever but cannot visualize vertical cell shape changes, for instance occurring during motile membrane blebbing. Here, we have integrated a mirror-based sideview system to monitor cell shape changes resulting from motile bleb behavior of Xenopus cranial neural crest cells (CNCs) during AFM elasticity and adhesion measurements. Using the sideview setup, we quantitatively investigate mechanical changes associated with bleb formation and compared cell elasticity values recorded during membrane bleb and non-bleb events. Bleb protrusions displayed significantly lower stiffness compared to the non-blebbing membrane in the same cell. Bleb stiffness values were comparable to values obtained from blebbistatin-treated cells, consistent with the absence of a functional actomyosin network in bleb protrusions. Furthermore, we show that membrane blebs forming within the cell-cell contact zone have a detrimental effect on cell-cell adhesion forces, suggesting that mechanical changes associated with bleb protrusions promote cell-cell detachment or prevent adhesion reinforcement. Incorporating a sideview setup into an AFM platform therefore provides a new tool to correlate changes in cell morphology with results from force spectroscopy experiments.

C Gonnermann1, J Kashef2 and CM Franz1

1Center for Functional Nanostructures, Karlsruhe Institut of Technology, Germany; 2Zoological Institute, Karlsruhe Institute of Technology, GermanyEmail: [email protected]

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ReferencesQuantitating membrane bleb stiffness using AFM force spectroscopy and an optical sideview setupC. Gonnermann, C. Huang, S Becker, D.R. Stamov, D. Wedlich, Jubin Kashef, C.M. FranzIntegrative Biology, 7, 356-363 (2015) Cadherin-11 localizes to focal adhesions and promotes cell-substrate adhesion in migrating neural crest cellsR. P. Langhe, T. Gudzenko, M. Bachmann, S. F. Becker, C. Gonnermann, C. Winter, G.Abbruzzese, D. Alfandari, M.-C. Kratzer, C. M. Franz, and Jubin KashefNature Communications, in press (2016)

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AFM study of diamond particles internalization by monitoring MCF7 cell membrane stiffness changes

Fluorescent nano-diamonds (fND) are attractive tools for nanoscale biological cellular imaging allowing both photoluminescence and magnetic resonance imaging [1]. Recent technological developments enable to fabricate bright fND particles of various sizes with high content of nitrogen-vacancy (NV) centres [2]. In this work we study internalization processes of NDs in the breast cancer MCF7 1DIV cell line using Atomic Force Microscopy imaging and force spectroscopy. fND particles of size range from 5 to 60 nm were used, prepared from Ib synthetic diamond, electron irradiated, annealed and plasma oxidized to create NV centers. Changes of cells stiffness were detected by AFM force measurements after introducing the fNDs into the cell medium. We observed an oscillating variation of cell membrane Young’s modulus while the cells become stiffer. We believe that repetitive uptake/release processes of fNDs are responsible for these mechanical changes. Contrarywise, more confluent MCF7 cells (3DIV) did not show any significant change in Young’s Modulus, as compare with control ones. Indeed, it has been shown that differences in nanoparticle uptake can arise from how close the cells are to one another (confluence) [3] and how old they are. Moreover, our results suggest that studies on fNDs uptake should consider the cell cycle, as in a cell population, the dose of internalized nanoparticles in each cell varies as the expression of membrane proteins vary during the cell cycle [4]. The cell cycle is a series of events that lead to cell division and replication, consisting of four phases: G1 (when the cell increases its size), S (the cell synthesizes DNA), G2 (the cell synthesizes proteins for cell division) and M (the cell divides and the two daughter cells enter the G1 phase). During each event, cellular processes can vary; meaning that the rate at which a cell takes up foreign material, as for instance nanoparticles, may depend on the phase the cell is in [4-6], modifying accordingly their elasticity. When nanoparticle internalization is studied, it is therefore crucial to resolve how the state of the different cells affects the uptake.

M. Martin1, M. Gulka2, 3, B. Varga1, 4, T. Cloitre1, P. Cígler5, M. Nesládek6, C. Gergely1

1Laboratoire Charles Coulomb (L2C), UMR 5221 CNRS-Université de Montpellier, Montpellier, F-France; 2CTU in Prague, Faculty of Biomedical Engineering, Sítná sq. 3105, 272 01, Kladno, Czech Republic; 3Institute of Physics, AS CR, v.v.i., Na Slovance 5, 185 00, Prague 8, Czech Republic; 4Institute of Biophysics, Biological Research Centre, 6726 Temesvári krt. 62, Szeged, Hungary; 5Institute of Organic Chemistry and Biochemistry AS CR, v.v.i. Flemingovo nam. 2, 166 10 Prague 6, Czech Republic; 6IMOMEC division, IMEC, Institute for Materials Research, University Hasselt, Diepenbeek, Belgium; [email protected]

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References[1] L. Moore, M. Nesladek et al., Nanoscale (2014).[2] J. Havlik, M. Gulka, M. Nesladek et al., Nanoscale (2013).[3] B. Snijder, et al. Nature 461, 520–523 (2009). [4] E. Boucrot and T. Kirchhausen, Proc. Natl Acad. Sci. USA 104, 7939–7944 (2007). [5] D. Raucher and M. P. Sheetz, J. Cell Biol. 144, 497–506 (1999). [6] J. K. Schweitzer, E. E. Burke, H. V Goodson and C. D’Souza-Schorey. J. Biol. Chem. 280, 41628–41635 (2005).

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Crowding redefines Endonuclease Recognition in Highly Dense DNA Nanoreactors

Our goal is to elucidate the biomolecular reactions in conditions that are more akin to the cell interior in terms of macromolecular crowding and confinement, and which cannot be accurately reproduced in solution due to solubilisation limits of crowding agents (R.J Ellis, 2001). In this poster, I will present the results of our study on the BamHI restriction enzyme reactions in nanoreactors consisting of self-assembled monolayers (SAMs) of short, dsDNA molecules of varying DNA density that are confined laterally by the presence of adjacent bio-inert alkylthiol SAMs over ultra-flat gold surfaces. We generate such SAM-based nanoreactors (SAM-NR) by means of an atomic force microscopy (AFM)-based nanolithography technique termed nanografting (M.Castronovo et al., 2011). We designed three distinct, 44-bp-long DNA molecules with recognition sites located in the middle. One is provided with the correct BamHI restriction site that ensures specificity for the enzyme (5’-GGATCC-3’, termed DNA-1), while the other two are provided with non-canonical sites (i.e. 5’-AGATCA-3’ and 5’-CGATCA-3’, termed DNA-2 and DNA-3 respectively). With such tools we studied BamHI action as a function of DNA density and sequence by measuring the height of the DNA monolayer before and after their reaction with BamHI in standard catalytic conditions, using AFM topography profiling, followed by correlation analysis of the individual height profiles to detect the enzyme recognition in all the different density domains of each SAM-NR. Our results show that BamHI cleaves the canonical site (DNA-1) in SAM-NR with height lower than 14 nm, above which the SAM-NR is in-accessible to BamHI due to steric hindrance. In addition, we observed two unprecedented behaviours for DNA-2 and DNA-3. For each DNA, we demonstrated three distinct regimes. At very low densities, BamHI cleaves none of the two. This is in agreement with its known high specificity for the canonical site (DNA-1) in dilute systems. For SAM-NR with thickness profiles higher than 6 nm, BamHI unexpectedly digests both non-canonical sites (DNA-2 and DNA-3). In such SAM-NR, however, the reaction reaches completion only within the lower-density domains. Finally, the enzymatic reaction is fully inhibited for SAM-NR with thicknesses higher than 12 nm for DNA-2, and 14 nm for DNA-3. Our interpretation is that the restriction enzyme is physically trapped within SAM-NR with thicknesses higher that 6 nm. Thus, the enzyme diffuse two-dimensionally in the SAM-NR and the non-canonical reactivity of DNA-2 and DNA-3 is likely an effect of nano-crowding .In conclusion, the use of nanoreactors allows studying unprecedented modes of protein-DNA recognition.

Abimbola Adedeji1,2,§, Dianne Choi3,§, Vincent Inverso 4,§, Shiv K. Redhu4, Marco Vidonis2,5, Luca Crevatin6, Allen W. Nicholson3,4, Giacinto Scoles1,2,3, Matteo Castronovo1,2,3

§ Equally contributing authors; 1 PhD School in Nanotechnology, Dept. of Physics, University of Trieste, Trieste, Italy; 2 Dept. of Medical and Biological Sciences, University of Udine, Udine, Italy; 3 Dept. of Biology, Temple University, Philadelphia, PA, USA; 4 Dept. of Chemistry, Temple University, Philadelphia, PA, USA; 5 Dept. of Chemistry, University of Rome “Tor Vergata”, Rome, Italy; 6 Dept. of Life Sciences, University of Trieste, Trieste, Italy; Corresponding author’s email: [email protected], [email protected]

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Fluorescence Tracking of Genome Release during Mechanical Unpacking of Single Viruses

Viruses package their genome in a robust protein coat to protect it during transmission between cells and organisms. In a reaction termed uncoating, the virus is progressively weakened during entry into cells. At the end of the uncoating process the genome separates, becomes transcriptionally active, and initiates the production of progeny. Here, we triggered the disruption of single human adenovirus capsids with atomic force microscopy and followed genome exposure by single-molecule fluorescence microscopy. This method allowed the comparison of immature (noninfectious) and mature (infectious) adenovirus particles. We observed two condensation states of the fluorescently labeled genome, a feature of the virus that may be related to infectivity (1). Beyond tracking the unpacking of virus genomes, this approach may find application in testing the cargo release of bioinspired delivery vehicles.

P. J. de Pablo1, A. Ortega-Esteban1, K. Bodensiek2, C. San Martín3, M. Suomalainen4, U. Greber4, I. Schaap2,5

1 Departamento de Física de la Materia Condensada, Universidad Autónoma de Madrid, Spain; 2III. Physikalisches Institut, Georg August Universität, Göttingen, Germany; 3Centro Nacional de Biotecnología CSIC, Madrid, Spain; 4Institute of Molecular Life Sciences, University of Zürich, Zürich, Switzerland; 5Institute of Biological Chemistry, Biophysics and Bioengineering School of Engineering and Physical Sciences Heriot Watt University Edinburgh EH14 4AS, UK

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References[1] Ortega-Esteban A, Bodensiek K, San Martin C, Suomalainen M, Greber UF, de Pablo PJ, Schaap IA (2015) Fluorescence Tracking of Genome Release during Mechanical Unpacking of Single Viruses. ACS Nano 9(11): 10571-10579.

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Structural insights into translation repression via AFM imaging of mRNA/protein complexes

Translation regulation plays a key role in the control of protein level, which encompasses regulation of mRNA translation initiation and ribosome processing by mRNA binding proteins. Translation regulation is of particular interest when cells are exposed to stress conditions: the expression of house-keeping proteins should be repressed while that of mandatory for the cell survival, should be enhanced. Such sorting functions have been suspected for stress granules (SGs) which are micrometric aggregates appearing in the cytoplasm of stressed cells and containing mRNA and numerous mRNA-binding proteins. The absence of membrane around these granules leaves mRNA and protein free to shuttle in and out of granules but also lead to their intrinsic instability. Due to this, they cannot be isolated for further characterization. We proposed recently to reconstitute artificially these granules in vitro using specific proteins found enriched in SGs like TIA-1 and to check, using AFM approach, if some RNA-binding proteins are able to release mRNA from these granules (Bounedjah et al. Nucleic Acids Research, 2014). We focused our study on YB-1, an mRNA-binding protein present in SGs, which forms isolated complexes with mRNA easily identifiable on AFM images. When YB-1 is added to mRNA/TIA-1 granules we observed the dissociation of the granules and the appearance of isolated mRNA/YB-1 complexes. These results were further confirmed in a cellular context after the overexpression of YB-1. YB-1 is also known to interact with specific mRNA sequences/structures and could potentially release specific mRNA from SGs. Moreover, when mRNA are saturated at elevated YB-1 concentration, (about 30 nucleotides per YB-1), translation is repressed. This suggests a link between SGs and translation regulation via YB-1. However, how YB-1 could exert its putative function in translation repression while a large amount of YB-1 is required to stop translation remains elusive. One possibility is that YB-1 does not bind to mRNAs homogeneously but rather accumulates on specific mRNA targets via cooperative binding. Using AFM in combination with gel mobility shift assays, we determined the characteristics of the binding of YB-1 to mRNA and the different YB-1 domains involved in this process (Kretov et al. Nucleic Acids Research, 2015). These results presented offer a novel view on translation repression orchestrated by YB-1 or other mRNA binding proteins.

D.A. Kretov, S. Abrakhi, D. Pastre, L.HamonLaboratoire Structure-Activité des Biomolécules Normales et Pathologiques, INSERM U1204 and Université d’Evry, Université Paris-Saclay, [email protected]

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Proteins sense different grain orientations in hydroxyapatite during adsorption

Protein adsorption is the first step of biofilm formation and can therefore be highly desirable or unwanted. Characterizing protein adsorption on different types of very controlled substrates enables us to gain insight into the governing forces [1]. In this new study, we used hydroxyapatite (HAP) pellets as a model system for tooth enamel. The pellets reach a density of > 97 % of the theoretical crystallographic density of HAP and have been produced by compacting and sintering commercially available HAP powder. They consist of micron-sized crystalline grains of different orientation. Atomic force microscopy (AFM) combined with electron backscatter diffraction (EBSD) measurements reveal the smoothness and the crystal orientation of the HAP grains on the surface of the pellets. On these surfaces, single molecule BSA adsorption experiments are performed in a microfluidic setup revealing that different grain orientations provoke different adsorption rates. These findings open a pathway to control protein adsorption.

T Faidt1, J Schmauch1, MJ Deckarm1, S Grandthyll1, F Müller1, K Jacobs1,*1Department of Experimental Physics, Saarland University, Germany*Correspondence: [email protected]

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References[1] H. Hähl et al., Langmuir 28 (2012) 7747.

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Probing mechanical properties of young and senescent human fibroblasts: a critical comparison of Force Volume and Peak Force QNM

AFM is used in biology to observe high-resolution topography of cells, bacteria or supported membranes1, to measure adhesion and specific interactions2 and to study cell elastic and viscoelastic properties3,4. A great effort is being devoted to the development of high speed AFMs, both for pure imaging5,6 and for force curves – based techniques7. To establish force maps, several thousands of curves should be acquired. Speeding up measurements increases their statistical weight (i.e. higher number of curves per cell and higher number of cells analyzed). Moreover, lower time for high resolution maps is particularly critical to analyze leaving cells. Recently, “fast” force mapping modes emerged, e.g. Quantitative Imaging (QI™) by JPK and Fast Force Volume (F-FV) and PeakForce QNM® (PF-QNM)8 by Bruker. While the first two are standard force curve – based techniques i.e. adopting linear Z ramping, PF-QNM uses a sinusoidal modulation of the base of the cantilever relative to the sample surface. PF-QNM provides precise force control and high working speed (i.e. ~1’ for the acquisition of a 10µm square map with 256x256 curves at 1 kHz) but still awaits to be validated for cell physics on many different biological systems. The study highlights the physical/methodological differences between FV and PF-QNM mainly related to the calibration of the real piezo displacement and to the synchronization of the force peak. We systematically analyzed the influence of the experimental setup, scanning parameters (like PF-QNM amplitude and frequency) and calibration procedure (touch and no-touch modalities) on the resulting PF-QNM curves and on the elasticity of eukaryotic cells. Shape of peak force curves and noise as a function of PF frequency and background subtraction are also reported along with how to properly import PF-QNM curves into external software. We will discuss the results obtained comparing these modes when analyzing the elasticity (Young’s modulus) of young and senescent human primary fibroblasts. After a certain number of duplication a cell population stops dividing: at this stage cells are considered as senescent. Senescent cells are involved into the development of many pathological conditions related to the aging process, and offer a suitable model to investigate important issues related to cancer9.

S. Bovio1, V. Dupres1, N. Martin2 C. Abbadie2 and F. Lafont1

1Cellular Microbiology and Physics of Infection Group, Center of Infection and Immunity of Lille - Institut Pasteur, CNRS UMR8204, INSERM U1019, regional Hosp. Center of Lille, University of Lille, France; 2M3T – Mechanisms of Tumorigenesis and Target Therapies, University of Lille, Institut Pasteur, UMR 8161 CNRS, Lille, France; [email protected]

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References1 Y. Dufrêne et al. Nature Methods 10, 847 (2013)2 D.J. Müller et al. Nature Chemical Biology 5, 383 (2009)3 K. Haase and A.E. Pelling J. R. Soc. Interface 12, 20140970 (2015)4 J. Alcaraz et al. Biophysical Journal 84, 2071 (2003)5 T. Ando Current Opinion in Structural Biology 28, 63 (2014)6 A. Colom et al. Nature Communications 4, 2155 (2013)7 F. Eghiaian et al. FEBS Letters 589, 1389 (2015)8 B. Pittenger et al. Bruker Application Note #128 (2011)9 J. Campisi Annu. Rev. Physiol. 75, 685 (2013)

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Detection of endothelial dysfunction in ex vivo vessels from ApoE/LDLR-/- mice: A multimodal approach

The functionality of the vascular endothelium is strongly influenced by the nanomechanics of the endothelial cortex, an actin-rich region 50-150 nm beneath the plasma membrane. A shift from G- to F-actin results in a stiff cortex and subsequently in reduced release of the vasodilating gas nitric oxide (NO), which is a hallmark for endothelial dysfunction and associated with cardiovascular diseases. Recently, the endothelial sodium channel EnNaC could be identified as being crucial for the mechanical stiffness in that it interacts with the cortical actin, i.e. the more EnNaC, the stiffer the cortex of the endothelial cells (EC). On the other hand, functional inhibition of EnNaC (i.e. Na+ influx into the cell) with amiloride or benzamil induces cortical softening (shift from F- to G-actin) and thus improves endothelial function. Interestingly, in a clinical trial we found that 50% of the probed ex vivo preparations from human vessels showed an abolished softening after amiloride exposure. Further, an association between impaired response to EnNaC inhibition and two predictors of cardiovascular risk (increased pulse wave velocity and impaired renal function) was identified in these patients. Thus, it is postulated that the regulation of EnNaC-dependent cortical plasticity is disturbed within endothelial dysfunction. However, the underlying mechanisms are not understood yet. To link EnNaC function, endothelial nanomechanics and the development of endothelial dysfunction a combined method was established to investigate (i) the mechanical properties of living ECs from ex vivo mouse aortae with an Atomic Force Microscope (AFM) and (ii) the biochemical composition with Raman spectroscopy (RS) within the very same aortic area. Aortae of either wildtype (WT) or ApoE/LDLR-/- mice were used, whereas the latter represent a reliable model of atherosclerosis and severe endothelial dysfunction. Ex vivo patches of dissected aortic rings were fixed onto glass-dishes with the ECs facing upwards and studied with a combined setup of AFM and RS. Initially, single force-distance-curves were performed on the ECs to detect the cortical stiffness. Subsequently, the biochemical composition was determined with RS. Within this method, the scattered light of a laser is detected, which is shifted in energy because of interactions between light and matter (Raman scattering). By plotting the shift intensity against the frequency a specific spectrum of the biochemical contents of the samples can be recorded. RS and AFM revealed an increased lipid content (+17%) and cortical stiffness values (+16%), respectively in the ApoE/LDLR-/- ECs compared to WT. Benzamil (1µM) decreased the cortical stiffness in WT ex vivo ECs, but not in the ApoE/LDLR-/-.

Martina Maase1, Anna Rygula3, Magdalena Sternak3, Agnieszka Serwadczak3, Marta Pacia2,3, Malgorzata Baranska2,3, Stefan Chlopicki3, Kristina Kusche-Vihrog1

1Institute of Physiology II, University of Münster, 48149 Münster, Germany; 2Faculty of Chemistry, Jagiellonian University, 30-060 Krakow, Poland; 3Jagiellonian Centre for Experimental Therapeutics (JCET), Jagiellonian University, 30-348 Krakow, Poland; Email corresponding author: [email protected]

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This finding supports the results obtained in human arteries, and gives another hint, that the regulation of EnNaC-dependent cortical plasticity is disturbed within endothelial dysfunction. Together, we identified two markers for endothelial dysfunction in the ApoE/LDLR-/- ECs, since hyperlipidemia is a known risk factor for the development of atherosclerosis and an increased cortical stiffness results in reduced NO production. Although the mechanisms behind these observations are not known yet, the established combination of AFM and RS allows to study various aspects of EC function/dysfunction. Thus it will be possible to shed light on the pathways leading to endothelial dysfunction and disturbed EnNaC regulation in the future.

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Early Impairment of Lung Mechanics in a Murine Model of Marfan Syndrome

Early morbidity and mortality in patients with Marfan syndrome (MFS) –a connective tissue disease caused by mutations in fibrillin-1 gene- are mainly caused by aorta aneurysm and rupture. However, the increase in the life expectancy of MFS patients recently achieved by reparatory surgery promotes clinical manifestations in other organs. Although some studies have reported respiratory alterations in MFS, our knowledge of how this connective tissue disease modifies lung mechanics is scarce. Hence, we assessed whether the stiffness of the whole lung and of its extracellular matrix (ECM) is affected in a well-characterized MFS mouse model (FBN1C1039G/+). We studied 5-week and 9-month old mice, whose ages are representative of early and late stages of the disease. The compliance of the whole lung was measured in vivo by conventional mechanical ventilation (100 breaths/min, room air, 10 ml/kg) and the viscoelastic properties of the ECM were evaluated in different regions of decellularized lung scaffold with atomic force microscopy (pyramidal cantilever, 0.03 N/m nominal spring constant and multifrequency oscillations). Histological sections, obtained from additional mice, were stained with hematoxylin-eosin and quantitative analyses were carried out measuring interseptal air spaces. At both ages, the lungs of MFS mice were significantly more compliant than in wild type (WT) mice (~35%). By contrast, no significant differences were found in local lung ECM viscoelastic properties. Moreover, histopathological lung evaluation showed a clear emphysematous-like pattern in MFS mice since alveolar space enlargement was significantly increased (36% and 65% at 5-week and 9-month respectively) compared with WT mice. These data suggest that the mechanism explaining the increased lung compliance in MFS is not a direct consequence of reduced ECM stiffness, but an emphysema-like alteration in the 3D structural organization of the lung.

Juan J. Uriarte1,2,3, Thayna Meirelles4, Darya Gorbenko del Blanco4, Paula N. Nonaka1,5, Noelia Campillo1,2,3, Elisabet Sarri4, Gustavo Egea4,6,7, Ramon Farré1,2,7, Daniel Navajas1,2,3.

1Unitat Biofísica i Bioenginyeria, Universitat de Barcelona, Barcelona, Spain2CIBER de Enfermedades Respiratorias, Madrid, Spain; 3Institute for Bioengineering of Catalonia, Barcelona, Spain; 4Departament de Biologia Cellular, Universitat de Barcelona, Barcelona, Spain; 5Nove de Julho University, Sao Paulo, Brazil; 6Institut de Nanociències i Nanotecnologia, Universitat de Barcelona, Barcelona, Spain; 7Institut d’Investigacions Biomèdiques August Pi Sunyer, Barcelona, Spain; Email: [email protected]

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Supported by Ministerio de Economia y Competitividad (PI11/00089 and PI14/00004)

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Biomolecular mechanisms in Alzheimer’s disease: investigation by high speed Atomic Force Microscopy and nanoInfrared spectroscopy of the interaction between toxic amyloid peptides and model membranes

Alzheimer’s disease is the most common neurodegenerative disease, leading to 50% of dementia cases, caused by the aggregation of an amyloid, the peptide in patient brain. Many studies link the toxicity of amyloids, as involved in Alzheimer disease, to the existence of various intermediate structures prior to fiber formation and /or their specific interaction with membranes.In this presentation, we focused on the interaction between membrane models and peptides and mutants more or less toxic as the presence of membrane can play the role of catalyst during the self-assembly of amyloid. Several mechanisms are reported for explaining the interaction between toxic amyloid and membranes [1-3] but lots of uncertainties are remaining to fully understand the whole process. As instance, the molecular mechanism of interaction of peptide with membranes is far from being elucidated and major contradictions exist depending on the choice of the peptides and/or the target membranes. If fluorescence and electronic microscopies are already used to study the interactions between amyloids and membranes [4], these methods of investigation did not enable to couple high resolution information, work in physiological environment and a suitable time-resolution at the biomolecular level. The development of new atomic force microscopy (AFM) modes such as nanoinfrared absorption spectroscopy [5] or high speed AFM now offers the possibility to get information related to the chemical and morphological composition of wild-type or mutant peptides, and biomimetic membranes and then, to observe the dynamics of interaction between peptides and membranes at the nanoscale [6]. In this study, new lipid composition GM1, cholesterol, sphingomyelin and POPC has been chosen to mimic neuronal membranes and nanoscale infrared spectroscopy and high speed AFM were used to assess to the morphology and the secondary structure of the peptides in presence of membrane and to observe the dynamic of this interaction. The results obtained show that the gangliosides GM1 and the cholesterol play a central role in the interaction of Aβ with membranes.

M Ewald1, S Henry2, N. Bercu1, C. Culin2, S Lecomte2, M. Molinari1

1LRN EA4682– Laboratoire de Recherche en Nanosciences, Université de Reims Champagne Ardenne, France; 2CBMN - UMR CNRS 5248 Chimie et Biologie des Membranes et Nanoobjets, Université de Bordeaux, [email protected]

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We were able to propose a model of the interaction mechanism: firstly accumulates on the GM1 domains present in the membrane via hydrogen bonding and then inserts the membrane in the cholesterol enriched domains. Cholesterol and gangliosides are required for the interaction of with membrane.

References[1] Williams T.L. and Serpell L.C. Membrane and surface interactions of Alzheimer’s Abeta peptide-insights into the mechanism of cytotoxicity. FEBS J., 278(20) :3905-3917, 2011.[2] Butterfield S.M. and Hilal A. Lashuel. Amyloidogenic protein-membrane interactions : mechanistic insight from model systems. Angew. Chem. Int. Ed. Engl., 49(33) :5628_5654, 2010.[3] Kotler S.A., Walsh P., Brender J.R., and Ramamoorthy A. Differences between amyloid-beta aggregation in solution and on the membrane : insights into elucidation of the mechanistic details of Alzheimer’s disease. Chem Soc Rev, 43(19) :6692-6700, 2014.[4] Hilal A. Lashuel. Membrane Permeabilization : A Common Mechanism in Protein-Misfolding Diseases. Sci. Aging Knowl. Environ., 2005(38) :pe28, September 2005.[5] Dazzi A., Prazeres R., Glotin F., and Ortega J. M. Local infrared microspectroscopy with subwavelength spatial resolution with an atomic force microscope tip used as a photothermal sensor. Opt Lett, 30(18) :2388_2390, 2005.[6] Ando T., Kodera N., Takai E., Maruyama D., Saito K. and A. Toda. 2001. Proc. Natl. Acad. Sci. USA 98 :12468.12472.

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Single molecule force spectroscopy study for curli-mediate bacterial adhesion

The microbial functional amyloids called curli are the major feature of complex extracellular materials (ECM) of Escherichia coli strains and implicated in many physiological and pathological processes of E. coli, including epithelial attachment and tissue invasion. Recent research indicates that amyloids of human and animal pathogens might facilitate colonization of host tissue by binding the ECM component fibronectin and subsequently activating the fibrionlytic and contact systems of haemostasis [1]. In a recent study we used scanning probe microscopy to determine the effects of curli on the topology and on the mechanical properties of live E. coli cells [2]. Here, so as to identify and characterize the curli binding site on fibronectin, various fibronectin constructs varying in size were coupled onto silanized silicon nitride AFM tips employing specially tailored chemistry protocols. We show that a specific interaction between the bacterial curli protein CsgA and the RGD domain of fibronectin leads to tight binding. Via single-cell force spectroscopy we measured distinct interaction forces on the surface between the isolated CsgA protein or the bacteria-producing CsgA curli and full length fibronectin, fibronectin domain III, and RGD peptide, respectively. These experiments demonstrate that the forces required to break adhesion are quantized and reflect formation of a dense collective network. These networks comprise at least 10 specific molecular bonds that lead to tight bacterial binding. Our results underline the significance of amyloid formation on bacterial surfaces as multi-bond structural components in curli-mediated bacterial adhesion to fibronectin.

YJ Oh1, M Hubauer-Brenner1,2, HJ Gruber1, Y Cui3, L Traxler1, S Park3,4, P Hinterdorfer1,2

1Institute of Biophysics, Johannes Kepler University Linz, Austria; 2Center for Advanced Bioanalysis, Linz, Austria; 3Mechanobiology Institute, National University of Singapore, Singapore; 4School of Mechanical Engineering, Sungkyunkwan University, Korea; ([email protected])

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References1) M. F. B. G. Gebbink, D. Claessen, B. Bouma, L. Dijkhuizen and H. A. B. Wösten, “Amyloids-a functional coat for microorganisms”, Nature reviews microbiology, 3 (2005) 333.2) Yoo Jin Oh, Yidan Cui, Hyunseok Kim, Yinhua Li, Peter Hinterdorfer, and Sungsu Park, “Characterization of Curli A production on living bacterial surfaces by scanning probe microscopy”, Biophysical Journal, 103 (2012) 1666.

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The impact of hyperglycemia on adhesion between endothelial and cancer cells

Epidemiological studies indicate that people with diabetes are at significantly higher risk for cancer. However, the links between those two diseases are not clear. Furthermore, it is suggested that some drugs used by diabetic patients can either increase, or reduce the risk of cancer [1].In this work, adhesive interactions between Primary Pulmonary Artery Endothelial Cells (PHAEC) and lung carcinoma cells (A549) are investigated by means of a single-cell AFM spectroscopy using a living cancer cell immobilized on a tip-less AFM cantilever probe. Such a cellular probe is used to perform force spectroscopy on the endothelial cells grown at normal glucose levels and in hyperglycemic conditions. The acquired force-distance curves provide information about the total adhesion and about the specific ligand-receptor interactions.Experiments are performed for the cells cultured in static and flow conditions, and for different high glucose exposures. A statistically significant increase of adhesion between the cancer and the endothelial cells are observed for the hyperglycemic cells. This effects is correlated with the impairment of the endothelial glycocalyx layer. In addition, the effect of hyperglycemia on the cell adhesion is compared with the effect of TNF-β and VEGF stimulations, that are well know pro-cancer factors. Finally, a role of the anti-diabetic drug Metformin on the cell adhesion is demonstrated.

Katarzyna Malek-Zietek, Marta Targosz-Korecka, and Marek SzymonskiCentre for Nanometer-Scale Science and Advanced Materials, NANOSAM, Faculty of Physics, Astronomy and Applied Computer Science, Jagiellonian University, Krakow, Poland

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[1] E. Giovannucci et al., Diabetes and Cancer: a consensus report, Diabetes Care 33, 1674 (2010).

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A Microfluidic Chip to Measure the Effect of Oxygen on Cell Mechanics Probed with AFM

Partial pressure of oxygen (PO2) in the cellular microenvironment is a key biophysical signal mediating cell energetic metabolism and other important processes including inflammation and immune response. Moreover, PO2 exhibits major changes among tissues. However, the effect of regional PO2 changes in cell biomechanical properties is poorly known. The objective of this work was to design a microfluidic polydimethylsiloxane (PDMS) chip to measure the effect of regional differences of PO2 on cell mechanics measured by atomic force microscopy (AFM). The chip consists of two gas channels (100 height, 15 mm length, 5 mm wide) separated 100 µm and covered with a 20 µm thin PDMS membrane. The membrane is sustained by PDMS pillars 50 µm in diameter and separated 150 µm forming a triangular pattern repeated throughout the whole gas channels. Each channel has a gas inlet and outlet allowing circulation of different gas mixtures. Owing to the high gas permeability of PDMS, cells cultured onto the membrane are exposed to the gas composition of the underneath channel. This design creates an open cell culture compatible with AFM. However, PDMS is a flexible polymer and small forces applied by the AFM tip could deflect the thin membrane resulting in potential indentation measurement errors. The complex geometry of the membrane sustained by pillars does not allow the application of theoretical models of membrane bending under concentrated forces. Therefore, to assess the effect of membrane deflection on AFM cell mechanical measurements we recorded force-indentation curves on top of a pillar and on the freestanding membrane at the centre of the triangular pillar pattern. By comparing curves recorded at both sites, the deflection of the membrane produced by the force applied by the tip was computed. A proportional force-deflection relationship was found with an effective stiff constant of 2.21 ± 0.01 m/N. Considering that the maximum force applied in AFM cell mechanical measurements is usually < 2 nN the maximum deflection of the membrane is < 4 nm which is within the range of AFM noise. Therefore, cell mechanics can be accurately measured with AFM on cells cultured on top of the chip membrane neglecting membrane deflection.

Ignasi Jorba1,2, Noelia Campillo1,2,3, Ramon Farré1,3,4, Daniel Navajas1,2,3

1Unitat de Biofísica i Bioenginyeria, Universitat de Barcelona, Barcelona, Spain2Institute for Bioengineering of Catalonia, Barcelona, Spain; 3CIBER de Enfermedades Respiratorias, Madrid, Spain; 4Institut d’Investigacions Biomèdiques August Pi i Sunyer, Barcelona, Spain; Email: [email protected]

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Mapping the viscoelastic creep behavior of cancer cells by atomic force microscopy

We present force clamp force mapping (FCFM), an AFM imaging technique, which spatially resolves the viscoelastic creep behavior of live cells. This technique is based on recording a two-dimensional array of conventional force-distance curves with an added constant force clamp phase. The creep response of the cell during the force clamp phase is characterized by a weak power-law dependency. Applying a power-law rheology model allows us to generate maps of two viscoelastic power-law parameters: modulus scaling parameter (“stiffness”) and power-law exponent (“viscosity”). Measurements on normal and cancerous human breast cell lines revealed large local variations of both power-law parameters across the cell surface. By averaging the local values over the cell area we obtained robust mean values for stiffness and viscosity. While the mean stiffness and the mean viscosity greatly varied between individual cells, they were highly correlated: softer cells exhibited a larger viscosity. On average, cancer cells were significantly softer and showed a more viscous-like behavior than normal cells.

N Schierbaum1, J Rheinlaender1, FM Hecht1, WH Goldmann2, B Fabry2, and TE Schäffer1

1IAP - Institute of Applied Physics, University of Tübingen, Germany; 2DP - Department of Physics, University of Erlangen-Nuremberg, Germany; [email protected]

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Towards higher-throughput platforms for cell mechanobiology

It remains extremely challenging to dissect the cooperative influence of multiple extracellular matrix parameters on cell behavior. This stems in part from a lack of easily deployable strategies for the combinatorial variation of matrix biochemical and biophysical properties. Here we describe a simple, high-throughput platform based on light-modulated hyaluronic acid hydrogels that enables imposition of mutually independent and spatially continuous gradients of ligand density and substrate stiffness. We characterize the resulting independent stiffness and adhesivity gradients using atomic force microscopy and immunoaffinity methods. We then validate this system by showing that this platform can support mechanosensitive differentiation of mesenchymal stem cells. We also use it to show that the oncogenic microRNA, miR18a, is nonlinearly regulated by matrix stiffness and fibronectin density in glioma cells. The parallelization of experiments enabled by this platform allows condensation of studies that would normally require hundreds of independent hydrogels to a single substrate. This system is a highly accessible, high-throughput technique to study the combinatorial variation of biophysical and biochemical signals in a single experimental paradigm.

S Kumar1

1Department of Bioengineering, Department of Chemical & Biomolecular Engineering, University of California, Berkeley, USA. [email protected]

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Quantitative measurements of the viscosity of biomimetic membranes using the AFM circular mode

Atomic Force Microscope (AFM) has opened new perspectives for investigations of phenomenological mechanisms at the nanoscale that are encountered in different fields such as material science, biology, tribology among others. In the recent years, new tools and modes have been added and adapted to the AFM in order to increase its characterization capabilities and accuracy. One of those new adaptations is the Circular mode, which is a novel scanning probe microscopy mode that offers new capabilities for surface investigations specially for measuring physical properties that require high scanning velocities and/or continuous displacements with no rest periods [1]. Its principle consists in scanning in a circular fashion at high scanning frequency (10-500Hz). Coupled with the force spectrum mode, it allows the measurement of friction coefficient or viscous friction.To ensure quantitative measurement of this new technique, we calibrate the lateral force using the original method developed by Ogletree [2] using a fused silica sample that was scratched by a Berkovich tip using a Nanoindenter. The sample ensures constant slope and constant friction coefficient on the two sides of the scratch increasing the reliability of the method. Furthermore, the circular displacements were calibrated at various frequencies (10-500Hz) by wearing a Mica surface leading to well define circular tracks.The calibrated AFM circular mode was used to quantify the friction force of supported lipid bilayers Dioleoylphosphatidylcholine (DOPC) deposited on mica in buffered conditions. Experiments show that the friction force was quasi independent of the load but proportional to the sliding velocity (from 225µm/s until 1125µm/s) suggesting that the friction force was mainly governed by viscous friction. Finally, the viscosity of the confined membrane was calculated to be = 0.1±0.014 µN.m-1.s. This study demonstrates that AFM circular mode offers a more reliable method for characterizing mechanical properties of lipid bilayers. In addition, this mode provides new advantages compared to conventional AFM nano characterization methods contributing new relevant insights about the influence of the sliding velocity on break-through force and allowing the easy and instantaneous quantitative measurement of nanomechanical properties of biomimetic membranes.

S Jaramillo Isaza1, R N Baiti2, K El-Kirat2, O Noël3, P.-E Mazeran1

1Roberval CNRS, UMR 7337 - Sorbonne university, Université de technologie de Compiègne, France; 2BMBI - Biomechanics and Bioengineering, Sorbonne university, Université de technologie de Compiègne, CNRS, UMR 7338, France3IMMM- Institut des Molécules et de Matériaux du Mans – CRNS, UMR 6283– Université du Maine, France; [email protected]

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References[1] Nasrallah et al., Rev. Sci. Instrum. 82 (2011) 113703[2] Ogletree et al., Rev. Sci. Instrum. 67 (1996) 3928-3306

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High-Resolution Studies of Cell Structure and Mechanics by Atomic Force Microscopy

Atomic force microscopy (AFM) provides the unique capability to obtain three-dimensional images of the morphology of individual living cells under physiological conditions without the need for labeling or staining. However, due to their flexible and dynamic nature, visualization of cellular structures by AFM with nanoscale resolution has remained challenging. Microvilli are common structures found on epithelial cells that increase the area of the apical surface thus enhancing the transmembrane transport capacity and also serve as mechanosensors. Changes in both the density and structure of microvilli are associated with various disease states. We have used a newly developed AFM probe, together with the low picoNewton imaging forces enabled by PeakForce Tapping mode to resolve individual microvilli structures on the surface of living cells for the first time by AFM. Our studies also revealed a direct relationship of the observed structure of the microvilli with the interaction force of the AFM probe.

Cell biology has recently seen a surge in mechanobiology-related research directed towards understanding how cells exert and respond to forces. Examining the effects of forces on cells has a wide-range of applications from understanding disease pathology to the development of tissue engineering devices. PeakForce Tapping not only allows direct examination of the nanoscale structure of cell membrane surfaces, it also provides fast, high-resolution quantitative measurements of the nanomechanical properties of live cells. We have used a combination of PeakForce Tapping (PeakForce QNM), force volume, and force spectroscopy techniques to examine both the elastic and viscoelastic properties of living cells. Through integration and synchronization with fluorescence microscopy, we attempt to correlate these mechanical properties to the organization and distribution of various subcellular structures with nanoscale resolution.

With regards to studying dynamic processes involved with cell mechanics, traditional AFM imaging has been restricted in this area of research due to the typically longer acquisition times required to obtain a single image (on the order of minutes). With recent advances in high-speed AFM imaging, where images are now obtained in a matter of seconds, we have successfully applied AFM imaging to investigate cell dynamics. Using a unique combination of high-resolution and high-speed AFM imaging, we have been able to directly observe the formation and advancement of individual lamellipodia and fillipodia, as well as reorganization events within the actin cytoskeleton, of individual living stem cells during the migration process.

AL Slade1, B Pittenger1, I Medalsy1, S Hu1, JE Shaw1, H Schillers2, M Radmacher3

1Bruker Nano Surfaces, Santa Barbara, CA, USA; 2Insitute of Physiology II, University of Münster, Münster, Germany; 3Institute for Biophysics, Universität Bremen, Bremen, Germany; ([email protected])

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Simultaneous advanced microscopies for live cell signaling dynamics investigations

We present a new type of combined microscopy for the life sciences based on the integration of differential spinning disk fluorescence optical sectioning microscopy and nanomechanical mapping Atomic Force Microscopy (AFM).[1] Our hardware platform creates the opportunity for simultaneous acquisition of membrane receptor recognition maps and fluorescence optical sectioning images of live cells, providing new experimental capabilities in cell signaling investigations.Simultaneous spatio-temporal generation of AFM and fluorescence microscopy data is made possible through time-independent illumination at low light intensities of the AFM cantilever that strongly reduces the interaction of AFM cantilever motion with fluorescence excitation light as compared to existing hardware integration platforms based on confocal laser scanning microscopy and AFM. Namely, fluorescence excitation light has the capability to induced cantilever heating and subsequent sample heating through heat conduction. We identify several system specific noise-sources that can interfere with live cell investigations and describe solutions to mitigate them.

Adelaide Miranda1, Marco Martins1, Pieter A. A. De Beule 1

1International Iberian Nanotechnology Laboratory, Avenida Mestre José Veiga s/n, 4715-330, Braga, Portugal; ([email protected])

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References[1] Miranda, A., Martins, M. & De Beule, P. A. A. Simultaneous differential spinning disk fluorescence optical sectioning microscopy and nanomechanical mapping atomic force microscopy. Rev. Sci. Instrum. 86, 093705 (2015).

85

Force-controlled Patch Clamp and Scanning Ion Conductance Microscopy with the FluidFM

Since its invention in the 1970s, the patch clamp technique is the gold standard in electrophysiology research and drug screening as it is the only tool enabling accurate investigation of ion channels which are pore-forming membrane proteins regulating transmembrane ionic flow and involved in many diseases. Although powerful and implemented in many laboratories, the patch clamp remains a labour-intensive and low-efficiency procedure due to the difficult positioning of the pipette onto the cell to create a good seal required for high-quality recordings (the “gigaseal”, corresponding to a seal resistance above 1 GΩ). Many innovation efforts are being made to reduce its complexity towards more automated systems, especially useful for drug screening, but conventional patch-clamp still remains unmatched in fundamental research due to its versatility. In our group, we addressed those hurdles by merging the patch-clamp and the Atomic Force Microscope (AFM) to take the advantage of its force-feedback to automatically and precisely position the probe onto the cell of interest. The combination was made possible using the Fluidic Force Microscope1 (FluidFM), a recently developed technology based on an AFM integrating a microchannel inside the cantilever: by combining the force feedback of an AFM with microfluidics, the FluidFM acts as a micropipette able to precisely control the forces applied on a cell. The potential of this tool was illustrated on freshly isolated adult mouse cardiomyocytes where the AFM force control can not only ensure a constant gentle contact with the contracting cells but also enables the simultaneous recording of membrane current and force development during the cardiomyocytes contraction, giving deeper insights on their correlation2. Several improvements on the setup still need to be carried out, especially regarding the formation of a gigaseal, as the current setup could only lead to resistances up to 100 MΩ, considerably limiting the quality of the patch clamp recordings. Currently, we are investigating different parameters which could improve the seal formation to reach a more reliable protocol.. The setup can also be adapted to carry out scanning ion conductance microscopy (SICM), a powerful technique enabling high-resolution noncontact imaging (Hansma et al., 1989). By adapting the scanning ion conductance microscopy on the FluidFM, the force sensitivity of its AFM allows a continuous monitoring of the forces applied on the cell, helping to further limit damages on the scanned cell (Dorwling-Carter et al., 2015). As a tool able of both SICM and patch-clamp, the FluidFM could further be used for “smart patch-clamp” (Gorelik et al., 2002), a technique that consists of a SICM pre-scan leading to a high-resolution topography of the cell useful to then place with a nanometer precision the probe to carry out patch-clamp in a region of interest.

L. Dorwling-Carter1, D. Ossola1, J. Vörös1, H. Abriel2, T. Zambelli1 1LBB – Laboratory of Biosensors and Bioelectronics, Institute for Biomedical Engineering, ETH Zürich, Zürich; 2Ion Channel Research Group, Department of Clinical Research, University of Bern; [email protected]

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References1.Meister et al., Nano Letters, 2009, 9:2501 2.Ossola et al., Nano Letters, 2015, 15 : 17433.Dorwling-Carter et al.,Physical Review Letters, 2015, 115:238103 4.Gorelik et al., Biophysical Journal, 2002, 83:3296

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Revealing contact formation characteristics of bacteria

Bacteria exhibit an outstanding ability to adhere to various kinds of surfaces. Details of contact formation, however, are hard to gain and single cell AFM force spectroscopy has proven to be a powerful tool to quantify the acting forces if combined with a clever choice of substrates. On hydrophobic surfaces, the hydrophobic interaction plays the main role for the adhesion of bacteria [1] and the contact formation process is dominated by the longest cell wall macromolecules. In our AFM study, we were able to observe the process of making contact by observing the snap-in process in detail [2]. To interpret the data, Monte Carlo simulations were set up, involving a simple model for a bacterium. The simulations yield strikingly matching results, corroborating the interpretation that the contact formation of S. aureus relies on thermally fluctuation cell wall proteins that tether to a surface and subsequently pull the bacterium to the surface. That way, e.g. S. aureus is able to attach to surfaces over distances far beyond the range of classic surface forces! Our results therefore suggest that the bacterial adhesion process in general, can be described by solely taking into account the tethered macromolecules between a bacterium and a surface.

K.Jacobs1, N. Thewes1, C. Spengler1, F. Nolle1 1Experimental Physics, Saarland University, Germany

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References[1] N. Thewes et al, Hydrophobic interaction governs unspecific adhesion of staphylococci: a single cell force spectroscopy study, Beilstein J. Nanotechnol. 2014, 5, 1501 – 1512[2] N. Thewes et al, Stochastic binding of Staphylococcus aureus to hydrophobic surfaces, Soft Matter 2015, 11, 8913 - 8919

Corresponding author: [email protected]

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Sphingomyelin-rich domains in bilayer models of the milk fat globule membrane: temperature governs structural and mechanical heterogeneity

The biological membrane enveloping the milk fat globules (MFGM) supplies consumers, especially suckling infants, with bioactive lipids and proteins, and constitutes the interface to digestion or immunity. Bioactive mechanisms depend on biological structure, which itself highly depend on temperature. However, the polar lipid assembly and biophysical properties of the MFGM are yet poorly known, especially in connection with the temperature history that milk can experience upon storage or processing and up to consumption, e.g. in human milk banks or in dietary intake of bovine milk. Noteworthy, in all mammalian milks, polar lipids of the MFGM include a significant proportion of species with a high phase transition temperature (Tm), especially milk sphingomyelin (MSM; ~30% w/w of the total polar lipids; Tm = 34°C – Murthy et al., 2015), that is held responsible for the formation of lipid domains at the surface of the milk fat globule (Lopez et al., 2010; Fig.1).

F. Guyomarc’h1,2, A.V.R. Murthy1,2, C. Lopez1,2 1INRA, UMR 1253 Science and Technology of Milk and Egg, Rennes, France; 2AGROCAMPUS OUEST, UMR 1253 Science and Technology of Milk and Egg, Rennes, France; [email protected]

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The objective of this study was therefore to investigate the polar lipid packing in hydrated bilayers prepared with a MFGM extract, and to follow cooling-induced changes at inter-molecular level using a combination of differential scanning calorimetry (DSC), wide-angle X-ray diffraction (XRD), atomic force microscopy (AFM) imaging and force spectroscopy. On cooling, a liquid disordered (ld) to solid ordered (so) phase transition of MSM (and other high-Tm polar lipids) in MFGM bilayers started at ~40°C and reached its maximum at 30.3°C. Using temperature-controlled AFM, phase separation was observed for temperatures below 35°C, with formation of so phase domains with a height difference H β1 nm from the continuous ld phase (Fig.2). In complement to AFM topographical images, indentation measurements to map the yield (or breakthrough) force over the imaged surfaces showed that the mechanical stability was significantly higher for the so phase domains than for the ld phase. Also, the mechanical stability of both the domains and the fluid phase increased with decreasing temperature, probably as a result of lower molecular agitation and increased ordering.

Fig.1. Observation of milk fat globule in raw milk using confocal laser scanning microscopy and fluorescent labelling.

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ReferencesLopez C., Madec M.-N., Jiménez-Flores R. (2010) Food Chemistry, 120: 22-33.Murthy A.V.R, Guyomarc’h F., Paboeuf G., Vié V., Lopez C. (2015) BBA: Biomembranes, 1848: 2308-2316.

AcknowledgementsThe authors thank the French Dairy Interbranch Organization (CNIEL, Paris) for funding A. V. R. Murthy’s post-doctoral fellowship. The MFP3D-BIO AFM was funded by FEDER, MENESR, INRA, CG35 and Rennes Métropole.

However, lipid packing, integrity and stability of the bilayers were adversely affected by fast cooling to 6°C or by cooling-rewarming cycle, which may have important consequences in handling milk samples in neonatal or food applications.For the first time, AFM was successfully used to report correlated structural and mechanical changes at the nanoscale, in hydrated multi-component bilayer models of a complex, biologically relevant membrane: that of the milk fat globule.

Fig.2. Typical 3D topographical images of hydrated bilayers of MFGM extract obtained at different temperatures upon cooling. Width of the green and blue arrows indicates the relative values of the breakthrough force on the domains and fluid phase, respectively.

89

Nanoscale Infrared Spectroscopy and functional imaging of proteins

Local chemical analysis is especially useful when inhomogeneous samples are studied, or very small quantity of proteins is available.Infrared (IR) absorption can be reliably detected with very high spatial resolution. Using tunable, pulsed Infrared laser as a source and contact AFM as a detector, one is able to measure local IR absorption by measuring the local thermal dilatation of the sample, and enhancing it using resonances of AFM cantilever. Functional imaging of local IR absorption at single wavelength can have 10 nm spatial resolution [2]. In addition local stiffness distribution is mapped using resonant frequency of AFM cantilever in contact with the sample.Two applications of nanoIR will be discussed:1) Light Harvesting Proteins (LHCII) are responsible for photosynthesis. Embedded in artificial lipid membrane, they form characteristic pillars, when extracted from the leaves grown in shadow, improving efficiency of photosynthesis. On the other hand, LHCII extracted from leaves grown in sunshine are not forming pillars, since enhancement of the efficiency is not needed [1].2) Amyloids are insoluble aggregates of proteins related to neurodegenerative diseases. Several examples of measurements of Amyloid fibrils will be equally presented, together with an assessment of the technique – nanoIR [3]. The nanoIR showed that ataxin-3 misfold after it aggregates, not before as would be expected by current views on aggregation [4]

AJ Kulik,1, FS Ruggeri1, WI Gruszecki2, G. Dietler1 1Institute of Physics of Living Matter, EPFL, CH-1015 Lausanne, Switzerland; 2Department of Biophysics, UMCS, PL-20-031 Lublin, Poland; [email protected]

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References[1] E. Janik et al., The Plant Cell, 2013, 25, 6, 2155-2170.[2] W.I. Gruszecki et al. Nanoscale, 2015,7, 14659-14662 DOI: 10.1039/c5nr03090k[3] T. Müller et al. Lab Chip, 2014, 14, 1315-1319[4] F.S. Ruggeri et al., Nature Communications 2015, 6, 7831, doi:10.1038/ncomms8831

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Real-time interactions of pharmaceutical compounds with lipid rafts: from nanoscale lipid organization to immunosensing

Biological membranes play a crucial role in maintaining cell homeostasis not only as a selective barrier between extra and intracellular media but also supporting numerous physiological functions such as inter and intracellular signaling and communication. To accomplish these functions it is crucial that the membrane is compartmentalized and organized into domains for which lipid composition is a driving factor. Lipid domains have different functions which rely on specific physical properties, in terms of size, rigidity or thickness. Of special biological relevance are lipid rafts, which are transient nanoscale lipid domains, enriched in cholesterol and sphingolipids that have offered numerous challenges for their in vivo investigation [1]. An elegant way of overcoming these hurdles is the use of multicomponent supported lipid bilayers (SLB), displaying different nanoscopic/microscopic phase separation, which are a validated in vitro model of lipid rafts. When prepared on mica, atomic force microscopy (AFM) is a determinant tool to disclose the membrane morphological changes, e.g. sub-nanomateric thickness variation, bilayer expansion, domain coalescence/disruption, that can be induced by different lipid compositions as well as the result of the action of externally added molecules of interest. When prepared on gold, multicomponent SLB can combine AFM with optical and electrochemical information [2].In this work, the importance of AFM will be illustrated through studies of real-time interactions of different classes of pharmaceutical compounds with lipid rafts on SLBs: anesthetics (ethanol) [3], excipients (cresol), and antifungal agents (nystatin).Concerning the use of gold, the first study where a clear depiction of lipid rafts was achieved on a metallic substrate will be shown [4]. Finally, a robust straightforward method to prepare biomimetic immunosensing interfaces on gold using an alkanethiol and raft lipids, preserving the natural fluidity and dynamics of the bilayer, is described [5]. As a consequence the whole arrangement is extremely stable regarding its use in a flow antibody-antigen detection protocol. Moreover, we show that this interface inhibits the nonspecific adsorption of proteins on the surface and allows AFM imaging of discrete antibody molecules and estimate their distribution on the membrane surface. This lipid-based biosensing platform is versatile and can be adapted to the biorecognition reaction of interest.

JT Marquês1, I Almeida1, TO Paiva, RFM de Almeida1, AS Viana1 1Centro de Química e Bioquímica. Faculdade de Ciências, Universidade de Lisboa, Edifício C8, Campo Grande, 1749-016 Lisboa, Portugal;[email protected]

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AcknowledgementsThis work was financed by the 7th Sino-Portugal Scientific and Technological Cooperation of 2013-2015 and by Portuguese national funds through FCT: IF/00808/2013/CP1159/CT0003, PEst 2015-2020 (UID/Multi/00612/2013), PTDC/BBB-BQB/6071/2014, PTDC/CTM NAN/0994/2014 and IF 2012/2013 initiatives (POPH, Fundo Social Europeu).References[1] J.T. Marquês, C.A.C. Antunes, F.C. Santos, R.F.M. de Almeida, Advances in Planar Lipid Bilayers and Liposomes, 22 (2015) 65-96.[2] J.T. Marquês, A.S. Viana, R.F.M. de Almeida, Langmuir, 30 (2014) 12627-12637.[3] J.T. Marquês, A.S. Viana, R.F. de Almeida, Biochim.Biophys.Acta, 1808 (2011) 405-414.[4] J.T. Marquês, R.F.M. De Almeida, A.S. Viana, Soft Matter, 8 (2012) 2007-2016.[5] I. Almeida, J.T. Marquês, W. Liu, Y. Niu, R.F.M. de Almeida, G. Jin, A.S. Viana, Colloids and Surfaces B: Biointerfaces,136 (2015) 997–100.

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The Architecture of Neutrophil Extracellular Traps revealed by AFM Imaging and Force Spectroscopy

Neutrophils are immune cells that make the first line of defense against pathogens. They are known for their ability to phagocyte foreign bodies, and to release granules that contain a cocktail of biocidal enzymes that effectively kill pathogens. More recently, they were found to engage in a suicidal pathway that, unlike apoptosis or necrosis, leads to the release of partially decondensed chromatin, or neutrophil extracellular traps (NETs). The emergence of NETs, or NETosis, results in their binding to pathogens, limiting the spread of the latter, thus mitigating their adverse effects. However, NETs have also been linked with deep-vein thrombosis and autoimmune diseases, which highlights the poorly selective nature of NETs and the imminent deleterious outcomes associated with them. Despite the fundamental role of NETs as part of an immune system response, we currently have a very poor understanding of their nanoscale properties, and how they explain their macroscopic functions.In this work, using a combination of fluorescence and atomic force microscopy, we set out to characterize NETosis with respect to its dynamics, as well as high detail imaging and mechanical properties of NETs. We found that NETosis arise from a two-step process, whereby nuclear dilation is then followed by extracellular extrusion of NETs over tens of microns. This process reveals a filamentous network with a mean thickness of 3±1 nm (s.d.), and a reticulate appearance comprising a 2D mesh with openings that span two orders of magnitude in size, from 10-8 to 10-6 m. The area of the openings follows a logarithmic frequency distribution with pores with areas up to 500×500 nm2

being the most observed. Interestingly, statistical analysis of the branches’ contour length typically revealed 120 nm filament stretches connecting each node of the mesh, a value that can be understood as stemming from the nucleosomal organization of DNA. Image analysis further shows that, although primarily consisting of DNA, NETs also have a substantial protein content. The presence of DNA could be further inferred from force spectroscopy, whereby the occurrence of force plateaus above 60 pN are expected to arise from DNA overstretch transitions of one or more molecules. Sawtooth force-distance profiles were routinely observed and their interpretation is consistent with the manipulation of a network of molecules. Proteolysis assays combined with force mapping revealed a highly significant 30% average drop (p<0.001) in the adhesion force and work associated with the manipulation of NETs. These results are consistent with the overall increased surface coverage of NETs upon proteolytic treatment.

Ricardo H. Pires 1,2,4, Stephan B. Felix 2, 4, and Mihaela Delcea 1,3,4

1ZIK-HIKE, University of Greifswald, Germany; 2Department of Internal Medicine B, Cardiology, University Medicine Greifswald, Germany; 3Institute for Immunology and Transfusion Medicine, University Medicine Greifswald, Germany; 4DZHK (German Centre for Cardiovascular Research), partner site Greifswald, Germany; [email protected]

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We conclude that NETs comprise a porous filament network containing openings with a scale compatible with the size of both viruses and bacteria. They consist of a DNA-protein assembly where proteins are not innocent bystanders but are active participants, contributing both the adhesion of foreign particles as well as to the structural integrity of NETs. From our observations, we anticipate that NETs may work as microscopic mechanical sieves that segregate particles mostly as a result of their size, rather than through their biological properties. Such behavior can help to explain their participation in capturing pathogens as well as in thrombus formation.

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Correlated atomic force microscopy and single molecule localization microscopy

Investigating the structure and function of biomolecules in vitro, in bacterial or mammalian cells, requires microscope methods with nanoscale resolution. Atomic force microscopy (AFM) creates topological images of biological samples in fluid. However, to correlate biological function to the nanoscale structure requires additional information, which single molecule localization microscopy (SMLM) can provide. In order to get correlated structural information and functional information by AFM and SMLM we built a low noise platform allowing correlative measurements by these respective methods. Characterization of this instrument in an individual as well as correlative approach demonstrates its high resolution imaging capabilities of both AFM and SMLM, enabling the full potential of both imaging modalities.In experiments local fluorescent labelling density of a biomolecule in vitro has been attributed to the structural information provided by AFM. Moreover we imaged a component of focal adhesions in mammalian cells as well as a fusion protein expressed in bacterial cells which are correlated with the high resolution structural information given by the AFM (Figure 1). Live correlated AFM/SMLM experiments on mammalian cells show the dynamics of the leading edge of the cell measured by AFM and the underlying dynamic changes of focal adhesions by SMLM. The temporal resolution achieved allows for observation of dynamic biological processes and optimal integration of both imaging modalities enables simultaneous imaging.This instrument enables correlated AFM and SMLM for live-cell experiments allowing the observation of dynamic biological processes. Structural changes can be correlated to changes of biomolecules at nanoscale resolution. This has the potential to enhance our understanding of biomolecules at work.

Pascal D. Odermatt1, Arun Shivanandan2, Hendrik Deschout2, Radek Jankele2, Adrian P. Nievergelt1, Lely Feletti2, Michael W. Davidson3, Aleksandra Radenovic2 and Georg E. Fantner1

1Laboratory for Bio- and Nano-Instrumentation, Institute of Bioengineering, School of Engineering, EPFL, 1015 Lausanne, Switzerland; 2Laboratory of Nanoscale Biology, Institute of Bioengineering, School of Engineering, EPFL, 1015 Lausanne, Switzerland; 3National High Magnetic Field Laboratory, Florida State University, Tallahassee, Florida, USA. Department of Biological Science, Florida State University, Tallahassee, Florida, USA; (E-mail of corresponding author: [email protected] and [email protected])

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Figure 1 Live-cell time-resolved AFM/PALM on mammalian cell. (a) CHO-K1 cell transiently expressing a paxillin-mEos2 construct imaged in its off-state under TIRF illumination. Overlaid is an AFM overview image from the upper part of the cell. The white square outlines the area zoomed in for the AFM time-series shown in b. (b) Time-resolved AFM sequence of the leading edge of the cell. The white square in the AFM image recorded after 9 min marks the area of subsequent PALM images shown in c. (c) Live-cell PALM time series showing the reorganization of the paxillin-mEos2 clusters. (d-g) Zoom-in of the squares outlined in c.

ReferencesP. D. Odermatt et al., High-Resolution Correlative Microscopy: Bridging the Gap between Single Molecule Localization Microscopy and Atomic Force Microscopy. Nano letters 15, 4896 (2015).

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Resolving the division process in Mycobacterium Smegmatis; from milliseconds to days Georg E. Fantner1, Pascal D. Odermatt1, Haig-Alexander Eskandarian1,2, Joëlle Ven1,2, Mélanie Hannebelle1 John McKinney2

1Interfaculty Institute for Bioengineering – Laboratory for Bio and Nanoinstrumentation, École Polytechnique Fédéral de Lausanne, 1015 Lausanne, Switzerland; 2Interfaculty Institute for Bioengineering – Laboratory of Microbiology and Microsystems, École Polytechnique Fédéral de Lausanne, 1015 Lausanne, Switzerland; Email: [email protected]

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Bacterial cell division occurs as a sequence of events for which many processes remain only partially understood. In most bacterial models, FtsZ-ring formation is believed to be the first event leading to division. Nucleoid segregation, septum formation, cytokinesis, and physical cell separation are all successive events in the process of division [2]. To date, many of these processes have been characterized using static methods. Establishing the sequence and possible dependence of these processes however requires time resolved live-cell imaging [3, 4], at high resolution. Here we present a concise time-sequence of events describing division of Mycobacterium smegmatis, a non pathogenic cousin of Mycobacterium tuberculosis. We used a combination of multi-day time-lapse atomic force microscopy (AFM), time-lapse fluorescence microscopy with real time measurements of the cell separation at timescales down to 10s of milliseconds. Combining the nanoscale 3D information from AFM and the biochemical specificity from fluorescence microscopy we have characterized cell division from the early stage pre-selection of division sites, to assembly and subsequent disassembly of the FtsZ ring, localization of Wag31 and cytokinesis all the way to the rapid cell separation. Contrary to what is believed to be true in many bacterial cell types, cell separation in Mycobacterium smegmatis isn’t a gradual event, but occurs abruptly within 10s of milliseconds, resembling more a mechanical fracture than a cellular remodeling process. Using mechanical stimulation, we demonstrate that the build-up of mechanical stress governs the time and place of cell separation. By applying additional mechanical stress, we were able to initiate cell separation at times in the cell cycle well before it would occur in the non-stimulated case. These observations suggest a new model for the late stages of cell division in Mycobacterium smegmatis, where cell mechanical properties and local stress concentration govern the timing and place of cell separation.

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Local hydration properties of single bacterial cells and spores by lift-mode Electrostatic Force MicroscopyMarc Van Der Hofstadt1, R. Fabregas1, M. C. Biagi1, L. Fumagalli2, A. Juárez3, G. Gomila1, 4

1IBEC – Institut de Bioenginyeria de Catalunya, Barcelona, Spain; 2School of Physics and Astronomy, University of Manchester, United Kingdom; 3Departament de Microbiologia, Universitat de Barcelona, Spain; 4Departament d’Electrònica, Universitat de Barcelona, Spain; Corresponding authors: [email protected] and [email protected]

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The resistance of biological samples to death by dehydration relies mainly on its capabilities to retain water molecules in their system. This is a special issue in medicine when regarding pathogenic bacterial cells and bacterial spores. The ability to retain water can be correlated to the biochemical components of the bacterial cell wall and to their global hydrophobic or hydrophilic behaviour. The hydration properties of bacterial cells and spores has been studied at the population level but not at the single bacterial cell level. In order to achieve it, we propose to assess the hydration properties of bacterial samples at the single cell level by means of Electrostatic Force Microscopy. This technique is sensitive to the local electrical permittivity of biological samples [1-3], and also to the presence of water, which shows a large dielectric constant value [2]. Using this technique, we have already been able to differentiate the re-hydration capabilities of gram positive and gram negative bacteria at the single bacterial cell level [2]. Here we push forward the limits of the developed technique to adapt it to bacterial samples (e.g. spores) not directly addressable by previous methods due to their highly non-planar geometry. To this end we have implemented a methodology to make lift mode Electrostatic Force Microscopy quantitative and capable of reconstructing images of local electric permittivity of biological samples, and hence of their local hydration properties and with nanoscale resolution. This approach includes image post-processing to remove topographic cross-talk contributions and 3D finite element numerical simulations, allowing one to visualize and quantify local electric permittivity of practically any biological sample at the whole image level. This opens new frontiers for the imaging and characterization of biological samples with nanoscale resolution.We have applied the methodology developed to vegetative bacterial cells and confirmed the results obtained earlier with a different approach [2]. Moreover, we have also applied it to single bacterial spores for the first time, and clearly showed their resistance to hydration. This result confirms the ability of bacterial spores to sustain harsh conditions, such as long periods in dry ambient, what is mainly due to their high hydrophobic wall (in comparison to vegetative cells) which prevents the entrance of water and hence the activation of the spore’s metabolism [4].

[1] A. Dols-Perez, G. Gramse, A. Calo, G. Gomila, L. Fumagalli, Nanoscale electric polarizability of ultrathin biolayers on insulator substrates by electrostatic force microscopy, Nanoscale 7, 18327-18336 (2015).[2] D. Esteban-Ferrer, M.A. Edwards, L. Fumagalli, A. Juarez, G. Gomila, Electric polarization properties of single bacteria measured with electrostatic force microscopy, ACS Nano 8(10), 9843-9849 (2014).[3] A. Cuervo, P. D. Dans, J. L. Carrascosa, M. Orozco, G. Gomila, L. Fumagalli, Direct measurement of the dielectric polarization properties of DNA, Proc. Nat. Acad. Sci. USA 11135, E3624-E3630 (2014).[4] K.M. Wiencek, N. A. Klapes, P. M. Foegeding, Hydrophobicity of Bacillus and Clostridium spores, Appl. Environ. Microbiol. 56, 2600-2605 (1990).

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The organization of membrane proteins in rod outer segments revealed by AFM imaging and SMFSSourav Maity1,2, Monica Mazzolini1, Vincent Torre1

1International School for Advanced Studies (SISSA-ISAS) via Bonomea 265, 34136 Trieste (Italy); 2Moleculaire Biofysica, Zernike Instituut, Rijksuniversiteit Groningen, Nejenborg 4, 9747 AG, Groningen (The Netherlands).corresponding author: [email protected]

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Rod cell outer segments (ROSs) harbor the phototransduction cascade, which involves membrane bound and cytosolic proteins. Membrane bound proteins are distributed on the membrane of disks and on the plasma membrane of ROS. The composition of these two membranes is different in their lipid and protein concentration and distribution. The most populated protein in both membranes is the G-protein coupled receptor rhodopsin. Another important protein playing an essential role in phototransduction cascade is the cyclic nucleotide gated (CNG) channel, which is exclusively found in the ROS plasma membrane. Here we used AFM imaging and Single molecule force spectroscopy (SMFS) to look for the spatial arrangement of membrane protein along the disk and plasma membrane of ROS, and to unfold membrane proteins from membrane patches of rod outer segments obtained from Xenopus laevis retinas. We found that the native CNG channels are organized along thin strings composed by a single line of CNG channels. We also found different ensembles of force-distance (F-D) curves which can be associated with unfolding of different native proteins from ROS plasma membrane such as those obtained from the unfolding of rhodopsin and of CNGA1 single subunits. Both the unfolding of rhodopsin and CNG channels shows a good agreement to what was observed previously, but with some significant differences: the unfolding of rhodopsin from the plasma-membrane of ROS shows a pattern of force peaks consistent with the unfolding of single individual transmembrane segments at each peak. In contrast, the unfolding of rhodopsin from disks is obtained from a pairwise unfolding of the two transmembrane segments at each force peak. These observations can describe the possible differences in Kinetics, energetics, and mechanics of rhodopsin molecules in the disk and plasma membrane. The unfolding of native subunits of CNG channels is similar to that observed for CNGA1 channels expressed in oocytes. This abstract also represents a method to study in a single experiment- a population of membrane proteins in their natural environment.

Molday, R.S. & Molday, L.L. Differences in the protein composition of bovine retinal rod outer segment disk and plasma membranes isolated by a ricin-gold-dextran density perturbation method. J. Cell. Biol. 105, 2589-2601 (1987).βBoesze-Battaglia, K. & Schimmel, R. Cell membrane lipid composition and distribution: implications for cell function and lessons learned from photoreceptors and platelets. J. Exp. Biol. 200, 2927-2936 (1997).βNemet, I., Tian, G. & Imanishi, Y. Organization of cGMP sensing structures on the rod photoreceptor outer segment plasma membrane. Channels 8, 528-535 (2014). doi: 10.4161/19336950.2014.973776.βSapra, T.K. et al. Detecting molecular interactions that stabilize native bovine rhodopsin. J. Mol. Biol. 358, 255-69 (2006).Kawamura S, Gerstung M, Colozo AT, Helenius J, Maeda A, Beerenwinkel N, Park PS, Müller DJ. Kinetic, energetic, and mechanical differences between dark-state rhodopsin and opsin. Structure 21, 426-37 (2013). βSourav Maity, Monica Mazzolini, Manuel Arcangeletti, Alejandro Valbuena, Paolo Fabris, Marco Lazzarino & Vincent Torre. Conformational rearrangements in the transmembrane domain of CNGA1 channels revealed by single-molecule force spectroscopy. Nat. Commun. 6, 7093 (2015). doi: 10.1038/ncomms8093.

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ESCRT-III spiral springs: polymerization and depolymerization observed by High-Speed AFM L. Redondo-Morata1, N. Chiaruttini2, A. Roux2, S. Scheuring1

1U1006 INSERM, Aix-Marseille Université, Marseille, France; 2University of Geneva, Department of Biochemistry, Geneva, Switzerland; Corresponding author: Simon Scheuring ([email protected])

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Among all the membrane vesicle budding processes, the machinery of the Endosomal Sorting Complex Required for Transport (ESCRT) is unique because it is the key player whenever the membrane budding takes place “away” from the cytoplasm, topologically into the opposite direction compared to clathrin or COP proteins. They were first studied in the formation of endosomes, but posteriorly its implication has been described in other events which require vesicle release, notably cell abscission, plasma membrane wound healing and virus release. ESCRT-III is the main responsible for the final vesicle fission. Snf7, the major component of ESCRT-III complex, exists in solution as an autoinhibited monomer. When it is recruited by ESCRT-II, it polymerizes on the membrane. In 2008, Hanson et al. observed that overexpression of Snf7 leads to membrane tubulation (1), pointing out that polymerized Snf7 could deform the membrane.However, it is still unclear how ESCRT-III deforms membranes, because it has been observed that ESCRT-III polymers on membranes may adopt a wide variety of shapes (1), and the molecular assembly of ESCRT-III does not lead to a unique structure (2). In this work (3), we aimed to understand how ESCRT-III polymerization could drive membrane deformation. For that we used High-Speed AFM (HS-AFM)(4), which allowed us to study membrane phenomena with unprecedented spatial and temporal resolution. When adding Snf7 to negatively charged lipid bilayers, we observed polymerization in filaments that assemble in concentric rings forming disk-like assemblies of about 120nm radius. The filaments dynamics suggest that concentric circles are formed by double-stranded filaments that are interconnected by thinner single-stranded filaments. To explore the existence of internal stress in the assemblies, we used the HS-AFM tip as a nano-dissector. Applying increased forces to break the assemblies, we observed that filaments curl spontaneously at 20-30 nm, implying that if they grow at a different radius, they are under mechanical stress. Our video imaging revealed the mechanism by which spiral nucleation occurs (Fig.1). Strikingly, when increasing from 2 to 3 concentric circles, the radius of the innermost circle is reduced from about 22nm to about 14nm, supporting our hypothesis that the preferred radius of curvature of polymerized Snf7 is about 25 nm, but lateral pressure from filaments under lower curvature induce pressure towards the inside of the assembly forcing compression. All in all, our data suggests that Snf7 spirals expansion leads to membrane deformation. We proposed a model for ESCRT-III mediated budding and fission of vesicles. In this model, polymerized Snf7 can deform to store significant elastic energy. This stored energy can be transferred to the lipid membrane to deform it and eventually lead to fission.

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Currently, we are studying the depolymerization process of Snf7, which is driven by Vps4, an AAA+ ATPase. It has been proposed that the enzyme induce conformational changes in Snf7, releasing the proteins from the assembled polymer. We want to gain insights in how the disassembly of Snf7 takes place at the molecular level.

Fig.1) High-speed AFM visualization of Snf7 assembly maturation.

1. Hanson et al., Journal of Cell Biology 180, 389-402 (2008).2. McCullough et al., Annual Review of Biochemistry, 82, 663 (2013).3. Chiaruttini et al., Cell 163, 866-879 (2015).4. Ando et al., Chemical Reviews 114, 3120-3188 (2014).

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Fluctuating Lipid Nanodomains Near Critical TransitionsGeorge R. Heath, Stephen D. Evans and Simon D. ConnellSchool of Physics and Astronomy, University of Leeds, LS2 9JT, United Kingdom; ([email protected])

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The lateral organization of lipids in biomembranes is fundamental to many cellular processes, and the dominant theory of so-called “lipid rafts” in the last decade is still one of micro-domains of two liquid phases co-existing at equilibrium. However, there is little evidence that these stable micro-domains exist in real cell membranes, with most data suggesting transient structures at a sub-optical length scale. In recent years this hypothesis has grown into a more dynamic description, encompassing modulated phases, compositional fluctuations and micro-emulsions. One compelling theory is that lipid rafts are a manifestation of critical fluctuations in the single phase close to a critical point in the phase diagram, and not in fact two phases in equilibrium. The static and dynamic behaviour of critical lipid mixtures has previously been characterised by fluorescence microscopy of giant vesicles, but only at length scales of >1 μm due to optical resolution limits. Here we present Fast Scan AFM data of the critical phase behaviour of a model cell membrane. Below the critical temperature (in the macroscopic phase separation regime) the boundaries of domains100 nm – several microns in size were observed to fluctuate with amplitudes of 10’s of nm’s. This motion has been quantitatively analysed to give line tensions in the range 1 pN at room temperature (similar to that observed in GUV’s), reducing linearly as temperature increases to a low of 0.02 pN close to the critical point. As temperature is increased whilst imaging we show the crossing of the critical temperature (TC) with real time high speed imaging, resulting in the breakdown of large domains into much smaller nanoscale fluctuations. The relationship of domain correlation length with temperature gives an exponent of exactly 1.0, indicating the 2D Ising Model of phase separation is valid. The fluctuations at and above TC result in domains of 5-100 nm with lifetimes of seconds to almost 1 minute, again dependent upon temperature. Smaller domains are short lived, whilst the largest domains are most persistent. Domains exist up to 10 degrees above TC and over a broad compositional space, removing one of the main objections for the lipid fluctuation hypothesis, namely that the critical region is far too confined to be of use to a cell. For direct comparison with our results we perform simple Monte Carlo 2D Ising model simulations which we analyse with the identical methods as our AFM movies. This shows closely analogous behaviour for line tensions, phase structure break down and domain lifetimes. Our data also provides a measure of the dynamics of lipid bilayers on a solid support, showing a crossover around the hydrodynamic radius from molecular diffusion to one of hydrodynamic flow that is coupled with the 3D bulk water. Our results provide the first direct insight into membrane nanodomain dynamics.

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On chip biodetection, analysis and nanometrology of blood microparticlesS. Obeid1, C. Decourbey1, A. Ceroi3, G. Mourey3, P. Ducoroy2, G. Belliot4, P. Saas3, W. Boireau1, C. Elie-Caille1*1School of Physics and Astronomy, University of Leeds, LS2 9JT, United 1 FEMTO-ST Institute, UFC, CNRS, ENSMM, UTBM, UBFC, 25030 Besançon, France; 2 CLIPP, Proteomic Platform, University of Burgundy, 21000 Dijon, France; 3 INSERM U1098, EFS Bourgogne Franche-Comté, UBFC, 25000 Besançon, France; 4 National Reference Center for Enteric Viruses, Public Hospital, Dijon, France; *[email protected]

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Blood microparticles (MPs) are small membrane vesicles (50 - 1000 nm) derived from different cells types. They are recognized to play an important role in various biological processes and are also recognized as potential biomarkers of various health disorders1. Currently, the technic of choice used for the detection of MPs is flow cytometry (FC)2, but the main drawback of FC is the lower detection limit (about 300 nm at best) ; consequently, only a small fraction of microvesicles can be detected. Our work aims to use the potential of surface plasmon resonance (SPR) method to detect, and qualify, specifically and without labeling, the MPs directly in their complex media, via their (immune)capture onto the biochip-surface using different specific ligands. Using two calibration standards of known size and concentration (Virus Like Particles (VLP) & modified melamin resin MF beads, corresponding to the minimum (50 nm) and the maximum (920 nm) size of MPs, respectively), we have shown that the capture level (2D quantification) of these entities by their specific ligands, is linearly related to their concentration in the sample (3D quantification). Dynamic ranges of concentrations ranging from 8x1010 to 2x1012 VLP/ml and 3,25x107 to 3,25x108 MF beads/ml, have been established for 2 min injection time. These concentration ranges match the typical range of MP natural concentrations in plasma3. Additionally, by varying the injection time, the dynamic range increases, which provides more sensibility for the detection of MPs in a wide concentration range. Moreover, through an atomic force microscopy (AFM) investigation of the SPR biochip after capture of calibration standards or MPs, we managed to correlate the capture level to the biochip surface density in particles. Thus, the combination between SPR and AFM approaches appears to be suitable for the metrological analysis of circulating MPs, which allows identification of their size subpopulations. Furthermore, an on-chip proteomic study was also engaged to discover specific proteomic profiling of MPs in different conditions. At last, we have adjusted these analytical platform based on a gold biochip to analyze, qualify and compare, from the nano- to the micro-scale, resting and activated platelet MPs. Such an original bioanalytical platform, coupling AFM to SPR, Mass Spectrometry and even Surface Enhanced Raman Spectroscopy4, is then proposed as an original multiscale approach, based on custom nanostructured gold biochip5, for a deep qualitative and quantitative characterization of a huge range of relevant, but often complex, biological fluids.

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1 Burnouf, T., et al, 2014. Platelet microparticles: Detection and assessment of their paradoxical functional roles in disease and regenerative medicine. Blood Rev. 28, 155–166. 2 Lacroix, R., et al, 2010. Standardization of platelet-derived microparticle enumeration by flow cytometry with calibrated beads: results of the International Society on Thrombosis and Haemostasis SSC Collaborative workshop. J. Thromb. Haemost. 8, 2571–2574.3 Maiolo, D., et al, 2015. Colorimetric Nanoplasmonic Assay To Determine Purity and Titrate Extracellular Vesicles. Anal. Chem. 87, 4168–4176.4 Aybeke E.N, et al, 2015. Homogeneous large-scale crystalline nanoparticle-covered substrate with high SERS performance. Nanotechnology, 26 (24), 245302.5 Elie-Caille C., et al, 2009. Preparation of flat gold terraces for protein chip developments. Micro & Nano Letters, 4(2), 88-94.

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The conformational equilibria of the prion protein characterized by AFM-based single-molecule force spectroscopyG Zuccheri1,2, A Raspadori3, V Vignali1, G Legname3, B Samorì1 1Department of Pharmacy and Biotechnologies, University of Bologna, Italy.2S3 Center, Institute of Nanoscience of the Italian CNR; 3Laboratory of Prion Biology, International School for Advanced Studies, Trieste, Italy; Email: [email protected]

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Neurodegenerative disorders, including prion diseases, are associated with protein aggregation. According to an emerging view, proteins involved in these diseases are conformationally heterogeneous, switching back-and-forth between functional and amyloidogenic/prionogenic conformations [1,2]. The prion protein (PrP) is a typical example of such class of proteins that can assume different conformations each possibly endowed with different properties. In its best characterized form, PrP displays a structured portion and an unstructured one. In peculiar conformations, PrP can lead to protein aggregation processes and it can become an infectious protein, able to lead to disease is animals. The characterization of these proteins by standard structural biological methods has proven to be extremely challenging, due to the contemporary presence of multiple conformations and to the transient nature of such conformations in the protein monomers. Single-molecule characterization techniques, such as single-molecule fluorescence spectroscopy or single-molecule force spectroscopy can sample the conformations of one molecule at a time, in a small population, and they can report on the presence of transient structures in a small fraction of the molecular population even if short-lived.We used Atomic Force Microscopy- Single Molecule Force Spectroscopy (AFM-SMFS) in order to probe the presence of mechanically stable structures on the truncated murine prion protein (MoPrP). We characterized its structural heterogeneity at the monomeric level by preparing and mechanically unfolding a protein chimera specifically designed and expressed for this study. Recording hundreds of unfolding processes on single molecules of PrP and characterizing the unfolding events, we showed that the protein was able to adopt several structures different from the native one, which we could also characterize. These could represent aggregation-prone structures. The experiments confirmed the intrinsic structural heterogeneity of the PrP protein. Experiments performed at different pH proved subtle differences in the molecular behaviour.Using different protein chimera comprising multimeric constructs of truncated MoPrP with different reciprocal orientations, we attempted to characterize the protein early oligomerization events. Measurements of the force-extension curves revealed a broad range of structures that involved more than one MoPrP molecule. Different reciprocal MoPrP orientation changed drastically the equilibrium between monomeric and oligomeric structures and sometimes marked the disappearance of the signature of

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the native PrP conformation in favour of alternative structures. These could have a role towards the of early events linked to protein toxicity and infectiousness. We demonstrated how MoPrP conformational equilibria and oligomerization processes can be studied at the single-molecule level with AFM single-molecule force spectroscopy, confirming the intrinsic structural heterogeneity of MoPrP.

[1] Dyson HJ, Wright PE Intrinsically unstructured proteins and their functions. Nat. Rev. Mol. Cell. Biol. 6: 197 208. (2005).[2] Uversky VN, Dunker AK Understanding protein non folding. Biochimica et biophysica acta. 1804:1231 1264 (2010).

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Cardiomyocyte syncytium combined with Atomic force microscopy, advanced setup of universal biosensor for phenotype screeningMartin Pesl,1,2, Jan Pribyl 2,3, Sarka Jelinkova1,2, Ivana Acimovic2, Anton Salykin,2, Petr Dvorak 1,2, Vladimir Rotrekl1,2, Petr Skladal 2,3

1International Clinical Research Center, St. Anne’s University Hospital, Brno, Czech Republic; 2Department of Biology, Faculty of Medicine, Masaryk University, Brno, Czech Republic; 3CEITEC MU, Masaryk University, Brno, Czech Republic; 4Department of Biochemistry, Faculty of Science, Masaryk University, Brno, Czech Republic;

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Intro: Cardiomyocyte contraction and relaxation are important parameters of cardiac function altered in most cardiac diseases. However we routinely model such diseases using cardiomyocytes derived from human embryonic stem cells and especially from patient specific induced pluripotent stem cells; it is the biosensing of contraction and relaxation which makes the models a great tool for drug development. We employ atomic force microscopy to analyze these parameters. In this novel approach we employ the direct contact of the cantilever with standardized relaxation and stress conditions with the stem cells derived cardiac syncytium. We present advanced experimental setup for quantification of the cardiomyocyte dynamic changes induced by external stimuli such as extreme temperature, calcium level and stress conditions or administered various drugs. Further we show novel automatized data evaluation process. Advantages of feedback-gain parameter previously described (2) are discussed.Methods: Cardiomyocytes were generated as previously described (1) using aggregation in agarose micro-wells allowing homogeneous cluster formation in size and shape (size of the cluster as well as cardiomyocyte content was quantified). Atomic force microscopy analysis was performed in Tyrode’s solution, in Calcium concentration gradients, temperature gradients and ion channel- and adrenergic receptor-modulator gradients.. For data postprocessing and statistical evaluation was involved specific script allowing automatized evaluation.Results: The active AFM mode has no effect on contraction frequency, while providing for correct contraction force recording. We found the optimal value corresponds to average recorded value of EBs contraction force. It was set to 6 nN, in our case, which allowed for the measurement of 95% of maximum contraction force (4.6 nN vs. 4.9 nN, when SP was 6 and 18 nN, respectively). Use of noncoated AFM probes eliminates thermal instability and allows measurements for hours and days continuously. Calcium gradients allowed us to study the excitation-contraction (un)coupling as well as arrythmogenicity of calcium extreme concentrations. Beat to beat surface mapping has shown comparable values all over accessible syncytium rendering the method robust and unbiased by the cantilever position.

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1 Pesl, M., Acimovic, I., Pribyl, J., Hezova, R., Vilotic, A., Fauconnier, J., Vrbsky, J., Kruzliak, P., Skladal, P., Kara, T., Rotrekl, V., Lacampagne, A., Dvorak, P., Meli, A.C., 2014. Forced aggregation and defined factors allow highly uniform-sized embryoid bodies and functional cardiomyocytes from human embryonic and induced pluripotent stem cells. Heart Vessels 29, 834–846. doi:10.1007/s00380-013-0436-9

2 Liu J, Sun N, Bruce MA, Wu JC, Butte MJ (2012) Atomic Force Mechanobiology of Pluripotent Stem Cell-Derived Cardiomyocytes. PLoS ONE 7(5): e37559.3 Pesl M, Pribyl J, Jelinkova S, Acimovic I, Salykin A, Meli AC, Dvorak P Rotrekl V, Skladal P. Atomic Force Microscopy combined with patient specific pluripotent stem cell derived cardiomyocytes as a biosensor tool for studying the biomechanical properties of human cardiomyocytes Biosensros and Biophysics 2016

The research has been funded by the grant of the Grant Agency of the Czech Republic (no. GA13-19910S), and supported by the project no. LQ1601 CEITEC and LQ1605 FNUSA-ICRC from the National Program of Sustainability II. Czech Republic

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posters’ list

Quantitative Description of Thermodynamic and Kinetic Properties of the Platelet factor 4/Heparin BondsTH Nguyen, M Decea, and A Greinacher

Influence of surface and subsurface modifications of a substrate on bacterial adhesionFriederike Nolle, Nicolas Thewes, Christian Spengler, Thomas Faidt, Peter Loskill, Karin Jacobs

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02

Direct mapping intercellular interactions: key steps for brain metastasis formation AG Végh, B Varga, C Fazakas, I Wilhelm, J Molnár, Z Szegletes, I Krizbai and G Váró

A novel SU-8 cantilever sensor system for pathogen detection C Soum, J Moran Meza, R de Miguel Viscasillas, J Normand, M Pham Van, F Thoyer, C. Feraudet-Tarisse, H Volland, T Berthelot, L Pham Van

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Effects of Bacterial LPS on the Biomechanics of Alveolar Epithelial Cells Measured by Atomic Force Microscopy Vinicius Rosa de Oliveira, Juan José Uriarte, Walter A. Zin, Ramon Farré, Isaac Almendros, Daniel Navajas

AFM investigation of Mesothelial Cells mechanics and their mechanosensing of ECM with different stiffness I Musella, S Fusco, V Panzetta, M De Menna, M Volante, I Rapa, PA Netti

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06

Fabrication of Microstructured composite scaffolds to increase osseointegration for Dental and Orthopedic Medicine B. Sakamoto, M. Silva, D. M. Zezell, A. F. G. Monte, A. Antunes

Pilot study about characterization by AFM imaging of chitosan artificial vessels manufactured to Cardiovascular Medicine performanceM. Silva, B. Sakamoto, D. Pasquini, A. Alencar, A. F. G. Monte, A. Antunes

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08

POSTER SESSION I

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Quantitative Nanohistology on the Disease Scleroderma Suggests Excessive Interfibrilar CrosslinksAP Strange, RJ Stratton, S Parekh, L Bozec

Internalization and cytotoxicity of zeolites in breast cancer cellsN Vilaça, F Santos, R Amorim, R Totovao, E Prasetyanto, Am Fonseca, L De Cola, F Baltazar, Ic Neves

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AFM Imaging of bacteria in liquid without immobilization: detection of intercellular nanotubesS Dhahri, C Marlière

Structural changes in plasmid DNA verified by gel electrophoresis and AFM: Sample radiation and damage detection (I)S. Meyer, M.-A. Schröter, M. B. Hahn, T. Solomun, H. Sturm, H.-J. Kunte

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TRF2-mediated control of telomere DNA topology as a mechanism for chromosome end protectionSabrina Pisano ,Delphine Benarroch-Popivker, Parminder Kaur, Hong Wang, Serge Bauwens, Vincent Fraisier, Arturo Londoño-Vallejo, Aaron Mendez-Bermudez, Liudmyla Lototska , Nadir Djerbi, Bei Pei, Eric Gilson, Marie-Josèphe Giraud-Panis

AFM study of convergent transcriptional complexes EV Dubrovin, ON Koroleva, IV Yaminsky, VL Drutsa

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Quantifying the impact of plant hormones on cell wall mechanical propertiesP Milani, A. Berquand, A. Boudaoud, O. Hamant

Parallel atomic force microscopy for rapid nanomechanical tissue diagnostics M Favre, G Weder, R Ischer, M Liley, H Heinzelmann, M Despont

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Nanocharacterization of chemical and mechanical properties of plant cell walls and lignocellulosic bioinspired assembliesMuraille L., Chabbert B., Bercu N.-B., Aguié-Béghin V., Molinari M.

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Nanoindentation studies on viral nanoparticles S. Maity, W. H. Roos

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POSTER SESSION III

POSTER SESSION II

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Morphological and nanomechanical characterization of neurons in co-culture with dental pulp stem cells B Varga, M Martin, O Romieu, T Cloitre, PY Collart Dutilleul, O Páll, F Cuisinier, F Scamps, C Gergely

Structural and nanomechanical properties of model lipid membranesB Gumí-Audenis, F Carlà, F Comin, F Sanz, L Costa, MI Giannotti

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Effect of Actin Organization on the Stiffness of Living Breast Cancer Cells Revealed by Peak Force Modulation Atomic Force MicroscopyA Calzado-Martín, M Encinar, JE Martínez-Gómez, A San-Paulo, J Tamayo, M Calleja

Mechanical Properties of the Drosophila Central Nervous SystemIgnasi Jorba, Katerina Karkali, Anand P. Singh, Timothy E. Saunders, Enrique Martin-Blanco, Daniel Navajas

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AFM Imaging on NanoParticles: Forms and Features Shu-wen W. Chen, Jean-Marie Teulon, Christian Godon, Jean-Luc Pellequer

Biomechanics of a bone-like 3D model for bone tissue regenerationN Ribeiro, SR Sousa, FJ Monteiro

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Stability and mechanical properties of conducting polymer films interfacing with biological systemsC. Duc, S. Bovio, S. Janel, F. Lafont, G. Maliaras, V. Senez, A. Vlandas

The structure of lipid membranes on rough surfacesFlorence Blachon, Frédéric Harb, Agnès Piednoir, Bernard Tinland, Thierry Charitat, Giovanna Fragneto, Olivier Pierre-Louis, Jean-Paul Rieu

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Structural changes in plasmid DNA verified by gel electrophoresis and AFM: Sample AFM preparation and imaging (II)M.-A. Schröter, S. Meyer, M. B. Hahn, T. Solomun, H.-J. Kunte, H. Sturm

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Mechanical properties of membranes composed of gel-phase or fluid-phase phospholipids probed by AFM on liposomesO. Et Thakafy, N. Delorme, C. Gaillard, V. Vié, J. Léonil, C. Lopez, F. Guyomarc’h

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POSTER SESSION IV

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Measuring the bacterial interaction area to abiotic surfaces by single-cell force spectroscopy on tailored samplesC Spengler, N Thewes, K Jacobs

Probing peptide-gold interaction at the single-molecule level through AFM-based methodsM Steffenhagen, J Landoulsi, E Maisonhaute

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In-vivo high resolution AFM topographic imaging of C.elegans reveals previously unreported surface structuresClara L Essmann, Muna Elmi, Mike Shaw, Giridhar M Anand, Vijay Pawar, Mandayam A Srinivasan

Watching the eukaryotic fatty acid synthase multienzymatic factory at workFriederike Benning, Habib Bukhari, Yusuke Sakiyama, Roderick Lim, Timm Maier

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Mechanical unfolding of HCN2 channelsAndrea Pedroni, Andrea Alfieri, Paolo Fabris, Anna Moroni, Loredana Casalis, Vincent Torre

AFM imaging of silica nanoparticles with and without the presence of adsorbed proteinsJean-Marie Teulon, Shu-wen W. Chen, Antoine Thill, Laurent Marichal, Jean Labarre, Jean-Luc Pellequer

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Opening and stabilization of Transendothelial Macroapertures in living cells probed by AFM elasticity measurements S. Janel, C. Stefani, D. Gonzalez-Rodriguez, A. Doye, Patricia Bassereau, E. Lemichez, F. Lafont

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Detecting nanoscale motion of biological systems

Petar Stupar, Giovanni Longo, Giovanni Dietler, Sandor Kasas36

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Quantitative Description of Thermodynamic and Kinetic Properties of the Platelet factor 4/Heparin Bonds

Heparin is the most important antithrombotic drug in hospitals. It binds to the endogenous tetrameric protein platelet factor 4 (PF4) forming PF4/heparin complexes which may cause a severe immune-mediated adverse drug reaction, so-called heparin-induced thrombocytopenia (HIT). Although new heparin drugs have been synthesized to reduce such a risk, detailed bond dynamics of the PF4/heparin complexes have not been clearly understood. In this study, single molecule force spectroscopy (SMFS) is utilized to characterize the interaction of PF4 with heparins of defined length (5-, 6-, 8-, 12-, and 16-mers).1 Analysis of the force-distance curves shows that PF4/heparin binding strength rises with increasing heparin length. In addition, two binding pathways in the PF4/short heparins (≤8-mers) and three binding pathways in the PF4/long heparins (≥8-mers) are identified. We provide a model for the PF4/heparin complexes in which short heparins bind to one PF4 tetramer, while long heparins bind to two PF4 tetramers. We propose that the interaction between long heparins and PF4s is not only due to charge differences as generally assumed, but also due to hydrophobic interaction between two PF4s which are brought closely to each other by long heparin. This complicated interaction induces PF4/heparin complexes more stable than other ligand-receptor interactions. Our results also reveal that the boundary between antigenic and non-antigenic heparins is between 8- and 12-mers. These observations are particularly important to understand processes in which PF4-heparin interactions are involved and to develop new heparin-derived drugs.

[1] Nguyen, T.H., Greinacher, A. & Delcea, M. Quantitative description of thermodynamic and kinetic properties of the platelet factor 4/heparin bonds. Nanoscale 7, 10130-10139 (2015).

TH Nguyen1,2*, M Decea1,2, and A Greinacher1

1Institut für Immunologie und Transfusionsmedizin, Universitätsmedizin Greifswald,17475 Greifswald, Germany; 2ZIK HIKE - Center for Innovation Competence, Humoral Immune Reactions in Cardiovascular Diseases, University of Greifswald, 17489 Greifswald, Germany.

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114

Influence of surface and subsurface modifications of a substrate on bacterial adhesion

Numerous studies concentrate on finding conditions for preventing biofilms. However, its main constituents, proteins and bacteria, exhibit clever strategies to adhere to various surfaces. Our experiments aim at determining nature and strength of the involved forces by a judicious choice of substrates: Keeping the surface roughness and chemistry constant, differences in the subsurface composition cause distinct changes in the adhesion forces due to a variation of the long-range van der Waals force. Keeping the surface chemistry constant and changing the roughness only also influences bacterial adhesion due to geometry constraints. Comparing hydrophobic and hydrophilic substrates of identical roughness and nearly identical van der Waals forces reveal the influence of short-range, e.g. hydrophobic forces. We review our recent experiments of bacterial adhesion (e.g. S. aureus, S. carnosus, Strep. oralis, Strep. mutans) as determined by single cell AFM force spectroscopy.

Friederike Nolle1, Nicolas Thewes1, Christian Spengler1, Thomas Faidt1, Peter Loskill1, Karin Jacobs1*

1Saarland University, Dept. of Experimental Physics, 66041 Saarbrücken, Germany; *Corresponding author: [email protected]

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Direct mapping intercellular interactions: key steps for brain metastasis formation

The most life-threatening aspect of cancer is metastasis; roughly 90% of cancer patient mortality is due to metastasis. It is still not understood why some primary tumors metastasize, thus have a worse outcome, compared to others that do not metastasize. In most cases, dissemination of cancer cells relies on lymphogenous or hematogenous routes. Among all metastatic foci, presence of brain metastasis is of very poor prognosis; the median survival time can be counted in months. Therefore, prevention or lowering their incidence would be highly desired both by patients and physicians. Since the central nervous system (CNS) lacks lymphatic circulation, metastatic cells invading the CNS must breach the blood-brain barrier. The key step in this process is the establishment of firm adhesion between the cancer cell and the cerebral endothelial layer. Using the atomic force microscope, as a high resolution force-spectrograph, our aim is to explore the connections among the cell morphology, cellular mechanics and biological function in the process of transendothelial migration of metastatic cancer cells. Our model relies on direct measurement of intercellular interactions between a living cancer cell and a confluent endothelial layer as substrate. By immobilization of a living cancer cell to an atomic force microscope’s cantilever, intercellular adhesion is directly measured at quasi-physiological conditions. Hereby we present our latest results using this cell decorated probe, to test the linkage establishment to a confluent layer of brain endothelial cells directly by means of single cell force spectroscopy. Adhesion dynamics and strength is characterized by several different parameters calculated. Moreover, spatial distribution of detachment strength is investigated for cancer cells of different origin. Pseudo colored three dimensional maps reveal highly valuable data on several nanomechanical parameters which play crucial role in intercellular interactions.These results highlight the importance of cellular mechanics in brain metastasis formation and emphasize the enormous potential towards exploration of intercellular dynamics related processes. Nevertheless, underlines the power and potential of the Atomic Force Microscope as a highly accurate nano-force tool in cellular biomechanics and life sciences.

AG Végh1, B Varga1, C Fazakas1, I Wilhelm1, J Molnár1, Z Szegletes1, I Krizbai1 and G Váró1

1BRC – Biological Research Centre of the Hungarian Academy of Sciences, Szeged, Hungary; (email of corresponding author: [email protected])

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A novel SU-8 cantilever sensor system for pathogen detection

SU-8, an epoxy based negative photoresist, has emerged as a structural material for microfabricated sensors due to its attractive mechanical and chemical properties. However, the use of SU-8 in BioMEMS application has been limited by the fact that immobilization of biomolecules on SU-8 surfaces often requires prior surface modification or use of an intermediate polymer. 1-3

Herein, we report a novel fabrication process for the functionalization of SU-8 surfaces with IgG proteins. Indeed, fabrication of SU-8 cantilevers was investigated such as non-cross linked epoxy rings are still present on the surface. Immobilization of antibodies to SU-8 surface was thus made possible by condensation of primary and secondary amine groups with the free epoxy groups. The change in SU-8 surface morphology before and after antibody immobilization was characterized and confirmed using both fluorescence microscopy and atomic force microscopy (AFM). With these analytical techniques, optimum antibody concentration and incubation time were investigated to achieve homogenous antibodies layer. This novel antibody immobilization process is simple, rapid and robust as ultrasonic treatment did not remove the grafted IgG proteins.To assess protein binding of this grafted antibody layer, we have developed a new sensor system using piezoresistive material.4 Indeed, gold strain gauge resistors were incorporated in the SU-8 surfaces in order to make them sensitive to changes in surface stress.5,6 A twin-cantilever structure was considered where one is the reference and the other is the measurement cantilever. In these preliminary assays, the model antigen selected to validate our procedure was ovalbumin (OVA), and the antibodies directed against its epitopes were murine monoclonal antibodies. Only the measurement cantilever is coated with anti-OVA antibodies, whereas the reference is coated with non-OVA sensitive antibodies. Our preliminary results showed that when the cantilevers were exposed to OVA antigen, the capture of the latter by the immobilized anti-OVA antibodies induced a surface stress on the cantilever which was translated into an electrical resistance change. The sensitivity of the SU-8 cantilevers was demonstrated in buffer solution and also in water samples. AFM images also confirmed the change in surface morphology after binding of OVA antigen to grafted antibodies. Measurements for pathogen detection using anti-Salmonella antibodies are currently under investigation.

C Soum1, J Moran Meza1, R de Miguel Viscasillas2, J Normand1, M Pham Van1, F Thoyer1, C. Feraudet-Tarisse3, H Volland3, T Berthelot4, L Pham Van1

1SPEC, CEA, CNRS, Université Paris-Saclay, CEA Saclay 91191 Gif-sur-Yvette - France; 2UMR 9187/Unité 1196 Institut Curie, Centre Universitaire 91405 Orsay - France; 3SPI, CEA Saclay 91191 Gif-sur-Yvette - France; 4NIMBE, CEA, CNRS, Université Paris-Saclay, CEA Saclay 91191 Gif-sur-Yvette - France; Corresponding author: [email protected]

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References:[1] Nordström M. et al., Sensors 8, 1595-1612 (2008).[2] Deepu A. et al., J Mater Sci: Mater Med 20, S25-S28 (2009).[3] Joshi M. et al., Appl Surf Sci 253, 3127-3132 (2007).[4] Polesel-Maris J. et al., “Biosensor and method of manufacturing such a biosensor”, FR2971851, WO2012114227 (2011).[5] Johansson A. et al., Sens Actuators, A 123-124, 111-115 (2005).[6] Johansson A. et al., Appl Phys Lett 89, 173505 (2006).

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Effects of Bacterial LPS on the Biomechanics of Alveolar Epithelial Cells Measured by Atomic Force Microscopy

Application of bacterial lipopolysaccharide (LPS) is a widely used model to induce acute lung injury (ALI) in animals. Available data regarding LPS-induced biomechanical changes on pulmonary epithelial cells are limited and conflicting. A better understanding of the effects of LPS on lung cell mechanics is crucial, since LPS could participate in the epithelial injury and disruption reported during mechanical ventilation. Our aim was to investigate the effects of LPS on biomechanical properties of lung epithelial cells using atomic force microscopy (AFM). Lung epithelial A549 cells were cultured at 80% confluence and AFM was employed to measure cell stiffness. Young’s modulus was measured every 5 min throughout 60 min of experiment after treatment with LPS from E. coli (Sigma Aldrich, L2880) (100 µg/mL). Measurements were carried out by using a 4.5 µm diameter spherical tip with a nominal spring constant k=0.03 N/m. Control cells were treated with culture medium and the values obtained were compared with LPS-treated cells for each time-point. Baseline measurements were taken prior the addition of LPS or culture medium. Differences between means were assessed with two-way repeated measures analysis of variance. Application of LPS induced a significant (p<0.05) Young’s modulus increase after 20 min (7.35 ± 1.40 kPa; mean ±SE, n=10) till 60 min (10.09 ± 1.90 kPa) in comparison to controls (4.14 ± 0.86 and 4.93 ± 0.98 kPa, respectively). The increase of lung epithelial cells stiffness by LPS challenge may increase the cytoskeleton centripetal forces applied to cell-cell adhesion, thereby facilitating the disruption of the pulmonary epithelial wall. Further studies focused on the effects of LPS-induce changes in cytoskeletal reorganization are needed.

Vinicius Rosa de Oliveira1,2, Juan José Uriarte1,3,4, Walter A. Zin1, Ramon Farré1,3,5, Isaac Almendros1,3, Daniel Navajas1,3,4

1Unitat Biofísica i Bioenginyeria, Universitat de Barcelona, Barcelona, Spain2Carlos Chagas Filho Institute of Biophysics, Federal University of Rio de Janeiro, Brazil;3CIBER de Enfermedades Respiratorias, Madrid, Spain; 4Institute for Bioengineering of Catalonia, Barcelona, Spain; 5Institut d’Investigacions Biomèdiques August Pi Sunyer, Barcelona, Spain; Email: [email protected]

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AFM investigation of Mesothelial Cells mechanics and their mechanosensing of ECM with different stiffness

Cells are highly dynamic systems that continuously change their chemical and physical characteristics. Many aspects of cellular physiology rely on the ability to control mechanical stimuli across the cell. Mechanical properties of cells, indeed, play an important role in many biological processes such as cell growth, migration, division and differentiation. Cell mechanics is mainly determined by the cytoskeleton [1].The cytoskeleton carries out three broad functions: it spatially organizes the content of the cell; it connects the cell physically and biochemically to the external environment; and it generates coordinated forces that enable the cell to move and change shape. Given the widespread role of the cytoskeleton in cellular function, changes in such a crucial structure lead to pathological condition. Many diseases have now been associated with abnormalities in cytoskeleton, including cancer [2-4]. Intracellular pathological state influences cytoskeletal structure and function, changing the cell mechanical properties. From a general point of view, cytoskeletal strength and organization increase as a cell becomes more differentiated. In particular, during cancer progression, malignant cells undergo a transdifferentiation process, becoming softer than mature fully differentiated cells. The increased deformability allows cancer cells to infiltrate and invade tissues, and migrate to distant sites. Consequently, measuring a cell’s rigidity provides information about its state and composition and may be viewed as a new mechanical marker. The decrease in cell stiffness with malignant transformation has been observed in a variety of cancers such as breast cancer, lung cancer, renal cancer, prostate cancer, oral cancer, skin cancer. [5-7] Furthermore, the decrease in cell stiffness seems to be greater in cells with higher malignancy and metastatic potential [8]. For a very detailed mechanical phenotyping of the tumors, it is necessary to consider the crosstalk between the cells and their surrounding extracellular matrix (ECM). Tissue mechanobiology is the result of a self-sustaining combination of interactions generating between cells and their microenvironments: i) the cell may modify the environment chemically by the metalloproteinases secretion [9,10] , or mechanically and topographically by the generation and the transmission of forces [11,12]; ii) the ECM, thanks its mechanical and topographic features, is able to induce structural modifications

I Musella1, S Fusco1*, V Panzetta1, M De Menna2, M Volante3, I Rapa3, PA Netti 1,4

1Center for Advanced Biomaterials for Health Care@CRIB Istituto Italiano di Tecnologia, Largo Barsanti e Matteucci n. 53, 80125 Napoli, Italy; 2 Department of Experimental and Clinic Medicine, University of Catanzaro Magna Graecia, Catanzaro, Italy; 3Department of Oncology, University of Turin, Regione Gonzole 10, 10043 Orbassano, Torino, Italy; 4 Interdisciplinary Research Centre on Biomaterials (CRIB), University of Napoli Federico II, P.le Tecchio 80, 80125 Napoli, Italy; *corresponding author (e-mail: [email protected])

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References1. Gardel ML1, Kasza KE, Brangwynne CP, Liu J, Weitz DA. Chapter 19 Mechanical Response of cytoskeleton network Methods Cell Biol. 89, 487–519 (2008).2. F.C.S. Ramaekers, F.T. Bosman. The cytoskeleton and disease. J. Pathol., 204 (2004), pp. 351–3543. Hall, A. The cytoskeleton and cancer. Cancer Metastasis Rev. 28, 5–14 (2009).4. C.M. Fife, J.A. McCarroll, M. Kavallaris. Movers and shakers: cell cytoskeleton in cancer metastasis. Br J Pharmacol (2014)5. Guck, J., Schinkinger, S., Lincoln, B., Wottawah, F., Ebert, S., Romeyke,M., et al. (2005). Optical deformability as an inherent cell marker for testing malignant transformation and metastatic competence. Biophys. J. 88, 3689–3698.6. Fuhrmann, A., Staunton, J. R., Nandakumar, V., Banyai, N., Davies, P. C. W., and Ros, R. (2011). AFM stiffness nanotomography of normal, metaplastic and dysplastic human esophageal cells. Phys. Biol. 8:015007.7. Plodinec, M., Loparic, M., Monnier, C. A., Obermann, E. C., Zanetti-Dallenbach, R., Oertle, P., et al. (2012). The nanomechanical signature of breast cancer. Nat. Nanotechnol. 7, 757–765.8. Swaminathan,V.,Mythreye, K.,O’Brien, E. T., Berchuck, A., Blobe, G. C., and Superfine, R. (2011). Mechanical Modeling the mechanics of cancer stiffness grades metastatic potential in patient tumor cells and in cancer cell lines. Cancer Res. 71, 5075–5080.9. Egeblad M, Werb Z. New functions for the matrix metalloproteinases in cancer progression. Nature Reviews Cancer. 2002;2:161-74.10. Deryugina EI, Quigley JP. Matrix metalloproteinases and tumor metastasis. Cancer and Metastasis Reviews. 2006;25:9-34.11. Malik R, Lelkes PI, Cukierman E. Biomechanical and biochemical remodeling of stromal extracellular matrix in cancer. Trends in biotechnology. 2015;33:230-6.12. Levental KR, Yu H, Kass L, Lakins JN, Egeblad M, Erler JT, et al. Matrix crosslinking forces tumor progression by enhancing integrin signaling. Cell. 2009;139:891-906.

of the cytoskeleton and, then, to trigger different cell functions as a responsive adaptation Thus, during tumor progression, not only cells, but also ECM is affected to transformation: cell softening is generally combined with matrix stiffening. For this reason, it could be intriguing to study if changes in ECM architecture and mechanics may be sufficient for cancer cell normalization and tumor regression. To this aim in this work we investigated how cancer modified the mechanics of mesothelial cells. Using AFM technique we quantified the mechanical properties of a benign human mesothelium cell line, Met5A, and three human mesothelioma cell lines (MSTO, REN, MPP-89) with different metastatic potentials. First, we assessed the major compliance of the cancer cells compared to the healthy ones. Then, we associated mechanical phenotype information to the metastatic potential of each cell line by pairing analyses of invasiveness and proliferation with mechanical classification. Finally, focusing the attention on the most aggressive cell line, the influence of ECM stiffness on cell mechanics and functions was outlined.

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Fabrication of Microstructured composite scaffolds to increase osseointegration for Dental and Orthopedic Medicine

Biomaterials-based therapies are a possibility to the repair and regeneration of bone failures caused by tumors. This is the critical problem for Dental and Orthopedic Medicine. Scaffolds are tridimensional structures that act as support to cell proliferation among them. In this work, we have fabricated scaffolds to osseointegration using chitosan, gelatin and microhydroxyapatite solutions and two distinct freeze-drying and cryogenic. Advantages for chitosan use are biocompatibility, biodegradability and non-toxicity. Hydroxyapatite (HA) is a primary inorganic component of bone. Studies in the literature have appointed that the composite scaffold fabricated with natural polymers added hydroxyapatite is effective to improve biological response. Our main objective was to compare freezing methodologies and to verify bone scaffolds degradation using SEM and AFM analysis. We have observed the cell proliferation mechanism in according to each method and alterations in polymeric solutions and hydroxyapatite concentration varying of 10 to 40 %. Authors have indicated that an ideal bone scaffold can be produced with macro and microstructures with a pore size varying of >100 µm to < 20 µm, respectively. Scanning electron microscopy (SEM) and Atomic force Microscopy results indicated that the bone composite scaffolds produced have a well-defined structure with pore size of < 20 µm (Fig. 1).

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Fig. 1: Micrograph of bone composite scaffold (GHapCH).

B. Sakamoto1, M. Silva1, D. M. Zezell2, A. F. G. Monte1, A. Antunes1 1INFIS – Instituto de Física- Universidade Federal de Uberlândia; 2CLA – Centro de Lasers e Aplicações- Instituto de Pesquisas Energéticas e Nucleares at Nuclear and Energy Research Institute (IPEN – CNEN/SP); [email protected]

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Pilot study about characterization by AFM imaging of chitosan artificial vessels manufactured to Cardiovascular Medicine performance

Biodegradable and biocompatible characteristics of chitosan permit their application for biomedical field and applications have been focused by manufacture of innumerous medical devices. Chitosan artificial vessels (CAV) were prepared according to two distinct methods: gelation and neutralization of chitosan solution 5 % (w/v) and freeze-drying. Glycerol was used as a plasticizer. The freeze-dried samples were immersed into 5 % (w/v) NaOH solution and equilibrated for 60 min to neutralize residual acetic acid, washed with distilled water and dried at room temperature. The effect of plasticizer on thermal behavior was verified in this investigation. The thermal behavior showed that occur a substantial mass reduction in order of 60 % to samples manufactured with CH solution. Samples produced with solution CH/GLY had mass reduced in order of 40 %. Internal fractures were observed in CAV prepared with CH. We

M. Silva1, B. Sakamoto1, D. Pasquini2, A. Alencar3, A. F. G. Monte1, A. Antunes1 1INFIS – Instituto de Física- Universidade Federal de Uberlândia; 2IQ – Instituto de Química- Universidade Federal de Uberlândia; 3IFUSP-Instituto de Física- Universidade de São Paulo; [email protected]

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Figure 1: SEM 3D view of CAV.

have observed an aspect of carbonization in the samples. We believe that scaffolds produced with chitosan (90%) and glycerol (10%) presented lower thermal degradation in comparison with pure CH solution. The CAV were put in a solution to simulate the human body environment (pH at ~ 7.40 at 36.5 °C). In the Fig.1 can be visualized the morphology of cross-section of CAV produced in this investigation. Physical characterization of the samples were performed using AFM and SEM.

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Quantitative Nanohistology on the Disease Scleroderma Suggests Excessive Interfibrilar Crosslinks

Fibrous collagens, found throughout the body, provide mechanical strength, elasticity and resilience to connective tissues. Disruption to the chemical, physical or mechanical behaviour of the collagen through disease can be life-threatening and pervasive. Histologically-sectioned biopsies of skin were taken under consent from patients with SSc, and from healthy control patients, and physisorbed to glass slides without fixation or embedding. These were first stained using picro sirrus red (PS), showing collagen fibril alignment under polarised light microscopy (LM). AFM imaging in contact mode in air was performed on different areas of the biopsies, informed by the LM images. Those areas were also indented using REFSPA cantilevers (k = 3 N/m), and modelled according to Oliver-Pharr to obtain Young’s modulus. Previously, we have shown areas of collagen fibril distortion suggestive of collagen stiffening though PS-enhanced LM. Mechanically, statistically significant changes in Young’s modulus were found in these regions by AFM, with SSc samples being stiffer than healthy samples. However, indentation on areas without the use of PS enhanced LM, did not show significant differences, showing the need for PS staining to indicate areas for mechanical testing. Consequently, we have performed mechanical tests using modified tipless (NP-O10) cantilevers and investigated In conclusion, a traffic light system was developed for grouping regions of collagen according their LM profiles after PS staining. This was then combined with mechanical indentation testing to indicate significant changes between SSc and control patients. It is hoped that this technique will soon be used in confirming early-stage SSc diagnoses.

AP Strange*, RJ Stratton, S Parekh, L Bozec1UCL Eastman Dental Institute, University College London, [email protected]

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124

Internalization and cytotoxicity of zeolites in breast cancer cells

Despite advances in therapy, cancer is still the leading cause of death worldwide. Motivated by that, in recent years, scientists have been exploring the medical applications of novel nanomaterials as a promising strategy in the development of anticancer therapies with the ability to overcome many of the limitations common of chemotherapeutic agents [1-3].Understanding the interactions of nanomaterials with biological systems, their rates of uptake/release and intracellular localization are critical goals in nanomedicine, aiming an efficient application of the nanomaterials [4, 5].Zeolites are solid inorganic crystalline nanomaterials with silicon, aluminum, and oxygen in 3D-dimensional structure and well-defined pore networks [6, 7]. Their structure allows the preparation of drug delivery systems (DDS), improving the biodistribution of chemotherapy drugs by protecting them from degradation, delivering them directly to the tumour site and/or preventing them from affecting healthy tissues [8, 2]. In the present work, we focused on the interaction of zeolites with breast cancer cells and epithelial mammary cells in order to evaluate the toxicity and cellular uptake of zeolites and to understand the mechanism underlying the endocytosis of zeolites. Thus, first we investigated the cytotoxicity of zeolites on normal and tumor cells and then we studied the interactions of zeolites with cells by using fluorescence microscopy, confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM).The results showed that the zeolites alone are non-toxic for both type of cells. The interaction studies showed that the cellular uptake is higher and faster in tumor cells than in normal cells and that the nanoparticles are mainly localized in the cytoplasm. These results support the potential of zeolites as carriers for cellular delivery.

N Vilaça1,2,3, F Santos2,3, R Amorim2,3, R Totovao4, E Prasetyanto4, AM Fonseca1, L De Cola4, F Baltazar2,3, IC Neves1

1Centre of Chemistry, Chemistry Department, University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal; 2Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, Campus Gualtar, Braga, Portugal; 3ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães, Portugal; 4Institut de science et d’ingénierie supramoléculaire (ISIS), Universit de Strasbourg, 8 Alle Gaspard Monge, 67083 Strasbourg (France)E-mail: [email protected]

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Acknowledgements: N.V. is recipients of PhD fellowship (SFRH/BD/97797/2013) from Fundação para a Ciência e a Tecnologia (FCT, Portugal). The research centers CQ/UM and Life and Health Sciences Research Institute (ICVS, University of Minho, Portugal) are financial supported by FCT, FEDER-COMPETE-QREN-EU.

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References[1] Sahay G., et al., Endocytosis of nanomedicines, J Control Release 145 (2010) 182-195.[2] Sagnella S.M., et al., Drug Delivery: Beyond active tumour targeting, Nanomedicine 10 (2014) 1131-1137.[3] Koo O.M., et al., Role of nanotechnology in targeted drug delivery and imaging: a concise review, Nanomedicine 1 (2005) 193-212.[4] Rahman M., et al., Protein-Nanoparticle Interactions - The Bio-Nano Interface, Springer Series in Biophysics 15 (2013) 21-44.[5] Moyano D.F., Rotello V.M., Nano Meets Biology: Structure and Function at the Nanoparticle Interface, Langmuir 27 (2011) 10376-10385.[6] Vilaça N., et al., Potentiation of 5-fluorouracil encapsulated in zeolites as drug delivery systems for in vitro models of colorectal carcinoma, Colloids and Surf B 112 (2013) 2013 237-244.[7] Lehman S.E., Larsen S.C., Zeolite and mesoporous silica nanomaterials: greener syntheses, environmental applications and biological toxicity, Environ. Sci.: Nano 1 (2014) 200-213.[8] Martinho O., et al., In vitro and in vivo studies of temozolomide loading in zeolite structures as drug delivery systems for glioblastoma, RSC Adv 5 (2015) 28219-28227.

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AFM Imaging of bacteria in liquid without immobilization: detection of intercellular nanotubes

Atomic Force Microscopy (AFM) is a very powerful tool allowing the investigation and exploration of biological individual structures as single-cell surface constituents at high resolution (Figure 1). In addition to the visualization of the sample studied, one of the most outstanding advantages of AFM is the possibility to map the micromechanical properties of the surface with high resolution in real time, measuring physical properties and providing, thereby, fundamental understanding of biological organization and functioning. Little is known about organization, biophysical properties of inter-bacterial communication, which plays a key role in establishing tight interactions between the bacterial cells, which is the basis of many important biological phenomena of great interest to the scientific community. We used AFM imaging to probe the nanoscale surface between bacterial cells. Atomic Force Microscopy (AFM) is a very powerful tool allowing the investigation and exploration of biological individual structures as single-cell surface constituents at high resolution (Figure 1). In addition to the visualization of the sample studied, one of the most outstanding advantages of AFM is the possibility to map the micromechanical properties of the surface with high resolution in real time, measuring physical properties and providing, thereby, fundamental understanding of biological organization and functioning. Little is known about organization, biophysical properties of inter-bacterial communication,

S Dhahri1,3, C Marlière2,3

1CRMN – Center For Research In Microelectronics & Nanotechnology, Sousse, Tunisia; 2ISMO - Institut des Sciences Moléculaires d’Orsay, University Paris-Sud, CNRS, Orsay, France; 3Géosciences Montpellier - University Montpellier 2, UMR CNRS 5243, Montpellier, France; [email protected]

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Figure1: AFM height images of Rhodococcus wratislaviensis in its

physiological medium.

which plays a key role in establishing tight interactions between the bacterial cells, which is the basis of many important biological phenomena of great interest to the scientific community. We used AFM imaging to probe the nanoscale surface between bacterial cells.

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We developed an original method, by the combination of a gentle sample preparation protocol and an AFM imaging procedure using a specific mode of imaging reducing lateral forces carried on the sample, based on fast and complete force-distance curves made at every pixel of the image. This novel method, enabled us to visualize bacterial intercellular links, nanotubes, under physiologival conditions by AFM in liquid without immobilization (figure 2). This is, to our knowledge, the first successful attempts to image intercellular bacterial links, nanotubes, on living bacteria by AFM in liquid medium without external immobilization. This exploratory study allowed us to observe the interaction between cells and our major observation was the presence of links (bridges, nanotubes) and their imaging AFM which is a big challenge in such in-vivo conditions. Present finding is of direct significance to the understanding of bacteria communication, adhesion. We believe that key challenge for further

Figure 2: AFM deflection image recorded in Qi mode, of the surface of Rhodococcus wratislaviensis showing the existence of links between bacterial cells.

bacterial communication research will be to track intercellular links dynamics over time, and to test the impact of different stimuli in many disciplines of (medicine - antimicrobial therapy, biofilm comprehension, bioremediation –environmental microbiology-, etc.).

KEYWORDS: AFM; Atomic Force Microscopy; Nanotubes; intercellular links, bacteria, liquid imaging; immobilization; cellular connections, Rhodococcus wratislaviensis , cyanobacteria; biofilms; Anabaenopsis circularis; Noctoc.

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Structural changes in plasmid DNA verified by gel electrophoresis and AFM:Sample radiation and damage detection (I)

Many microorganisms possess circular molecules of plasmid DNA. Their conformation may vary from more compact to relaxed forms. Conformational changes have important consequences for the physiological role of plasmids and will influence plasmid transcription, replication and their susceptibility to environmental stresses [1]. Radiation is a common form of stress and can cause structural damage to DNA such as single (SSB) and double strand breaks (DSB) [2]. Such structural damage will also effect plasmid conformation, which can be visualized by agarose gel electrophoresis and atomic force microcopy (AFM) [3]. Recently, it has been indicated that secondary low-energy electrons play an important role in biological damage [4].Therefore, our studies focused on irradiating the plasmid pUC19 with low-energy electrons for the first time under physiological conditions. Using the agarose gel electrophoresis, we identified single strand breaks and double strand breaks within the plasmid pUC19 after electron radiation. Apart from that, tapping mode AFM (TM-AFM) verified our results from gel electrophoresis and revealed additional information concerning the plasmid structure.

S. Meyer*1,2, M.-A. Schröter 1, M. B. Hahn1,3, T. Solomun1, H. Sturm1,4, H.-J. Kunte1

1Federal Institute for Materials Research and Testing, D-12205 Berlin, Germany2Institute of Biochemistry and Biology, University of Potsdam, D-14476 Potsdam, Germany; 3Department of Physics, Free University Berlin, D-14195 Berlin, Germany; 4Technical University Berlin, D-10587 Berlin, Germany* [email protected]

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[1] Berg, J. M., Tymoczko, J. L., Stryer, L. (2007), Biochemie (6. Aufl.). Berlin: Springer Spektrum.[2] Ward, J. F. (1985), Biochemistry of DNA Lesions. Radiation Research, 104, pp. 103-111.[3] Lyubchenko, Y.L., Shlyakhtenko L. S. (1997), Visualization of supercoiled DNA with atomic force microscopy in situ, PNAS, 94, pp. 496–501.[4] Sanche, L. (2009), Biological Chemistry: Beyond Radical Thinking. Nature, 461, pp. 358-359.

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TRF2-mediated control of telomere DNA topology as a mechanism for chromosome end protection

In eukaryotes, the evolution of linear chromosomes led to the appearance of specialized ribonucleoprotein complexes named telomeres that protect chromosome ends against end-to-end fusions and inappropriate activation of the DNA damage response (DDR). A specific telomeric protein TRF2 (Telomeric Repeat binding Factor 2) has a key role in protecting chromosome ends by promoting a DNA lasso-like structure, the t-loop, which caps the very end of telomeres. We showed, measuring DNA contour lengths and volumes of human TRF2-DNA complexes by Atomic Force Microscopy (AFM), that TRF2 wraps a constant number of ~90 bp of DNA per homodimer of one of its sub-domain named TRFH. The DNA wrapping around the TRFH domain was also confirmed by Dual Resonance frequency Enhanced Electrostatic force Microscopy (DREEM). This wrapping ability of TRF2 is closely related to its capacity to recognize and modify telomeric DNA topology. We identified the amino acid residues of TRFH domain involved in DNA wrapping and, hence, constructed a mutant form of TRF2, named Top-less. This mutant still binds telomeric DNA, but exhibits a compromised DNA wrapping activity. We found that in TRF2-depleted human cells, the over-expression of Top-less triggers DDR and alters telomere DNA topology. Using Stochastic Optical Reconstruction Microscopy (STORM) we could demonstrate that Top-less inhibits t-loops formation in cells, a finding that makes the link between wrapping, topology and telomere protection. To summarize, our results show that t-loop formation is tightly linked to TRF2-dependent DNA wrapping. This TRF2 ability to control DNA topology contributes to telomere protection and chromosome stability.

Sabrina Pisano1 ,Delphine Benarroch-Popivker1, Parminder Kaur2, Hong Wang2, Serge Bauwens1 ,Vincent Fraisier3, Arturo Londoño-Vallejo4, Aaron Mendez-Bermudez1,5, Liudmyla Lototska1 , Nadir Djerbi1, Bei Pei1, Eric Gilson1,5,8# and Marie-Josèphe Giraud-Panis1#

1IRCAN – Institute for Research on Cancer and Aging, Nice; CNRS UMR7284/INSERM U1081 University of Nice Sophia Antipolis; Faculty of Medicine; Nice, France; 2Physics Department, North Carolina State University, Raleigh, North Carolina, NC 27695, USA; 3UMR 144 CNRS Institut Curie, Cell and Tissue Imaging Platform (PICT-IBiSA), Nikon Imaging Centre, Paris, France; 4UMR3244, Telomeres and Cancer Laboratory, Institut Curie, Paris 75248, France; 5 International laboratory in Hematology and Cancer, Shanghai Jiao Tong University School of Medicine/Ruijin hospital/CNRS/INSERM/Nice University, associated with the “Pôle sino-français de recherche en sciences du vivant et génomique », Shanghai Ruijin Hospital, Shanghai, P.R. China. 8Department of Genetics, CHU; Nice, France; # corresponding authors :[email protected]; [email protected]

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AFM study of convergent transcriptional complexes

Transcription interference (TI), which takes place during convergent transcription, represents one of the factors regulating gene expression in the cells on a fundamental level [1]. Though several mechanisms of TI have been proposed [1-2] and confirmed by observation of particular transcriptional complexes by atomic force microscopy (AFM) [3], the reasons underlying these mechanisms are not always well understood. In the present work, using AFM, we studied the influence of the interpromoter distance on the formation of convergent transcriptional complexes on the DNA templates (plasmids) with closely spaced promoters, specially constructed for this study. We have shown that small variation of the interpromoter distance results in the formation of open complexes of different morphology and with different probability. In vitro run-off transcription assays have demonstrated varied transcription activity of these plasmids, which correlated with the observation frequency of convergent open complexes. For the interpretation of the observed results we have proposed model of TI, which considers a mutual configuration of the convergent promoters.

EV Dubrovin, ON Koroleva, IV Yaminsky, VL DrutsaLomonosov Moscow State University, Russian Federation; [email protected]

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This work is supported by Russian Foundation for Basic Research (grant 15-32-20629).

1. Palmer, A. C.; Egan, J. B.; Shearwin, K. E. Transcription 2011, 2, 9-14.2. Gullerova, M.; Proudfoot, N. J. Nat. Struct. Mol. Biol. 2012, 19, 1193-1201.3. Crampton, N.; Bonass, W. A.; Kirkham, J.; Rivetti, C.; Thomson, N. H. Nucl. Acids Res. 2006, 34, 5416-5425.

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Quantifying the impact of plant hormones on cell wall mechanical properties

Plant hormones have a key role in development, notably though their regulatory role on gene expression and the effectors of growth. Auxin in particular, plays a critical role in cell growth, affecting both cell division and cell expansion, but also in cell differentiation and cell identity. A key issue is to be able to quantify the local changes in mechanical properties in response to such inputs, to relate the molecular control of development to shape changes. Even though recent work has put forward the obvious correlation between plant development and the mechanical properties of the cell wall, less than five groups have used AFM to probe the changes in mechanical properties in situ. It was previously demonstrated that auxin plays a major role in the shape and mechanical properties in the meristem, the plant stem cell niche [Milani et al., Plant J. 2011]. More recently, the use of PeakForce QNM (PFQNM) on arabidopsis pavement cells allowed demonstrating that stress patterns that control the microtubule orientation lead to the reinforcement of the cell wall parallel to the microtubule network [Sampathkumar et al. eLife 2014]. Here we use a simpler, single cell, system to investigate the impact of auxin treatment on plant cell mechanical properties. More specifically, we use BY-2 cells, the most commonly used model cell line in plant research around the world (a bit like Hela cells in cancer research), mainly because of its unique ability to grow very rapidly (average doubling time of 13 hours in usual culture conditions), and to be easily synchronized and transfected [Nagata et al., Int. Rev. Cytol. 1992]. Using a PFQNM-based approach, we demonstrate that auxin tends to dramatically soften both core and edge parts of individual BY-2 cells. This strategy opens several prospects: this type of approach can be extended to different hormone treatments ; it would also be interesting to carry a dynamic study of the local changes in the cytoskeleton organization over time. More generally, this type of research work can serve as a proof of principle for animal cell models, where signalling pathways are better described than in plants, leading to exciting perspectives in biomedical research.

P Milani1,A. Berquand2, A. Boudaoud1 and O. Hamant1

1Ecole Normale Supérieure de Lyon, France; 2 Laboratoire de Recherche en Nanosciences, Université Champagne-Ardenne, Reims, France; (authors 1 and 2 contributed equally to the work); corresponding author: [email protected]

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Example of Confocal/AFM correlation showing differences in Young’s modulus 72h after the beginning of Auxin treatment at the shoot meristem in Arabidopsis [Sassi et al., Current Biol. 2014].

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Parallel atomic force microscopy for rapid nanomechanical tissue diagnostics

Introduction: Change in cell stiffness is a characteristic of cancer cells [1]. Molecular and structural changes in the extracellular matrix and cellular architecture of living tissue occur during cancer initiation, progression and metastasis [2]. Typical nanomechanical profiles have been correlated well with immunohistopathological analysis of normal, benign and malignant human breast tissue. These distinct nanomechanical signatures of breast cancer can be used as diagnostic tool. Atomic force microscopy (AFM) is the ideal instrument to investigate the nanomechanical properties of breast biopsy samples in a reliable way (Figure 1). AFM analysis of a single biopsy sample requires the acquisition of around 10’000 force displacement curves for a statistically meaningful set of data. Currently, one of the major limitations for a routine use of AFM analysis in hospitals is the diagnostic time that takes several hours: it is too slow. CSEM is developing a parallel AFM for rapid nanomechanical tissue diagnostics.

M Favre, G Weder, R Ischer, M Liley, H Heinzelmann, M DespontCSEM SA – Swiss Center for Electronics and Microtechnology. Neuchâtel, Switzerland; Corresponding author: [email protected]

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Figure 1: Nanomechanical analysis of breast tissue by a single AFM cantilever (left) and typical stiffness profiles of healthy and cancer tissues (right).

Methods: To reduce AFM analysis time, CSEM is developing an AFM instrument using a one-dimensional array of eight cantilevers. In combination with a commercial AFM-based diagnostic tool called ARTIDIS® - Automated and Reliable Tissue DIagnosticS - this will improve the speed of nanomechanical analysis of tissue from several hours to some tens of minutes. The standard procedure starts with removal of a breast biopsy from the suspicious lesion. Multiple stiffness maps are then acquired across the entire biopsy using a single AFM cantilever. Here the replacement of this single cantilever by a cantilever

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References1. Cross et al, Nature Nanotechnology, 2:780-783, 2007.2. Plodinec et al, Nature Nanotechnology, 7:757-765, 2012.

AcknowledgementsThis work is partially funded by the Swiss research program Nano-Tera.

Figure 3: Demonstrator showing the proof of concept of parallel AFM read-out (left) and its integration in a prototype based on the ARTIDIS® platform (right).

Figure 2: Schematic of parallel atomic force microscopy (left) and optical picture of a cantilever array (right) for rapid nanomechanical analysis of tissue sample.

array - 8 cantilevers operating in parallel - involves an innovative parallel read-out of the probes. The method consists in reading a cantilever array with a VCSEL array (Vertical Cavity Surface Emitting Laser). Each laser beam is focused at the end of one cantilever of the array, and the reflected light is detected using a photodetector (Figure 2).

Results: Major technical challenges include an optical system that allows precise alignment and illumination of eight AFM cantilevers by eight semiconductors lasers (VCSELs), the speed of acquisition eight times faster than current systems, and the approach between the cantilever array and the tissue sample. The first stage of the development provided a proof-of-principle, with ultrafast read-out of a 1D cantilever array and parallel force spectroscopy demonstrated on biogels (Figure 3, left). The second stage is actually focused on the integration of the cantilever array read-out in a commercially available AFM head. (Figure 3, right).

Discussion: The major innovation of this work is to reduce the analysis time of the tissue nanomechanical profile for clinical use in breast cancer diagnosis. This new instrument may also contribute to a better understanding of biomechanics in cancer metastasis.

135

Nanocharacterization of chemical and mechanical properties of plant cell walls and lignocellulosic bioinspired assemblies

Lignocellulosic resources have been recognized to be the most promising sustainable and renewable source of materials for the elaboration of composites [1]. Notably, plant cell walls have are multilayered materials resulting from the assembly of different biopolymers: cellulose, hemicelluloses and lignins. These components are interconnected through a variety of covalent and non-covalent bonds that make a highly organized network. This structural organization plays a key role in the regulation of plant cell growth, mechanical properties and biodegradation.Chemical and structural variations within cell wall layers will not only affect the mechanical properties of the fibers but also the different preparation steps such as the defibration, and the interface properties between the fibers and the matrix in the composites [2,3]. Therefore, anticipating and controlling the mechanical properties of the composites implies to evaluate a multi-scale analysis of the morphological and physicochemical properties of the fibers [4]. Nanoscale investigation of the chemical and physical properties of the cell wall could provide a more comprehensive view on the molecular and supramolecular mechanisms that promote their physicochemical properties and reactivity is still challenging.In order to get information of the fiber properties at nanoscale level, the potentialities of atomic force microscopy (AFM) were assessed in different modes while comparing plant cell walls and of bioinspired polymer films. To this end, non-lignified and lignified systems were obtained using cellulose nanocrystals, hemicelluloses and lignin as single, binary and ternary nanocomposites [5]. Thanks to the use of adhesion measurements via tip functionalization with lignocellulosic polymers [6], of nanoInfrared absorption, nanomechanical measurements, and by comparison between native cell walls (poplar, hemp) and lignocellulosic bioinspired films, we will try to understand the relationship between the composition and the supramolecular organization of lignocellulosic polymers and their nanoscale properties.

Muraille L.1,2,3, Chabbert B.2,3, Bercu N.-B.1, Aguié-Béghin V.2,3, Molinari M.1 1LRN EA4682– Laboratoire de Recherche en Nanosciences, Université de Reims Champagne Ardenne, France; 2Inra, UMR 614 Fractionnement des Agroressources et Environnement, Reims, France; 3Université de Reims Champagne Ardenne, Inra, UMR 614 Fractionnement des Agroressources et Environnement, Reims, France; [email protected]

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[1] Thakur V.K., Thakur M.K., Gupta, R.K. Review: Raw Natural Fiber-Based Polymer Composites (2014) International Journal of Polymer Analysis and Characterization. 19, 256-271.[2] Salmen L., Burgert I. (2009) Cell Wall Features with Regard to Mechanical Performance. A Review Cost Action E35 2004-2008: Wood Machining - Micromechanics and Fracture. Holzforschung 63, 121-129. [3] Burgert I., Keplinger T. (2013) Plant micro- and nanomechanics: experimental techniques for plant cell-wall analysis, Journal of Experimental Botany,64, 4635-4649.[4] Le Duigou, A., C. Baley, Y. Grohens, P. Davies, J. Y. Cognard, R. Creach’cadec, and L. Sohier. “A Multi-Scale Study of the Interface between Natural Fibres and a Biopolymer.” Composites Part a-Applied Science and Manufacturing 65, (2014): 161-168. [5] Muraille L., Pernes M., Habrant A., Serimaa R., Molinari M., Aguié-Béghin V., Chabbert B. Impact of lignin on water sorption properties of bioinspired self-assemblies of lignocellulosic polymers, 2015, European Polymer Journal, 64, 21–35. [6] Estephan E., Aguié-Béghin V., Muraille L., Molinari M. (2011) Substrate and film structure impacts on adhesion properties between lignocellulosic polymers. MRS Symposium Proceedings, MRS2011, Boston, USA.

137

Nanoindentation studies on viral nanoparticles

Here we present AFM nanoindentation studies on the mechanical properties of various viral capsids. Combining these measurements with high resolution imaging of the viral nano-particles before and after indentation it can be observed how single capsomeres (the viral structural units) are removed out of the icosahedral capsid lattice. This reveals the fundamental building blocks of the capsid. By biochemically removing the 12 vertices (the pentons) of capsids, we test the effects on the material properties of an icosahedron with missing vertices. In the light of changes in the particle spring constant and breaking force we discuss the influence of the icosahedral corner points on the mechanical stability of the particle. Next we discuss the complex influence of the genome on viral stability and ways how capsid proteins can be rigidified. Finally we show how mechanics and infectivity is linked to each other in an intricate approach using host cell proteins, revealing the interaction mechanism of these proteins with viral particles and how their mechanical interaction with viruses influences infectivity.

S. Maity, W. H. RoosMoleculaire Biofysica, Zernike Instituut, Rijksuniversiteit Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands; corresponding author: [email protected]

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J. Snijder, C. Uetrecht, R. Rose, R. Sanchez, G. Marti, J. Agirre, D. M. Guerin, G. J. Wuite, A. J. R. Heck, W. H. RoosProbing the biophysical interplay between a viral genome and its capsidNature Chemistry, 2013, Vol. 5, 502-509M. Baclayon, G. K. Shoemaker, C. Uetrecht, S. Crawford, M. Estes, B. Prasad, A. J. Heck, G. J. Wuite, W. H. RoosPre-stress strenghtens the shell of Norwalk Virus NanoparticlesNano Letters, 2011, Vol. 11, 4865-4869

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Morphological and nanomechanical characterization of neurons in co-culture with dental pulp stem cells

Peripheral nerve injuries are serious traumas resulting in loss of motor and sensory functions of nerves. The post-traumatic neuropathies are often chronic and mostly resistant to current treatments. Our objective is to develop new strategies to regenerate nerve after trauma by biomimetic approaches and go towards in vitro neurogenesis. For this we use neural crest derived multipotent dental pulp stem cells (DPSC) in co-culture to promote growth of neurons by the expressed neurotrophic factors. We are monitoring the cellular and molecular mechanisms leading to neurons re-growth by multiple biophysical techniques. In order to explore nanomechanical and morphological characteristics of GFP mouse derived DPSC and sensory neurons from mice dorsal root ganglia (DRG), separately and in co-culture, atomic force microscopy was employed. We showed that the mechanical properties of the soma and sub cellular growth cone regions correlate well with the amount, organization and dynamics of the underlying cytoskeletal structure [1,2]. Here AFM in various imaging modes was applied to acquire highly resolved images, showing the fine cytoskeletal structure of the cells in co-culture with DPSC. Using force spectroscopy to assess membrane elasticity on the soma and neurite growth cones of live cells/neurons provides spatially–resolved force maps of the nanomechanical characteristics of cells/neurons. The studies have been completed with optical imaging of neurite outgrowth and DPSC via differential interference contrast microscopy and fluorescence microscopy.Micropattern guided adhesion of neurons will be applied, allowing to follow neurogenesis via directed growth of the neurons in co-culture with DPSC. These measurements contribute to a better understanding of neurogenesis and developing new nerve regeneration strategies.

B Varga1,2, M Martin1, O Romieu1,3, T Cloitre1, PY Collart Dutilleul3, O Páll3, F Cuisinier3, F Scamps4, C Gergely1

1Laboratoire Charles Coulomb (L2C), UMR 5221 CNRS - Université de Montpellier, Montpellier, France; 2Institute of Biophysics, Biological Research Centre, HAS, 6726 Temesvári krt. 62, Szeged, Hungary; 3Bio-engineering Nanoscience Laboratory, EA 4203 Université de Montpellier, Montpellier, France; 4INSERM U 1051 INM - Hôpital St Eloi, 80, rue Augustine Fliche, 34091 Montpellier, France

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Key words: atomic force microscopy, AFM, nanomechanic, elasticity, sensory neuron, DPSC, dental pulp stem cell, live cell, force map, force spectroscopy.Corresponding author: Bela VARGA, [email protected][1] O. Benzina, V. Szabo, O. Lucas, MB. Saab, T. Cloitre, F. Scamps, C. Gergely and M. MartinJ. Biomedical Optics 18(10), 106014 (2013)[2] M. Martin, O.Benzina, V. Szabó, A. G. Végh, O. Lucas, T. Cloitre, F. Scamps and C. GergelyPlos One , (2013) 8 (2), 1.

139

Structural and nanomechanical properties of model lipid membranes

The composition and the function of biological membranes are altered in a large number of common diseases, including neurodegenerative diseases, cancer, diabetes, etc. The lipid phase is indeed increasingly appreciated as an active player in cellular events originated at the membrane. Changes in the membrane properties can affect receptor functioning, protein-membrane and protein-protein associations, as well as ion and small-molecule gradients. Galactosylceramides (GalCer) are glycosphingolipids (GSLs) bound to a monosaccharide group which are primarily found in neuronal tissues and are the major GSL component in the central nervous system. GalCer are responsible for inducing extensive hydrogen bonds that yield their alignment and accumulation in the outer leaflet of the lipid membrane together with cholesterol (Chol) in rafts [1].Using Atomic Force Microscopy (AFM) and AFM-based Force Spectroscopy (FS) (figure 1) to assess the nanomechanics of phospholipid membranes, we are able to evaluate the influence of different constituent of Supported Lipid Bilayers (SLBs) into their stability and function. By indenting with the AFM tip through the SLB, we can assess the maximum force the bilayer is able to stand before breaking, breakthrough force (Fb). This value is directly related to the lipid lateral organization and composition in the membrane in particular environmental conditions [2]. Having even a high spatial range sensitivity and versatility, however, the lateral and vertical resolution obtained by AFM might be inferior to the X-Ray (XR) techniques depending on the density, the order and the composition of the sample. We have therefore decided to investigate SLBs both by AFM and grazing-incidence XR techniques (Reflectometry (XRR) and Surface diffraction). By combining AFM and XR we can obtain not only information about the morphological (figure 2a) and mechanical properties (figure 2b), but also the electronic vertical structure by means of XRR (figure 2c) [3].DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) and DLPC (1,2-didodecanoyl-sn-glycero-3-phosphocholine) model SLBs were characterized by AFM and FS to assess the influence of GalCer on the mechanical properties, as well as the GalCer distribution into the SLBs upon the insertion of Chol as a third component [1]. Comparing the results for both phospholipid systems, we determine the relevance of the physical state of the lipid at the working temperature and the enhanced nanomechanical stability

B Gumí-Audenis1,2,3,4, F Carlà2, F Comin2, F Sanz1,3,4 , L Costa2 , MI Giannotti1,3,4

1IBEC – Institut de Bioenginyeria de Cataluna, Barcelona, Spain; 2ESRF – European Synchrotron Radiation Facility, Grenoble, France; 3UB – Physical Chemistry Department, Universitat de Barcelona, Spain; 4CIBER – CIBER de Bioenginiería, Biomateriales y Nanomedicina, Madrid, Spain;e-mail: [email protected], [email protected]

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after the addition of GalCer. Finally, we present a custom AFM for XR end-stations (figure 2d) able to work with biological samples under physiological conditions and that has been tested to investigate SLBs in an X-Ray synchrotron beamline [3]. This combination also allowed the characterization of the radiation damage induced by the beam on the sample to be studied.

1. B. Gumí-Audenis et al. Soft Matter, 11, 5447-5454 (2015)2. L. Redondo-Morata et al. Atomic Force Microscopy in Liquid, ed. A. M. Baró and R. G. Reifenberger, Wiley-VCH (2012)3. B. Gumí-Audenis et al. Journal of Synchrotron Radiation, 22, 1364-1371 (2015)

Figure 1. AFM and FS on a DLPC:GalCer (80:20 mol%) SLB. a) Topography; b) Profile section; c) Fb distribution; d) Fmap

Figure 2. DPPC SLB results using the custom AFM for X-Rays end-stations: a) AFM topography; b) FS curves; c) XRR (Inset: Scattering Density Profile); d) Custom AFM for X-Rays end-stations mounted at ID03 (ESRF)

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Effect of Actin Organization on the Stiffness of Living Breast Cancer Cells Revealed by Peak Force Modulation Atomic Force Microscopy

Cell stiffness is mainly defined by the arrangement of the cytoskeleton, which in turn controls a number of cellular functions [1]. Any alteration of its properties not only results in the alteration of physiological functions, but can also give rise to a number of human diseases, including cancer [2, 3]. Correlating cell stiffness with the cytoskeletal conformation of cells requires the ability to obtain high resolution apparent Young’s modulus images where specific cytoskeletal structures can be identified. Namely, Peak Force modulation (PFM) mode uses the maximum repulsive force registered during each actuation cycle of the probe as the feedback parameter, enabling quantitative measurements of mechanical properties at high speed and high resolution [4]. Here we use PFM mode for comparative elasticity imaging of three human epithelial breast cell lines with different degree of malignancy; MCF-10A (healthy), MCF-7 (tumorigenic, non-invasive) and MDA-MB-231 (tumorigenic, invasive). We establish the optimum specific image acquisition and analysis parameters for simultaneous high resolution topography and apparent Young’s modulus imaging of living cells in culture media. Furthermore, we describe systematic differences found in stiffness maps from healthy and tumor cells. These differences are resolved as a consequence of the unique high-resolution capability of PFM mode, and they provide crucial information for understanding differences in mechanical characteristics between normal and cancerous cells. The origin of these differences is investigated by complementary immunofluorescence assays, and a correlation is found between the features detected in the apparent Young’s modulus images and the cytoskeleton arrangement. The results show that the combination of PFM and fluorescence microscopy imaging allows establishing a correlation between the arrangement of actin cytoskeleton and the distribution of the apparent Young’s Modulus on cell surface. Healthy cells exhibit well-defined parallel actin stress fibers that correspond to higher apparent Young modulus than those found in both tumor lines, where less prominent and shorter filaments are found on the cell surface. The scarce long actin bundles detected by fluorescence in both tumor cell lines are mainly confined to cell borders or ventral regions, but no long stress fibers are detected in cortical regions by PFM.

A Calzado-Martín, M Encinar, JE Martínez-Gómez, A San-Paulo, J Tamayo, M Calleja IMM-CSIC, Madrid Microelectronics Centre, Isaac Newton 8, 28760, Madrid, Spain; Corresponding author: [email protected]

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References[1] Fletcher, D. A., & Mullins, R. D. (2010). Nature, 463(7280), 485-492.[2] Lu J, Steeg PS, Price JE, Krishnamurthy S, Mani SA, Reuben J, et al. Cancer Res. 2009:15;69(12):4951-3.[3] Suresh, S., et al. (2005). Acta biomaterialia, 1(1), 15-30.[4] Pittenger, B., Erina, N., Su, C., 2010. Bruker Application Note #128.[5] Lodish H, Berk A, Zipursky SL, et al. Molecular Cell Biology. 4th edition. New York: W. H. Freeman; 2000. Section 18.5, Actin and Myosin in Nonmuscle Cells.

We attribute the increase of stiffness of the stress fibers as a result of their intrinsic structure, where actin is coupled with motor proteins such as non-muscle myosin, and integrin-based adhesions, that confer mechanical tension to the bundles [5].

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Mechanical Properties of the Drosophila Central Nervous System

Cell functions can be controlled through purely mechanical means and the impact of mechanical forces is decisive in how tissues coordinate the unfolding of their developmental program to build functional and structurally optimized organs. Understanding not only how cells generate forces, but also how cells in turn respond to forces is of fundamental importance in biology. One neglected aspect of current initiatives exploring the brain is the study of nervous system biomechanics. To tackle this subject, we are studying the development of the Drosophila embryonic Central Nervous System (CNS), a model that allows imaging in vivo and where significant genetic tools are available. Within our general aim of establishing a 4D mechanical map of the developing brain, here we characterized the stiffness of the CNS of Drosophila embryos measured by atomic force microscopy (AFM). Embryos at stage 16 (n=5) were placed on top of positively charged glass slides to immobilize them on a rigid substrate. The embryos were immersed in PBS solution and dissected to expose the CNS allowing AFM measurements. Force-indentation curves were obtained with a custom-built AFM mounted on an inverted optical microscope. A 20 µm diameter polystyrene bead was glued (2-part epoxy) to the end of a tipless cantilever (nominal spring constant k= 0.01 N/m). For each embryo, the Young’s modulus (E) was measured at 3 positions along the antero-posterior axis in the midline and in lateral regions (left and right separated 20 µm from the midline). Five force-indentation curves (ramp amplitude of 20 µm and a frequency of 1 Hz) were recorded at each measurement point. E was computed by fitting force-indentation curves for a maximum indentation of 4 µm with the Hertz contact model for a sphere indenting an infinite elastic half-space. E of the midline was 237 ± 63 Pa (mean ± standard deviation, n=15) and 109 ± 44 Pa and 120 ± 36 Pa for each lateral region, respectively. Midline stiffness was significantly higher than those of lateral domains (p < 10-6). No statistical differences were found between the lateral domains (p > 0.4). The Drosophila embryonic CNS is thus extremely soft in correspondence with other measurements in nervous tissues from different organisms or individual neurons. Further, our results indicate that the most central regions of the embryonic CNS, where most axons bundle, are more rigid than the lateral domains, where most of the somata are found. This suggests a role for the fasciculated axons acting as a suspension cable network providing structural support to the developing nervous system.

Ignasi Jorba1,2, Katerina Karkali3, Anand P. Singh4, Timothy E. Saunders4, Enrique Martin-Blanco3, Daniel Navajas1,2,5

1Unitat de Biofísica i Bioenginyeria, Universitat de Barcelona, Barcelona, Spain2Institute for Bioengineering of Catalonia, Barcelona, Spain; 3Instituto de Biologia Molecular de Barcelona (IBMB-CSIC), Barcelona, Spain; 4Mechanobiology Institute, National University of Singapore, Singapore; 5CIBER de Enfermedades Respiratorias, Madrid, Spain; Email: [email protected]

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AFM Imaging on NanoParticles: Forms and Features

Based on nomenclature, nanoparticles of metallic oxide have at least one physical dimension greater than single crystals and up to one hundred of nanometers. Their structures and properties are significantly different from those of atomic scaled or bulk materials.Recently, these nanoparticles, such as SiO2, TiO2, Ag, etc., have been massively used in modern industrial technologies due to their special size and form. However, their usage also poses several serious health risks. Therefore, it is urgent and necessary of investigating the connection of this problem with biochemical-physical properties of the nano-metallic oxide.Determination of nanoparticle size is the first stage to provide information on characterizing chemical behaviors of the oxide in the toxicological test. In this work, we used atomic force microscopy (AFM) to directly measure the size of single agglomerated nanoparticles.

Shu-wen W. Chen1, Jean-Marie Teulon1, Christian Godon2, Jean-Luc Pellequer1,*1IBS, Univ. Grenoble Alpes/CNRS/CEA, 71 avenue des Martyrs, F-38044 Grenoble, France; 2CEA, iBEB, LBDP, F-13108 Saint Paul lez Durance, France* [email protected]

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Several factors influencing the accuracy of size determination will be addressed, including adhesion of nanoparticles with materials of substrate plate (e.g., mica), form of nanoparticles, or artifacts of probing tip on the topography of nanoparticles. In addition, we have performed contrast enhancement on the raw images to manifest surface features for comparing detailed structures either in various assemblies of same metal oxide, or in different metal oxides in same environments. Examples of AFM imaged nanoparticles of SiO2 with

raw images (left) and processed ones (right).

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Biomechanics of a bone-like 3D model for bone tissue regeneration

Electrospun collagen nanofibers and electrospray hydroxyapatite crystals biocomposites were produced in a membrane-like shape suitable for bone regeneration applications using non-toxic and non-denaturing technology.[ref] Morphological results assessed by scanning electron microscopy and atomic force microscopy (AFM) showed a mesh of collagen nanofibers embedded with crystals of HA, which fiber diameters within the nanometer range (30 nm), thus significantly lower than those reported in the literature, over 200 nm. The mechanical properties assessed by nanoindentation using AFM exhibited elastic moduli between 0.3 and 2 GPa. Fourier transformed infrared spectra analysis confirmed the collagenous integrity as well as the presence of nanoHA in composite. Parameters of biocompatibility, cell adhesion, proliferation and metabolic activity have been evaluated on an in vitro setup regarding of MC3T3-E1 cells. The network architecture allows cell access to both collagen nanofibers and HA crystals as in natural bone environment. The inclusion of nanoHA agglomerates by electrospraying to type I collagen nanofibers improved adhesion and metabolic activity of MC3T3-E1 osteoblasts. These unique properties suggest that this new nanostructured collagen-nanoHA composite can be suitable for bone tissue applications such as bone defects healing, or as a functionally membrane for guided bone tissue regeneration and the treatment of further bone diseases.

N Ribeiro1,2,3, SR Sousa1,2,4, FJ Monteiro1,2,4

1i3S – Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Portugal; 2INEB - Instituto de Engenharia Biomédica, Universidade do Porto, Portugal; 3FEUP – Faculdade de Engenharia da Universidade do Porto, Portugal; 4ISEP – Instituto Superior de Engenharia do Porto, Instituto Politécnico do Porto, Portugal; corresponding author: [email protected]

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[ref] N. Ribeiro, S. R. Sousa, C. A. van Blitterswijk, L. Moroni, and F. J. Monteiro, A biocomposite of collagen nanofibers and nanohydroxyapatite for bone regeneration, Biofabrication. 6, 035015 (2014).

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Stability and mechanical properties of conducting polymer films interfacing with biological systems

Offering a better mechanical interface with tissue and leading to low impedance contacts, conducting polymers are increasingly used to interface electronics with biology. Among the different conducting soft materials, the poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS) is becoming the polymer of choice in biological devices [1] such as neural implants. Despite its increasing use, little is known about the dynamic evolution of its interface and its mechanical behavior under water.The issue of PEDOT/PSS aging in solution has early on been identified as an important challenge in popularizing the use of this material in real life applications. We therefore study using AFM the thickness loss and roughness evolution display by PEDOT/PSS as a function of time and combine this study with a robust wettability characterization approach. We observe a thickness loss of 29%, a roughness rise of about 19% and an increase of the hydrophobia of the surface. The addition of the cross-linker GOPS to stabilize the film is commonly seen in the literature [2]. We confirm that the cross-linker reduces and slows down those effects, for example the thickness loss is reduced to 4.7%.A clear gap is however apparent in the community concerning the impact this chemical has on other properties of PEDOT/PSS. While the evolution of conductivity as a function of molar percentage has been measured, very little is known concerning other parameters which are crucial in interfacing cells with these devices. We once again turned to AFM to measure the evolution of mechanical parameters in liquid. First, we focus on the ability that PEDOT/PSS has to fill itself with water and swell. This hydrogel-like behavior is important when considering the interaction cells will have with the film. We show that while contact measurements are able to give accurate values of thickness in dry films, they fail in wet film due to the force exerted by the tip in this mode. Tapping on the other hand yields reproducible measurements enabling us to report that pure PEDOT/PSS film swell by 660% ± 90 while the addition of 1%wt GOPS results in a tenfold reduction of this value (40% ± 1).

C. Duc,1, S. Bovio,2 , S. Janel,2, F. Lafont,2, G. Maliaras,3, V. Senez,1, A. Vlandas,11BioMEMS Group, IEMN – Institut d’Electronique de Microélectronique et de Nanotechnologie, CNRS/Université de Lille 1, France; 2 IBL - Cellular Microbiology and Physics of Infection Group, CNRS UMR, 8204, INSERM U1019, Institut Pasteur de Lille, Lille University, Lille, France; 3 Department of Bioelectronics, Ecole Nationale Supérieure des Mines CMP-EMSE, MOC, 13541 Gardanne, France; [email protected]

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Intrigued by the strong effect GOPS has on the film properties, we conduct a further study aiming at characterizing the nano-mechanical response of our films with GOPS concentration between 0% and 1%. Very little is currently know about the mechanical properties of PEDOT/PSS films with only one study reporting values obtained in humidity controlled conditions [3] and another in liquid using Peakforce and thus presenting very small indentation values [4]. While the film show a fairly homogeneous mechanical response at various points of the surface (Figure 1), we see a strong variation of the average Young’s modulus of our films from 133kPa ± 13 to 172 MPa ± 19 for GOPS concentration 0% and 1% (Figure 2).We believe that this variation of the mechanical properties will have considerable impact on cell behavior at the interface with the sensors and actuators currently being developed using PEDOT/PSS.

References:[1] A.Elschner et al. PEDOT, Principles and Applications of an Intrinsically Conductive Polymer, CRC Press, 2010, 113–166.[2] S. Zhang et al. APL Mater., 2015, 3, 014911.[3] U. Lang U. et al. Synthetic Metals, 2009, 159, 473–479.[4] R. T. Hassarati et al. Journal of Polymer Science Part B: Polymer Physics, 2014, 52, 666–675.

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The structure of lipid membranes on rough surfacesFlorence Blachon1, Frédéric Harb2, Agnès Piednoir1, Bernard Tinland3, Thierry Charitat4, Giovanna Fragneto5, Olivier Pierre-Louis1, Jean-Paul Rieu1

1ILM – Institut Lumière et Matière, France; 2Faculty of Sciences, Section II, Lebanese University, Lebanon; 3CINaM-CNRS – Centre Interdisciplinaire de Nanoscience de Marseille, France; 4ICS – Institut Charles Sadron, Université de Strasbourg, France; 5ILL – Institut Laue-Langevin, France;

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Supported lipid bilayers have been extensively used as model systems for cell membranes offering the possibility of applying surface sensitive techniques such as atomic force microscopy (AFM), fluorescence microscopy or X-rays and neutron scattering techniques [1]. Most of these techniques require atomically flat surfaces. But in vivo, the surfaces to which lipid layers interact are generally rough. For instance, it was proposed that phospholipid multi-bilayers, which are localized on the rough cartilage surface [2], may explain the exceptional lubricant properties of joints [3]. Some experiments suggested that bilayers might smooth substrate roughness [4]. The purpose of this work is to investigate the structure of lipidic membrane on rough surface.We worked on two different surfaces with a root mean squared (RMS) roughness between 0.1 and 11 nm : silicon wafers etched using RIE (reactive-ion etching) present sharp asperities (Fig. 1.B), while N-BK7, a borosilicate crown glass shows deep holes after a wet chemical etching process (Fig. 1.A).

Figure 1 : (A) 5x5 μm² AFM image of a borosilicate crown glass BK7 (Melles-Griot) etched in a 1:1 sodium-hydroxide:ethanol solution in a US bath (RMS =5.41 nm). (B) 1x1 μm² AFM image in air (tapping mode) of silicon wafer surface etched using Oxford

Supported lipid bilayers of DMPC or DPPC were prepared with Langmuir-Blodgett/Langmuir-Schaeffer deposition technique on such substrates. We performed Fluorescence Recovery after Patterned Photobleaching (FRAPP) experiments to measure the apparent diffusion coefficient D of lipids as a function of roughness and temperature. We observe that D strongly depends

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References:[1] Giovanna Fragneto, Thierry Charitat, Jean Daillant, Eur Biophys J 2012, 41 (863–874)[2] A. V. Sarma, G. L. Powell, et M. LaBerge, J. Orthop. Res. 2001, vol 19 n°4 (671-676)[3] A.-M. Trunfio-Sfarghiu, Y. Berthier, et J.-P. Rieu, Langmuir 2008 , vol 24 n°16 (8765-8771)[4] M-C. Corneci, F. Dekkiche, et J.-P. Rieu, Tribol. Int.[5] O. Pierre-Louis, Phys. Rev. E. 2008, vol. 78 n°2 (p. 021603)

on roughness in the fluid phase, but not in the gel phase. These results suggest an unbinding transition for supported phospholipid bilayers on rough surface, as predicted or suggested by current theoretical models [5]. However, AFM and neutron specular reflectivity studies showed unambiguously that bilayers follow the main substrate corrugations. Fine AFM topographic analysis of bare substrates with super-sharp tip showed that the hidden area remains low with increasing roughness, but the proportion of the sample with highly curved surfaces increases significantly with roughness. Simple simulations of Brownian particle diffusion on flat surfaces with obstacles or defects indicate that these defects might explain the strong dependence between D and roughness in the fluid phase.

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Structural changes in plasmid DNA verified by gel electrophoresis and AFM:Sample AFM preparation and imaging (II)M.-A. Schröter *1, S. Meyer 1,2,M. B. Hahn1,3, T. Solomun1, H.-J. Kunte1, H. Sturm1,4

1Federal Institute for Materials Research and Testing, D-12205 Berlin, Germany2Institute of Biochemistry and Biology, University of Potsdam , D-14476, Potsdam, Germany; 3Department of Physics, Free University Berlin, D-14195 Berlin, Germany; 4Technical University Berlin, D-10587 Berlin, Germany*[email protected]

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Tapping mode AFM (TM-AFM) is a standard technique to image biomolecules and cells avoiding damage to susceptible samples [1]. For preparing AFM samples we fixed (untreated and radiated) plasmids chemically on ultra-smooth mica silanized with APTES [2,3]. Hence, we established a reproducible method to cover the plasmids area-wide on mica substrate.In our AFM study we worked with amplitude as well as frequency feedback control. To achieve high spatial resolution cantilevers with DLC (diamond-like carbon) whiskers at the tip were used which have diameters of about 2-3 nm. The recorded images were examined concerning the contour length, the conformation and the writhing number of the plasmids. We found that the measured contour length is in accordance with the number of base pairs. Apart from that, we indicate different structures of plasmids from our AFM images, which were assigned to our plasmid data from agarose gel-electrophoresis.

References:[1] Lyubchenko, Y.L., Shlyakhtenko, L.S., Ando, T. (2011) Imaging of nucleic acids with atomic force microscopy Methods, 54, 274–283.[2] Xiao, Z., Cao, L., Zhu, D., Lu, Z. (2011) Atomic Force Microscopy in Biomedical Research, Braga P.C., Ricci D. (Eds.), Springer Verlag Berlin, pp.425-435.[3] Adamcik, J., Jeon, J.-H., Karczewski, K.J., Metzler, R., Dietler, G. (2012), Quantifying supercoiling-induced denaturation bubbles in DNA, Soft Matter, 8, 8651.

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Mechanical properties of membranes composed of gel-phase or fluid-phase phospholipids probed by AFM on liposomesO. Et Thakafy1,2, N. Delorme3, C. Gaillard4, V. Vié5, J. Léonil1,2, C. Lopez1,2, F. Guyomarc’h1,2 1INRA, UMR 1253 Science and Technology of Milk and Egg, Rennes, France2AGROCAMPUS OUEST, UMR 1253 Science and Technology of Milk and Egg, Rennes, France; 3Université du Maine, UMR CNRS 6283 Institut des Molécules et Matériaux du Mans, Le Mans, France; 4INRA, UR 1268 Biopolymères Interactions Assemblages, Nantes, France; 5Université de Rennes 1, UMR CNRS 6251 Institut de Physique de Rennes, Rennes, France; [email protected]

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Milk fat globules are the biological entities secreted by all female mammals to ensure many functions in the gastro-intestinal tract of newborns (e.g. bring energy and bioactive molecules, protection against viruses and bacteria). Milk fat globules are enveloped by a biological membrane (the Milk Fat Globule Membrane, MFGM) with a similar composition as that of the plasmatic membrane of the lactating cells (Keenan et al., 1970). However, it remains poorly known despite its importance in nutrition. Phospholipids with saturated fatty acids, and in particular milk sphingomyelin (MSM; ~30% w/w of milk phospholipids) can segregate in the plane of the MFGM and form ordered (gel or Lo) phase domains dispersed in a fluid phase of unsaturated phospholipids (Fig 1 ; Lopez et al., 2010; Murthy et al., 2015). Do these ordered phase domains create a mechanical heterogeneity in the plane of the MFGM ? AFM force spectroscopy experiments performed with model supported bilayers (2-dimensional organization), evidenced a mechanical heterogeneity in the plane of the membrane, associated with the occurrence of stiff MSM domains in the

Fig.1. CLSM image of the biological membrane surrounding fat globules in milk.

Fig.2. Single-lipid liposomes deposited onto gold substrate in hydrated conditions at 20°C, imaged by AFM in contact mode then indented.

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gel phase, dispersed in a continuous fluid phase of unsaturated phospholipids, such as dioleoylphosphatidycholine (DOPC) (Guyomarc’h et al., 2014). Such mechanical heterogeneity in the MFGM could have important consequences on the biological function of the milk fat globule, e.g. on digestion or on their physical stability upon processes. Our strategy is now to prepare liposomes (3-dimensional organization such as milk fat globules) and to evaluate the respective mechanical properties of gel-phase MSM and fluid-phase DOPC bilayers, using AFM. Liposomes were produced in calcium-containing aqueous environment using sonication or extrusion at 60°C, then cooled to 20°C. The morphology of the gel-phase MSM liposomes and fluid-phase DOPC liposomes were characterized by cryoTEM. For AFM experiments, the liposomes were immobilized onto flat substrates of chemically modified gold and imaged in aqueous buffer. Indentation measurements were performed at 20°C at the apex of individual liposomes (Fig. 2) and data was interpreted in terms of the respective membrane stiffness values of the MSM or DOPC curved bilayers. When applicable, a model based on the shell theory is used to infer the Young’s modulus of either type of membrane (Delorme and Fery, 2006). The results are intended to serve as reference values for further investigations on membranes containing both gel phase and fluid phase phospholipids.

References:Delorme N., Fery A. (2006) Physical Review E 74: 030901RGuyomarc’h F., Zou S., Chen M., Milhiet P-E., Godefroy C., Vié V., Lopez C. (2014) Langmuir 30: 6516-6524.Kennan T.W., Morré D.J., Olson D.E., Yunghans W.N., Stuart P. (1970) Journal of Cell Biology, 44: 80-93.Lopez C., Madec M.-N., Jiménez-Flores R. (2010) Food Chemistry, 120: 22-33.Murthy A.V.R, Guyomarc’h F., Paboeuf G., Vié V., Lopez C. (2015) Biochimica and Biophysica Acta: Biomembranes, 1848: 2308-2316.

AcknowledgementsThe authors thank INRA and Région Bretagne for funding O. Et Thakafy’s PhD fellowship. The Asylum Research MFP3D-BIO AFM was funded by FEDER, the French Ministry of Education and Research, INRA, Conseil Général 35 and Rennes Métropole.

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Measuring the bacterial interaction area to abiotic surfaces by single-cell force spectroscopy on tailored samplesC Spengler1, N Thewes1, K Jacobs1 1UDS – Department of Experimental Physics, Saarland University, Saarbruecken, Germany; (email: [email protected])

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Bacteria adhere to virtually every surface and promote the formation of – desirable or unwanted - biofilms. Therefore, in many fields, like engineering, medicine, and biology, understanding bacterial adhesion is of great interest in order to support or inhibit the formation of biofilms. Consequently, there exist different models that describe the process of bacterial adhesion. In these models, besides apparent direct values, like the adhesion force and distance, also more indirect quantities, like the size of the interaction area between bacterial cell and surface, play an crucial role to understand the underlying mechanisms. We present a method to measure the radius of this circular interaction area for Staphylococci by taking advantage of the fact that the adhesion force of these cells differs strongly between surfaces with different surface energies [1]. The measurement is done by collecting multiple force/distance curves with single-cell AFM probes at a very sharp interface between hydrophilic silicon and a hydrophobic self assembling monolayer of silanes. The measured radii of the interaction area range from some tens of nanometers up to almost 300 nm depending on the exerted force trigger. These values also give new insights into the properties and distribution of surface molecules in the bacterial cell wall.

References:[1] N. Thewes, P. Loskill, P. Jung, H. Peisker, M. Bischoff, M. Herrmann, K. Jacobs, “Hydrophobic interaction governs unspecific adhesion of staphylococci: a single cell force spectroscopy study”; Beilstein J. Nanotechnol. 5 (2014) 1501

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Probing peptide-gold interaction at the single-molecule level through AFM-based methodsM Steffenhagen1,2, J Landoulsi 1, E Maisonhaute2 1Sorbonne Universités, UPMC Paris 6, UMR 7197, CNRS, Laboratoire de Réactivité des Surfaces, F-75005 Paris, France; 2 Sorbonne Universités, UPMC Paris 6, UMR 8235, CNRS, Laboratoire Interface et Systèmes Electrochimiques, F-75005 Paris, France; [email protected]

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Biomaterials have received increasing interest in material science due to the high versatility of biological molecules and their aptitude to exhibit unique specific interactions with other macromolecules and inorganic entities. Indeed, recent progresses in combinatorial biology have allowed the identification of peptide sequences which strongly interact with inorganic surfaces, conferring biological functions to synthetic materials. The specificity of these peptide binders has been exploited in various applications such as regenerative medicine, crystal growth regulation and molecular electronics [1]. Behind these potential applications, questions arise as to (i) the extraction of values quantifying site-specificitc recognition by the peptide and (ii) the assignment of the targeted recognition to structural properties. This issue can be approached by probing events at the single-molecule level.For this purpose, we used atomic force microscopy (AFM) to probe the interaction between a C(KAAA)2KC tailor-made oligopeptide and a gold surface and examined the evolution of the unbinding force as a function of the loading rate by means of dynamic force spectroscopy. Interestingly, our results revealed a similar behaviour to that generally observed for biological receptor-ligand complexes. Owing to the composition of the peptide and its conformation in phosphate buffer solution, several chemical groups may be involved in the peptide-surface interaction, including -COOH, -NH2 and -SH. We profoundly examined the occurrence of this interfacial processes in different situations obtained by varying (i) the composition of the gold surface via surface functionalization, (ii) the pH of the aqueous media and (iii) the degree of β-helicity in 20% TFE aqueous solution (confirmed by circular dichoism). The energetic of peptide-gold surface interaction was characterized by means of the widespread Bell-Evans approach. A second approach was conducted to ensure the reliability of gold-peptide interaction through conductance measurements using C-AFM break junctions. This method has proven to be an effective way for targeting metal-molecule junctions for single events since each conduction channel can be assigned to a specific current signature [2]. Depending on the anchoring site, multiples sets of conductance were expected to arise in accordance to the site-specific sensitivity of the electrical contact.

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Our results showed clear contributions of single event rupture with different sets correlated with their multiple bond rupture, giving an insight to the involved headgroup. Through the description of different situations, the interest of this methodology is detailed and its limitation is discussed.

References:[1] Kanata S. et al., Analytical Sciences 29(405-409) 2013[2] Venkataraman L. et al., Nature 442(904-907) 2006

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In-vivo high resolution AFM topographic imaging of C.elegans reveals previously unreported surface structuresClara L Essmann1*; Muna Elmi1; Mike Shaw1,2; Giridhar M Anand1,3; Vijay Pawar1; Mandayam A Srinivasan1,4

1Department of Computer Science, University College London, London, UK;2Analytical Science Division, National Physical Laboratory, Teddington, UK;3Massachusetts Institute of Technology, Cambridge, MA 02139, USA; 4Department of Mechanical Engineering and Research Laboratory of Electronics, MIT, Cambridge, USA; (*corresponding author: [email protected])

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The model organism C.elegans is used for a wide range of medical research applications including drug screening and the study of host-pathogen interactions; both applications highly depend on the integrity of the nematode’s cuticle. Mutations affecting both drug adsorption and pathogen clearance have been proposed to relate to changes in the cuticle structure, but never visually examined in high resolution. Scanning electron microscopy studies have described the gross surface structure of the worm’s cuticle, while TEM sections reveal the structure of the cuticle sub-layers. However, AFM offers significant advantages over SEM including the acquisition of quantitative 3D-images and biomechanical information. More importantly, for in-vivo biological imaging, AFM does not require sample dehydration or labeling. We have developed an AFM-protocol that allows us to acquire topographic images of the nematode’s cuticle at nanometer spatial resolution under physiological conditions. We apply our method to reveal surface structures that remain unreported by SEM in the cuticle of known collagen mutants whilst simultaneously investigating the biomechanical properties of individual surface components.

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Watching the eukaryotic fatty acid synthase multienzymatic factory at workFriederike Benning1,*, Habib Bukhari1, Yusuke Sakiyama1,2, Roderick Lim1,2, Timm Maier1

1Biozentrum, University of Basel, Switzerland; 2Swiss Nanoscience Institute, University of Basel, Switzerland; *corresponding author email: [email protected]

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Multienzymes are giant biosynthetic factories responsible for a variety of vital catalytic processes, particular in eukaryotic cells. As opposed to multisubunit complexes, a multienzyme integrates all required enzymatic protein functions as domains into a giant polypeptide chain, which may further oligomerize to form the functional biological assembly. The eukaryotic fatty acid synthases (FAS) are giant multienzymes, which catalyze the de-novo biosynthesis of fatty acids from carbohydrate-derived precursors. They comprise at least seven types of functional domains and catalyze more than 40 individual reaction steps for the biosynthesis of palmitic acid. Metazoan FAS is active as an X-shaped homodimer with two lateral reaction clefts, which is structurally and functionally segregated into a condensing and a modifying region. Efficient coupling of the individual reactions is facilitated by a large degree of conformational flexibility, as well as an elastically linked carrier protein domain to which the growing fatty acid chain is tethered during biosynthesis (1, 2, 3).Static structural information from crystallographic analysis or electron microscopy of metazoan FAS is available (1, 4). However, the crucial interplay and coupling of protein conformational dynamics and product biosynthesis remains poorly characterized at the spatial and temporal level. Here, we describe ongoing approaches for immobilizing FAS and visualizing it using high-speed AFM with the ultimate aim of real-time monitoring of protein dynamics during catalysis.

References:(1) Maier, T., M. Leibundgut, and N. Ban, The crystal structure of a mammalian fatty acid synthase. Science, 2008. 321(5894): 1315-22. (2) Brignole, E.J., S. Smith, and F.J. Asturias, Conformational flexibility of metazoan fatty acid synthase enables catalysis. Nat. Struct. Mol. Biol., 2009. 16(2): 190-7.(3) Maier, T., et al., Structure and function of eukaryotic fatty acid synthases. Q. Rev. Biophys., 2010. 43(3): 373-422. (4) Maier, T., S. Jenni, and N. Ban, Architecture of mammalian fatty acid synthase at 4.5 A resolution. Science, 2006. 311(5765): 1258-62

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Mechanical unfolding of HCN2 channelsAndrea Pedroni1, Andrea Alfieri2, Paolo Fabris1, Anna Moroni2, Loredana Casalis3 and Vincent Torre1

1International School for Advanced Studies (SISSA) Neuroscience Area, via Bonomea 265, 34136 Trieste (Italy); 2Università degli studi di Milano, via Celoria 26, Milano (Italy); 3CBM S.c.r.l., Area Science Park, Basovizza 34149 Trieste (Italy); Andrea Pedroni: [email protected]; Vincent Torre: [email protected]

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Hyperpolarization-activated and Cyclic Nucleotide-gated (HCN) channels belong to the voltage gated superfamily of ionic channels. The gating of HCN channels is unique, because HCN channels open upon hyperpolarization and not depolarization, as it usually occurs in Na and K channels. The gating of HCN channels is also regulated by cyclic nucleotides. HCN channels are tetramers and each subunit is composed by a cytosolic Cyclic Nucleotide Binding Domain (CNBD), connected by the C-linker to the six transmembrane domains (TMDs), which form the pore region and the Voltage Sensor Domain (VSD). The structural and molecular basis for the function of HCN channels is not fully understood and the crystal structure not yet solved. We have used Single Molecule Force Spectroscopy (SMFS) to study structural properties of HCN2 channels in the presence and in the absence of cyclic adenosine monophosphate (cAMP). We have analyzed the purified CNBD construct, functionalized with a histidine tag at the C-terminus, two cysteines at the N-terminus, which was then deposited on gold surfaces. The Force vs Distance (F-D) curves from the construct were used as a finger-print to recognize F-D curves obtained from the unfolding of the whole HCN2 channels, expressed in the membrane of Xenopus laevis oocytes.Our results show different unfolding pathways of the C-linker and CNBD construct, both in the presence and in the absence of the cAMP. In the absence of the cAMP the unfolding of the C-linker region shows a higher variability respect to CNBD. Moreover, the unfolding of the construct in the presence of the cAMP reveals a higher stability throughout the entire molecule, suggesting an involvement of the C-linker in the structural rearrangement underlying the regulatory function of the cAMP. The F-D curves from the entire channel, both in the presence and in the absence of the cAMP, show a conserved pattern of peaks, representing a template for the unfolding of the transmembrane core of the other members of the voltage gated superfamily. In the presence of cAMP, we observed a different unfolding of the pore region and of the N-terminus, possibly indicating different intersubunit interactions. Indeed, in the presence of the cAMP, several dozens of F-D traces exhibiting the sequential unfolding of consecutive subunits were observed.

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AFM imaging of silica nanoparticles with and without the presence of adsorbed proteinsJean-Marie Teulon1, Shu-wen W. Chen1, Antoine Thill2, Laurent Marichal3, Jean Labarre3, and Jean-Luc Pellequer1

1IBS, Univ. Grenoble Alpes/CNRS/CEA, 71 avenue des Martyrs, F-38044 Grenoble, France; 2CEA, IRAMIS, LIONS, F-91191 Gif-sur-Yvette, France; 3CEA, iBITECs, LSOC, F-91191 Gif-sur-Yvette, France; * [email protected]

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In physiological environment, nanoparticles (NPs) quickly become covered by proteins. Biodistribution of NPs are modified due to the type of proteins adsorbed on NPs. Such targeting is under intense investigation either to understand toxicology effects of NPs or use NPs as vectors to deliver cargo into a specific tissue or organ. Protein bound to NPs form the so-called “hard corona”. Because of the large difference in density between proteins and NPs, it is difficult to observe the presence of proteins at the surface of NPs. Besides, the putative impact of the behavior of NPs in presence of proteins is not well understood.In this work, we present results of measuring NPs sizes in the presence or absence of proteins. Spherical SiO2 NP was selected. Size characterization was performed using atomic force microscopy (AFM). Imaging of NPs was performed using the PeakForce tapping mode in ambient conditions with NP deposited on freshly cleaved mica. Raw SiO2 NP were imaged from 25 different samples in four independent experiments and totaling 519 isolated NP used to build its height distribution. The major peak in height distribution is 23 nm (ranging from 13 to 29 nm). No significant agglomerate or broken NPs was observed.Total yeast extract (10 mg/ml) was incubated and mixing with SiO2 NP during 2h at room temperature. NPs were centrifuged for 5 min at 5000 RPM and the supernatant was discarded. Pelleted NPs were re-suspended in water and deposited on mica in a super-dry cabinet overnight. Samples were imaged using the same conditions as raw NPs. A total of 896 isolated and agglomerated NPs were identified in 29 different samples in four independent experiments. Striking differences are observed: first, very large agglomerates are found when NPs were in contact with yeast extract, 2) a bimodal NP height distribution is observed with a major peak at 24 nm and a minor peak at 11 nm. Large agglomerates are likely due to protein bridging two NPs at a time and thus lead to increase precipitation of NPs. Height of large agglomerates were obtained by measuring NP located at the border of the agglomerate; thus, it was possible to measure their height similarly to what is done with isolated NPs. A significant quantity of isolated NPs are observed but their average height is much smaller than the initial height of raw SiO2 NP (11 nm). At the moment, it is not known whether these small NPs are degradation from raw NPs due to the presence of proteins or they come from a selection by proteins from the pool

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of raw NP which were not visible during the imaging of raw SiO2 NP. This work has been repeated on other type of purified proteins: bovine serum albumin (BSA) and hemoglobin. With hemoglobin, both large agglomerate and smaller diameter SiO2 NP were observed. However, with BSA no reduced diameter NPs are observed but the presence of large agglomerate is still observed.

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Opening and stabilization of Transendothelial Macroapertures in living cells probed by AFM elasticity measurements S. Janel1, C. Stefani2,3, D. Gonzalez-Rodriguez4, A. Doye2, Patricia Bassereau5, E. Lemichez2, F. Lafont1

1Cellular Microbiology and Physics of Infection Group, Center for Infection and Immunity of Lille, CNRS UM8204, INSERM U1019, Institut Pasteur de, Lille, Lille regional Hosp. Centr., Lille Univ., 59021 Lille, France; 2INSERM, U1065, Université de Nice-Sophia Antipolis, Centre Méditerranéen de Médecine Moléculaire (C3M), Microbial toxins in host-pathogen interactions, Nice, France; 3Immunology program, Benaroya Research Institute at Virginia Mason, Seattle, WA, USA; 4Laboratoire de Chimie et Physique (LCP-A2MC), Université de Lorraine - Metz, 1 boulevard Arago, 57078 Metz, France; 5 Sorbonne Universités, UPMC Univ Paris 06, 75005, Paris, France;

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Transendothelial cell macroaperture (TEM)[1] tunnels are important physiological regulators of the endothelial barrier. They are induced by several protein toxins from pathogenic bacteria such as Staphylococcus aureus EDIN, Bacillus anthracis Edema toxin or Clostridium botulinum C3 exotoxin. They are also involved in processes such as leukocytes transcellular diapedesis and probably many more migration phenomena. TEM dynamic comprises three phases: opening of the TEM due to a destabilisation of the cytoskeleton and dewetting[2], stabilization of the TEM width at a given size ranging from several microns to dozens of microns, and closure by induction of actin-driven waves. This spectacular process happens within minutes and closure is essential to prevent cellular leakage and animal death.We have already described the opening of TEMs by introducing a theoretical model considering the TEM formation as a cellular form of dewetting, and by showing the stabilization of TEM width by a local tension along the edge of the TEM[2]. We have observed by using AFM in topography mode the height profile around the edge, giving crucial evidence for the dewetting process[2]. We also previously described dendritic network of actin filaments induced by MIM-driven Arp2/3-dependent actin polymerization[3] for the closure.We herein deepen our biophysical understanding of the stabilization step by applying fast force mapping (Quantitative Imaging & Fast Force Volume) on living cells in order to probe key components: stiff actomyosin cable encircling TEMs, and the surrounding cell structure.

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References:[1] L. Boyer, A. Doye, M. Rolando, G. Flatau, P. Munro, P. Gounon, R. Clément, C. Pulcini, M. R. Popoff, A. Mettouchi, L. Landraud, O. Dussurget, and E. Lemichez, “Induction of transient macroapertures in endothelial cells through RhoA inhibition by Staphylococcus aureus factors.,” J. Cell Biol., vol. 173, no. 5, pp. 809–819, 2006.[2] D. Gonzalez-Rodriguez, M. P. Maddugoda, C. Stefani, S. S. Janel, F. Lafont, D. Cuvelier, E. Lemichez, and F. F. Brochard-Wyart, “Cellular dewetting: opening of macroapertures in endothelial cells.,” Phys. Rev. Lett., vol. 108, no. 21, p. 218105, May 2012.[3] M. P. Maddugoda, C. Stefani, D. Gonzalez-Rodriguez, J. Saarikangas, S. Torrino, S. Janel, P. Munro, A. Doye, F. Prodon, M. Aurrand-Lions, P. L. Goossens, F. Lafont, P. Bassereau, P. Lappalainen, F. Brochard, and E. Lemichez, “cAMP signaling by anthrax edema toxin induces transendothelial cell tunnels, which are resealed by MIM via Arp2/3-driven actin polymerization.,” Cell Host Microbe, vol. 10, no. 5, pp. 464–74, Nov. 2011.

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Detecting nanoscale motion of biological systems Petar Stupar1, Giovanni Longo1, Giovanni Dietler1, Sandor Kasas1

1Laboratory of Physics of Living Matter – EPFL, Lausanne, SwitzerlandCorresponding author: [email protected]

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Recently, the versatility of Atomic Force Microscopy technique has been demonstrated by the development of numerous new sensing modes. The applications of AFM-based nano-mechanical sensing have been mostly limited to measurements of resonance frequency shifts, to determine the presence of very small masses, or the static determination of the stress deflections induced by the presence of specific ligands [1]. Here, we present a recently developed nano-mechanical sensor that can characterize biological systems, transducing the small fluctuations that define living systems, into a measurable mechanical change in the cantilever [2, 3, 4]. The intensity of such movements is an indication of the viability of living specimens and conveys information related to their metabolic activity. Briefly, the technique consists of immobilizing the biological specimens of interest on a cantilever sensor, which is inserted into an analysis chamber, where its spontaneous oscillation are recorded as a function of time. The oscillations of the cantilever are quantified by measuring the signal’s variance while different working conditions are produced by exchanging the liquid in the analysis chamber. In case of bacterial and mammalian cells, upon the injection of nutrients the amplitude of the oscillations increases, whereas the exposure to inhibiting or lethal agents stops the movements of the cantilever. Numerous experiments recorded on various bacteria and mammalian cells will be presented. The sensitivity and the temporal resolution of this technique permit to predict its potential application to a vast number of fields, such as cellular and molecular biology, microbiology, drug development, high-speed pharmaceutical evaluation or molecule conformational monitoring.

References:[1] Tamayo, Javier, Priscila M. Kosaka, José J. Ruz, Álvaro San Paulo, and Montserrat Calleja. “Biosensors Based on Nanomechanical Systems.” Chemical Society Reviews 42, no. 3 (January 16, 2013): 1287–1311.[2] Longo, G., L. Alonso-Sarduy, L. Marques Rio, A. Bizzini, A. Trampuz, J. Notz, G. Dietler, and S. Kasas. “Rapid Detection of Bacterial Resistance to Antibiotics Using AFM Cantilevers as Nanomechanical Sensors.” Nature Nanotechnology 8, no. 7 (July 2013): 522–26. [3] Alonso-Sarduy, Livan, Paolo De Los Rios, Fabrizio Benedetti, Dusan Vobornik, Giovanni Dietler, Sandor Kasas, and Giovanni Longo. “Real-Time Monitoring of Protein Conformational Changes Using a Nano-Mechanical Sensor.” PloS One 9, no. 7 (2014): e103674.[4] Kasas, Sandor, Francesco Simone Ruggeri, Carine Benadiba, Caroline Maillard, Petar Stupar, Hélène Tournu, Giovanni Dietler, and Giovanni Longo. “Detecting nanoscale vibrations as signature of life.” Proceedings of the National Academy of Sciences 112, no. 2 (2015): 378-381.

partici-pants’list

165

participants’list Abimbola Feyisara AdedejiUniversity of TriesteItaly

Adam EnglerUC San DiegoUSA

Adam StrangeUCLUnited Kingdom

Alexander DuleboBrukerGermany

Alexandre BerquandUniversité de ReimsFrance

Alicia Calzado MartínMadrid Microelectronics CentreSwitzerland

Alma Pérez PerrinoInstituto de Materiales de Madrid-CSICSwitzerland

Ana Paula PêgoINEB, i3SPortugal

Andrea AntunesInstituto de Física- Universidade Federal de UberlândiaBrasil

Andrea PedroniScuola Internazionale Superiore di Studi AvanzatiItaly

Andrzej KulikEPFL - Laboratory of Physics of Living MatterSwitzerland

Anthony Le CigneUniversity of Reims Champagne-ArdenneFrance

Antoine DujardinBrukerFrance

Arkady BitlerPhysics Department and Nanotechnology Institute, Bar Ilan UniversityIsrael

Attila-Gergely VeghBRC HASHungary

Bart HoogenboomLondon Centre for Nanotechnology, University College LondonUnited Kingdom

Bartlomiej ZapotocznyJagiellonian UniversityPoland

166

Béla VargaLaboratoire Charles CoulombCNRS - Université de MontpellierFrance

Boris RittwegerBrukerGermany

Caroline DucIEMN - BioMEMSFrance

Celine Elie-CailleFEMTO-ST Institute UMR 6174France

Christian SpenglerSaarland UniversityGermany

Claire SoumCEAFrance

Clara EssmannUniversity Collegen LondonUnited Kingdom

Clemens M. FranzKarlsruhe Institute of TechnologyGermany

Cristina RibeiroINEB, i3SPortugal

Daniel NavajasUniversity of Barcelona/ IBECSpain

Drew MurrayBrukerUnited Kingdom

Eleonore LambertUniversity of Reims Champagne-ArdenneFrance

Evgeny DubrovinLomonosov Moscow State UniversityRussia

Fabian HerrmannInsitute of Pharmaceutical Biology and Phytochemistry, University of MunsterGermany

Fanny GuyomarchINRAFrance

Favre MélanieCSEM SASwitzerland

Fernando Jorge MonteiroINEB, i3SPortugal

Filomena A. CarvalhoIMMPortugal

Florence BlachonInstitut Lumière et Matière de LyonFrance

Friederike BenningBiozentrum, University of BaselSwitzerland

Friederike NolleSaarland UniversityGermany

Georg FantnerÉcole Polytechnique Fédéral de LausanneSwitzerland

Giacinto ScolesUniversity of UdineItaly

Giampaolo ZuccheriUniversity of BolognaItaly

167

Guido CaluoriInternational Clinical Research Center of St. Anne\’s University Hospital Brno (FNUSA-ICRC)Czech Republic

Hartmut StadlerBrukerGermany

Heinz SturmBAM - Federal Institute for Materials ResearchGermany

Hermann SchillersInstitute of Physiology II, University MünsterGermany

Ida MusellaIstituto Italiano di TecnologiaItaly

Ignasi JorbaInstitute for Bioengineering of CataloniaSpain

Isabel Freitas AmaralINEB, i3SPortugal

Iurii EfremovBirck Nanotechnology Center, Purdue UniversityUSA

Janel SébastienCNRS UR8204 CIILFrance

Jean-Luc PellequerInstitut de Biologie StructuraleFrance

Jean-Marie TeulonCEA/ DSVFrance

Jérôme DahlgrenBrukerFrance

Joaquim MarquêsFaculdade de Ciências, Universidade de LisboaPortugal

Juan José UriarteInstitute for Bioengineering of CataloniaSpain

Karin JacobsSaarland UniversityGermany

Katarzyna Malek-ZietekJagiellonian UniversityPoland

Livie Dorwling-CarterETH Zürich_Laboratory of Biosensors and BioelectronicsSwitzerland

Loic Hamon, Lab SABNP- INSERM U1204France

Lorena Redondo-MorataInserm U1006 / Université Aix-MarseilleFrance

Magali Phaner-GoutorbeInstitut des Nanotechnologies INL - Ecole Centrale de LyonFrance

Malgorzata LekkaInstitute of Nuclear Physics, Polish Academy of SciencesPoland

Manfred RadmacherInstitut für Biophysik, Universität BremenGermany

168

Manuela BrásINEB, i3SPortugal

Marc B. HahnBundesanstalt für Materialforschung und -prüfungGermany

Marc SerranoInstitut de Bioenginyeria de CatalunyaSpain

Marco TortoneseBrukerUSA

Marek SzymonskiJagiellonian UniversityPoland

María Gómez Lázaroi3S, INEBPortugal

Maria-Astrid SchröterBundesanstalt für Materialforschung und -prüfungGermany

Marie SteffenhagenUniversité Pierre et Marie CurieFrance

Marie-Lise PerraultBrukerFrance

Marina I. GiannottiCIBER-BBN/Institute for Bioengineering of CataloniaSpain

Mário BarbosaINEB, i3SPortugal

Marta Martin-FernandezUniversité de MontpellierFrance

Martin PeslThe International Clinical Research Center of St. Anne\’s University Hospital BrnoCzech Republic

Martina MaaseInstitute of Physiology IIGermany

Matthias LauerRoche Innovation Center Basel F. Hoffmann-La Roche LtdSwitzerland

Michael MolinariUnivertite de Reims Chamagne ArdenneFrance

Natália VilaçaUniversity of MinhoPortugal

Nicola GalvanettoScuola Internazionale Superiore di Studi AvanzatiItaly

Nicolas Bogdan BercuLaboratoire de Recherche en NanosciencesFrance

Nicolas SchierbaumInstitute of Applied Physics, University of TübingenGermany

Nicolas ThewesSaarland UniversityGermany

Nilza Ribeiroi3S, INEBPortugal

169

Nuno C. SantosIMM, FMULPortugal

Orsolya PállUniversity of MontpellierFrance

Oumaima Et- ThakafyINRAFrance

Pascal OdermattÉcole Polytechnique Fédérale LausanneSwitzerland

Pedro GranjaINEB, i3SPortugal

Pedro J. de PabloUniversidad Autónoma de MadridSpain

Pedro MorenoINEB, i3SPortugal

Pedro TempradoTelstarSpain

Petar StuparSwiss Federal Institute of Technology LausanneSwitzerland

Peter HinterdorferJohannes Kepler University Linz, Institute of BiophysicsAustria

Phil WilliamsThe University of NottinghamUnited Kingdom

Pierre ParotCEA CadaracheFrance

Pieter A. BeuleInternational Iberian Nanotechnology LaboratoryPortugal

Raphaël CosteLaboratoire de Recherche en NanoscienceFrance

Ricardo PiresZIK-HIKE, University of GreifswaldGermany

Sabrina PisanoIRCAN INSERM U1081 - CNRS UMR7284 - UNSFrance

Samia DhahriCRMN - Center of Research on Microelectronics and NanotechnologyTunisia

Samuel Lesko, BrukerFrance

Sanjay KumarUniversity of CaliforniaUSA

Sebastian JaramilloSorbonne University - Université de technologie de CompiègneFrance

Simon D. ConnellUniversity of LeedsUnited Kingdom

Simon ScheuringINSERM / Aix-Marseille UniversitéFrance

170

Simone BovioCNRSFrance

Sourav MaityZernike Institute, University of GroningenThe Netherlands

Susana R. SousaINEB, i3SPortugal

Susann MeyerFederal Institute for Materials Research and TestingGermany

Thi-Huong NguyenInstitute for Immunology and Transfusion Medicine, University Medicine GreifswaldGermany

Thomas FaidtSaarland UniversityGermany

Thomas MuellerBrukerUSA

Tihomir SolomunBAM Federal Institute for Materials Research and TestingGermany

Vadim V. KumeikoFar Eastern Federal UniversityRussia

Valeria PrystopiukInstitute of Physiology IIGermany

Verena HofschröerInstitute of Physiology IIGermany

Vlandas AlexisIEMN - Université Lille 1 – CNRSFrance

Yoojin OhInstitute of Biophysics, Johannes Kepler University LinzAustria

Zuzanna PietralikAdam Mickiewicz University in Poznan, Faculty of Physics, Department of Macromolecular PhysicsPoland

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