MOLECULAR BIOLOGY OF THE CELL

2012 - 2013

(From 14 January to 15 February, 2013)

Directors of the course

Roberto BRUZZONE HKU - Pasteur Research Centre

Hong Kong

Philippe CHAVRIER

Institut Curie - Research Section Paris

Head of Studies

Chiara ZURZOLO

Institut Pasteur Paris

Location Centre d'Enseignement de l'Institut Pasteur

Pavillon « Louis Martin » 28, rue du Docteur Roux

75724 Paris Cedex 15

Lectures: Room No 1 Practical Work: Room on the first floor

DESCRIPTION OF THE COURSE

The Molecular Biology of the Cell course is an intensive laboratory and lecture course of five

weeks divided into weekly modules, each focusing on a cutting-edge aspect of cell biology. It is

composed of lectures given by internationally renowned scientists, and of two practical sessions

organized together with teams from the Curie and the Pasteur Institutes. The main topics of the

course alternate between the cell biology of infection, cancer and intracellular trafficking

emphasizing new experimental approaches. The availability of the core Imaging Platform at

Institut Pasteur will introduce students to advanced techniques for the dynamic visualization of

cells in health and disease.

The maximum number of participants is 24, which includes a selected number of Master 2

students from the University of Paris 6, Paris 7 and Paris XI and foreign postgraduate students

in an interactive classroom setting. The course is intended to be a platform of excellence in

which students can meet and closely interact with worldwide top-level scientists to discuss,

exchange ideas and establish valuable contacts in the perspective of establishing a network of

young cell biologists at an early stage in their careers. Students will be able to understand the

importance of basic research and of a broad interdisciplinary approach to improve human

health. We also expect to provide orientations and mentoring to help students in their future

career.

The 2012-2013 course includes two practical sessions of one week each, focused respectively on

endocytosis and signaling and on tracking bacterial invasion and identification of virulence

factors. At the end of each session students are expected to present and discuss their

experiments. At the end of the course there is an exam consisting in the presentation of an

imaginary follow-up project based on the critical analysis of a recent scientific article. Students

submit a written document (4-5pages) and then discuss their project following a slide

presentation. The final evaluation is based on the participation in the course, the presentation of

the practical work and the final exam.

Practical work 1

MECHANISMS OF ENDOCYTOSIS

Endocytosis plays a fundamental role in cellular homeostasis and is required for the cellular

uptake of nutrients, controls plasma membrane turnover, the signaling response to hormones

and growth factors, antigen presentation or transport of molecules through epithelia. Moreover,

many infectious virus, bacteria, or protozoa take advantage of endocytosis to infect cells directly

or indirectly via toxins with intracellular activities.

Shiga toxin is getting endocytosed in a clathrin independent manner and is reaching the

endoplasmatic reticulum via the retrograde route.

First, the students will study retrograde trafficking of Shiga toxin and learn how localized

perturbation of cellular machinery can influence trafficking of Shiga toxin.

Second, the students will use a quantitative assay to study endocytosis of Shiga toxin and

transferrin, which are clathrin-independent and clathrin-dependent cargos, respectively.

Third, a lipid model membrane (giant unilamellar vesicles, GUVs) will be introduced and the

students will learn how to generate GUVs, study phase separation and STxB driven membrane

mechanics.

Practical work 2

CYTOSKELETAL REGULATION DURING ASTROCYTE POLARIZATION AND MIGRATION

Cell migration not only participates in morphogenetic movements during development, but also

contributes to tissue renewal, immune responses, wound healing and cancer cell dissemination

in adult organisms. Migration is a polarized cellular process, which opposes a protrusive front

edge to a retracting trailing edge. From the front to the rear, cytoskeletal rearrangements play a

key role in promoting the different processes leading to cell migration.

The students will use primary astrocytes in a scratch-induced migration assay to

- Identify the changes in morphology and in cytoskeletal organization in migrating astrocytes.

- Determine how alteration of cytoskeletal rearrangements modulate astrocyte migration

- Assess the role of RhoGTPases in the regulation of the cytoskeletal elements and adhesion

complexes during astrocyte migration and conclude on their functions during astrocyte

polarization and directed migration

* * * * *

1st WEEK

GENERAL INTRODUCTION

THE ORGANIZATION OF THE CELL

Doctoral School module 1 “Organization of the cell”

Monday, 14 January Introduction 9:00 - 16:00 Welcome and administrative matter Institut Pasteur Registration Office

► Presentation of the scope and overall Roberto BRUZZONE organization of the course (HKU-Pasteur Research Centre)

► Presentation of the practical sessions with Philippe CHAVRIER organizing teams (Institut Curie, France) ► Presentation of the exam and paper assignment Chiara ZURZOLO (Institut Pasteur, France) Student presentations 16:00 -18:00 Chromatin assembly from nucleosome Geneviève ALMOUZNI to nuclear organization (Institut Curie, France)

Tuesday, 15 January Nucleus and Epigenetics

INSTITUT CURIE DAY Location : Amphithéâtre Lacassagne,

Institut Curie 26 rue d’Ulm 75005 Paris

9:00 - 11:00 Chromosomes dynamics and epigenetic changes Edith HEARD during X inactivation (Institut Curie, France)

11:00 - 13:00 Nuclear organization during tissue differentiation: Susan GASSER dealing with heterochromatin (FMI, Switzerland) 13:00 - 14:15 Lunch for students and speakers (Chez Marie) 14:30 - 16:30 The epigenome: images from reality Valerio ORLANDO (Dulbecco Telethon Institute, Italy) 16:30 - 17:15 Epigenetics during bacterial infections Mélanie HAMON (Institut Pasteur, France)

Wednesday, 16 January Cell division and cytoskeleton 9:00 - 10:30 How cells divide Helder MAIATO (IBMC, Lisbon, Portugal) 11:00 - 12:30 Cytoskeleton and membrane dynamics in the final Arnaud ECHARD steps of cell division (Institut Pasteur, France)

14:00 - 15:30 Actin dynamics and cell migration Alexis GAUTREAU (CNRS, France)

16:00 - 17:30 Posttranslational modifications of tubulins: more Carsten JANKE than simple switches (Institut Curie, France) Thursday, 17 January Frontiers in microscopy 8:45 - 10:00 Quantitative biologicals imaging Jean-Christophe OLIVO-MARIN (Institut Pasteur, France) 10:15 - 11:15 The targeting of host cell compartments Jost ENNINGA by intracellular bacterial pathogens (Institut Pasteur, France) 11:45 - 12:45 Dissecting immune responses using intravital Philippe BOUSSO two-photon imaging (Institut Pasteur, France)

14:30 - 15:30 Intravital microscopy: a power tool to study Roberto WEIGERT biological processes in live animals (National Institute of Health, USA)

15:45 - 16:45 Dynamic imaging of cellular function David DIGREGORIO (Institut Pasteur, France)

17:15 - 18:15 Following single molecules in live and fixed cells Xavier DARZACQ to understand transcription regulation (Ecole Normale Supérieure, France)

Friday, 18 January Institut Pasteur - Institut Curie Day

Location: Auditorium Centre François Jacob (building 26) 13:30 - 14:30 Single-molecule biology and visualization of Tom KIRCHHAUSEN endocytosis (Harvard Medical School, USA)

14:30 – 15:30 Intravital microscopy as a tool to study membrane Roberto WEIGERT traffic and cell biology in live animals (National Institute of Health, USA)

15:30 - 16:00 Coffee Break 16:00 - 16:35 Glycosphingolipid function in the initiation Ludger JOHANNES of clathrin-independent endocytosis (Institut Curie, France)

16:35 - 17:10 Cortical remodelling and cell division Arnaud ECHARD (Institut Pasteur, France)

17:10 - 18:10 Endosomal sorting: from mechanisms of membrane Peter CULLEN deformation and cargo selection, through too (University of Bristol, UK) functional outcomes for organism development 18:15 - 19:00 Cocktail

* * * * *

2nd WEEK

MECHANISM OF INTRACELLULAR TRAFFICKING, ENDOCYTOSIS AND BACTERIAL INFECTION

Practical work: L. Johannes

Doctoral School module 2 “Membrane mechanics of endocytosis”

Monday, 21 January

9:00 - 11:00 Molecular mechanism of clathrin coat Tom KIRCHHAUSEN formation (Harvard Medical School, USA)

11:00 -12:00 Endocytosis and retrograde trafficking: Ludger JOHANNES mechanisms and applications (Institut Curie, France)

13:30 - 19:00 Practical work 1: Ludger JOHANNES, Guylène K’OUAS Mechanisms of uptake into cells: and Christian WUNDER Shiga toxin endocytosis and trafficking via the retrograde route Covalent conjugation of Shiga toxin with a fluorescent probe and chromatographic purification

Tuesday, 22 January

9:00 - 11:00 Multiple functions of myosin motor proteins Folma BUSS

in membrane trafficking pathways (University Cambridge, UK)

11:00 - 19:00 Practical work 1 (continuation): Study the retrograde transport of fluorescent Shiga toxin from the plasma membrane to the endoplasmatic reticulum, including perturbation by ATP-depletion or the Retro-2 inhibitor. Immunofluorescence staining and microscopy.

Wednesday, 23 January 9:00 - 11:30 Some physical aspects of membrane trafficking Patricia BASSEREAU (Institut Curie, France)

11:30 - 19:00 Practical work 1 (continuation): Quantitative analysis of endocytosis (Shiga toxin and transferrin) by ELISA

Thursday, 24 January

9:00 - 11:30 The fascinating strategies used by Listeria Pascale COSSART during infection (Institut Pasteur, France)

11:30 - 19:00 Practical work 1 (continuation): Continuation of ELISA and microscopy of immunofluorescence samples (day2)

Friday, 25 January 9:00 - 11:30 Transendothelial leukocyte migration: integration of Ronen ALON adhesive and chemotactic cues under shear forces (Weizmann Inst. of Science, Israel) 11:30 - 19:00 Practical work 1(continuation): Generation of giant unilamellar vesicles (GUVs) by electroformation. Study of phase separation and STxB driven membrane mechanics on GUVs.

* * * * *

3rd WEEK

CELL MIGRATION, CYTOSKELETON AND TUMOR INVASION

Practical work: S. Etienne-Manneville

Doctoral School module 3 “Cell migration, cytoskeleton and tumor invasion”

Monday, 28 January 8:30 - 9:30 Practical work 2: “Cytoskeletal regulation Sandrine ETIENNE-MANNEVILLE during astrocyte polarization and migration” and Guylène K’OUAS Day 1 morning: cell treatment and scratch assay

9:30 - 11:30 Rho GTPase signaling and cell migration Anne RIDLEY (King’s College London, UK)

12:00 - 13:00 seminar BCI Roles of Rho GTPases in cancer cell invasion Anne RIDLEY 14.00 - 18.00 Practical work 2 (continuation): Sandrine ETIENNE-MANNEVILLE Day 1 afternoon: and Guylène K’OUAS - si RNA nucleofection - End of the scratch assay. Cell fixation - Beginning of videomicroscopy (Gr 2, 6, 10)

Tuesday, 29 January 9:00 - 11:30 Astrocyte polarization and Sandrine ETIENNE-MANNEVILLE

migration (Institut Pasteur, France)

13:00 - 19:00 Practical work 2 (continuation) Day 2:

- Immunofluorescence - Preparation of primary astrocytes - Beginning of videomicroscopy (Gr 4, 8, 12)

Wednesday, 30 January 9:00 - 11:30 Neuronal migration in the developing Oscar MARIN PARRA brain (Instituto de Neurociencias, UMH, Spain)

13:00 - 19:00 Practical work 2 (continuation) Day 3 - Microscopy - Result analysis - Beginning of videomicroscopy (Gr 1, 5, 9)

Thursday, 31 January 9:00 - 9:30 Practical work 2 (continuation) Day 4 morning

Scratch assay on nucleofected cells

9:30 - 12:00 Cell biology of tumor cell invasion Philippe CHAVRIER (Institut Curie, France)

13:30 - 19:00 Practical work 2 (continuation) Day 4 afternoon

- Cell lysis, electrophoresis and transfert - End of the scratch assay. Cell fixation. - Beginning of videomicroscopy (Gr 3, 7, 11)

Friday, 01 February 9:00 - 11:30 Using vaccinia virus a tool to dissect cytoskeletal Michael WAY dynamics and signalling networks (Cancer Research, UK)

13:00 - 19:00 Practical work 2 (continuation) Day 5

- Western blot - Immunofluorescence

* * * * *

4th WEEK

ANALYTICAL DISSECTION OF THE PRACTICAL WORKSHOPS

Monday, 4 February 9:00 - 12:00 Practical work 2 (end): microscopy, result analysis and preparation of general discussion 12:00 - 13:00 Seminar BCI The diaries of a seasoned traveller: Giampietro SCHIAVO axonal transport and sorting of signalling endosomes (Cancer Research, UK)

14:00 - 18:00 Practical work 2 (end): microscopy, result analysis and preparation of general discussion Tuesday, 5 February 9:00 - 10:30 Motor proteins Giampietro SCHIAVO (Cancer Research, UK)

11:00 - 13:00 General discussion of Practicals 2 Giampietro SCHIAVO (presentations of students) (Discussion animator)

14:00 - 16:00 General discussion of Practicals 2 (presentations of students) Wednesday, 6 February 9:00 - 19:00 Practical work 1(end): Microscopy of immunofluorescence samples (day 2).

11:00 - 13:00 Optogenetic dissection of neural circuits function Filippo DEL BENE and development (Institut Curie, France)

Thursday, 7 February 9:00 - 10:30 A career around science: bringing Katja-Stina MAGNUSSON science out of the lab (ISARIC, UK)

11:00 - 12:30 On scientific writing and the making of a presentation Roberto BRUZZONE (Pasteur Research Center, Hong Kong)

Friday, 8 February 9:00 - 11:00 Dynamics and mechanics of intracellular Christophe LAMAZE signaling: role of caveolae (Institut Curie, France) 11:30 - 13:00 General discussion of Practicals 1 Christophe LAMAZE (presentations of students) (Discussion animator) (Institut Curie, France)

14:00 - 16:00 General discussion of Practicals 1

5th WEEK

PROJECT SUBMISSION AND EXAMS

Monday 11 to Tuesday 12 February Project preparation. The written project has to be submitted to the Course Commitee by Tuesday night. Wednesday 13 to Thursday 14 February Preparation of the oral exam Friday, 15 February 9:00 - 18:00 Final examination 18:00 Closing party

* * * * *

Detailed description of the examination

Oral exam on Friday 15 February, 2013 (mark on a 1-20 scale, coefficient 1):

Critical analysis of a scientific article and presentation of an imaginary 3-year research project as follow-up of the results of the article. Presentation: 13 minutes, questions: 7 minutes, total duration: 20 minutes Organization of the oral presentation: - The presentation is open to the public - Slides (Powerpoint or other supported formats) The scientific articles will be given to the students at the end of the first week of the course. As follow-up of the article received each student will write a fictional project in a 4/5 page document that should be submitted to the members of the jury no later than Tuesday 12 February at 20:00. This document should include: - Summary of the article (max 1 page) - Aims and description of the project (max 3 pages including figures if appropriate) - References (max 1 page)

Description détaillée de l’examen Examen oral le vendredi 15 février 2013 (note sur 20, coefficient 1) :

Présentation critique d’un article et discussion d’un projet fictif sur 3 ans découlant de ces résultats. Présentation : 13 minutes, questions du jury : 7 minutes, durée totale : 20 minutes.

Organisation de la présentation orale : - Exposé public de chaque étudiant devant le jury - Diapositives (logiciel Powerpoint ou autre format compatible) Les articles scientifiques seront donnés aux étudiants à la fin de la première semaine de cours. Le projet fictif est présenté dans un document de 4/5 pages à remettre au jury au plus tard le mardi 12 février à 20h00, comprenant : - Résumé de l’article (max 1 page) - Objectifs et description du projet (max 3 pages, figures incluses) - Bibliographie (max 1 page)

* * * * *

Centre d’Enseignement

MOLECULAR BIOLOGY OF THE CELL COURSE

2012 - 2013

ADDRESS DETAILS

* * * * *

Name First name email Institution Address City Country Telephone

Mr BRUZZONE Roberto bruzzone@hku.hkHKU-Pasteur

Research Centre

1/F Dexter HC Man Building,

8 Sassoon Road

Pokfulam,Hong Kong

Hong Kong 852-28168403

Mr CHAVRIER Philippe philippe.chavrier@curie.fr

Membrane & Actin Dynamics Lab,

CNRS/UMR144,Institut Curie

26, rue d'Ulm75248 Paris Cedex 05

France 33-(0)156246359

Ms ZURZOLO Chiara chiara.zurzolo@pasteur.frMembrane Trafficking and

Pathogenesis Unit,Institut Pasteur

25/28 rue du Dr Roux

75724 Paris Cedex 15

France 33-(0)145688277

Ms ALMOUZNI Geneviève genevieve.almouzni@curie.frUMR 218 CNRSInstitut Curie

26, rue d'Ulm75248 Paris Cedex 05

France 33-(0)156246701

Mr ALON Ronen ronen.alon@weizmann.ac.ilDept. of Immunology,

Weizmann Institute of ScienceWolfson Bldg.,

Room 330Rehovot 76100 Israel 972-8 934 2482

Ms BASSEREAU Patricia patricia.bassereau@curie.frPhysico-chimie Curie,

Institut Curie Research Center26, rue d'Ulm

75248 Paris Cedex 05

France

Ms BOUSSO Philippe philippe.bousso@pasteur.frUnité des Dynamiques des

Réponses Immunes,Institut Pasteur

25/28 rue du Dr Roux

75724 Paris Cedex 15

France 33-(0)1 45 68 85 51

Ms BUSS Folma fb1@mole.bio.cam.ac.uk

University of Cambridge,Cambridge Institute for

Medical Research,Wellcome Trust/MRC Building,

Hills RoadCambridge CB2

0XYUK 44-1223 763348

Ms COSSART Pascale pascale.cossart@pasteur.frUnité des Interactions

Bactéries-Cellules,Institut Pasteur

25/28 rue du Dr Roux

75724 Paris Cedex 15

France 33-(0)1 45 68 88 41

Mr CULLEN Peter Peter.Cullen@bristol.ac.uk

Henry Wellcome Integrated Signaling Laboratories,

Department of Chemistry,School of Medical Sciences,

University of Bristol

Bristol BS8 1TD UK 44-117 3312193

ORGANIZERS OF THE COURSE

LECTURERS

Name First name email Institution Address City Country Telephone

Mr DARZACQ Xavier darzacq@ens.fr

Functional Imaging of Transcription,

Institut de Biologie de l'Ecole Normale Supérieure,

CNRS UMR 8197

46, rue d'Ulm75230 Paris Cedex 05

France

Mr DEL BENE Filippo filippo.delbene@curie.fr

Génétique & Biologie du Développement,

CNRS UMR3215 / INSERM U934,

Institut Curie

26, rue d'Ulm75248 Paris Cedex 05

France

Mr DIGREGORIO David david.digregorio@pasteur.frUnit of Dynamic Neuronal

Imaging,Institut Pasteur

25/28 rue du Dr Roux

75724 Paris Cedex 15

France 33-(0)1 45 68 80 54

Mr ECHARD Arnaud arnaud.echard@pasteur.frMembrane Traffic & Cell

Division LabInstitut Pasteur

25/28 rue du Dr Roux

75724 Paris Cedex 15

France 33-(0)1 44 38 94 09

Mr ENNINGA Jost jost.enninga@pasteur.frGroupe Dynamique

Interactions Hôte-Pathogène,Institut Pasteur

25/28 rue du Dr Roux

75724 Paris Cedex 15

France 33-(0)1 44 38 94 13

MsETIENNE-

MANNEVILLESandrine

sandrine.etienne-manneville@pasteur.fr

Unité de Polarité Cellulaire, Migration & Cancer,

Institut Pasteur

25/28 rue du Dr Roux

75724 Paris Cedex 15

France 33-(0)1 40 61 39 05

Ms GASSER Susan susan.gasser@fmi.chFriedrich Miescher Institute for

Biomedical ResearchMaulbeerstrasse

664058 Basel Switzerland 41-61 697 5025

Mr GAUTREAU Alexis alexis.gautreau@lebs.cnrs-gif.frLaboratoire d'Enzymologie et

Biochimie Structurales,UPR3082 CNRS

Bât. 34,Avenue de la

Terrasse

91198 Gif-sur-Yvette Cedex

France 33-(0)1 69 82 35 08

Ms HAMON Mélanie melanie.hamon@pasteur.frUnité des Interactions

Bactéries-Cellules,Institut Pasteur

25/28 rue du Dr Roux

75724 Paris Cedex 15

France 33-(0)145688995

Ms HEARD Edith Edith.Heard@curie.frINSERM U934 / CNRM

UMR3215,Institut Curie

26, rue d'Ulm75248 Paris Cedex 05

France 33-(0)156246829

Name First name email Institution Address City Country Telephone

Mr JANKE Carsten Carsten.Janke@curie.fr

Institut Curie,Dept. Signalling, Neurobiology

& Cancer,CNRS UMR3306 - INSERM

U1005

Bat 110,Centre

Universitaire

91405 Orsay Cedex

France 33-(0)1 69 86 31 27

Mr JOHANNES Ludger ludger.johannes@curie.fr

Laboratoire Trafic, Signalisation & Criblage

Intracellulaire,UMR 144,

Institut Curie

26, rue d'Ulm75248 Paris Cedex 05

France 33-(0)1 56 24 63 51

Mr KIRCHHAUSEN Tom kirchhausen@crystal.harvard.eduImmune Disease Institute,

Harvard Medical School

W. Alpert Building,

Room 133,200 Longwood

Ave.

Boston,MA 02115

USA 1-617 713 8682

Mr LAMAZE Christophe christophe.lamaze@curie.frUMR 144 Curie / CNRS,

Institut Curie26, rue d'Ulm

75248 Paris Cedex 05

France 33-(0)1 56 24 69 66

Ms MAGNUSSON Kajsa-Stina kajsa-stina.magnusson@ndm.ox.ac.uk

ISARIC Project Manager,University of Oxford,

Nuffield Department of Medicine

Churchill Hospital,Old Road

Oxford OX3 7LJ UK

Mr MAIATO Helder Maiato@ibmc.up.pt

Chromosome Instability & Dynamics Lab.,

Instituto de Biologia Molecular e Celular,

Universidade do Porto

Rue do Campo Alegre 823

4150-180 Porto Portugal 351-22 6074 900

Mr MARIN PARRA Oscar o.marin@umh.esInstituto de Neurociencias,

UMH-CSICAvda. Ramon y

cajal s/n03550 Sant Joan

d'AlacantSpain 34-965 91 9415

Mr OLIVO-MARIN Jean-Christophejean-christophe.olivo-

marin@pasteur.fr

Unité d'Analyse d'Images Quantitative,

Institut Pasteur

25/28 rue du Dr Roux

75724 Paris Cedex 15

France 33-(0)1 45 68 85 06

Mr ORLANDO Valerio vorlando@dti.telethon.itDulbecco Telethon Institute,

IRCCS Santa LuciaVia del Fosso di

Fiorano 6400143 Roma Italy 39-06 501 703260

Name First name email Institution Address City Country Telephone

Ms RIDLEY Anne anne.ridley@kcl.ac.uk

King's College London,Randall Division of Cell and

Molecular Biophysics,New Hunt's House

Guy's Campus London SE1 1UL UK 44-(0)20 7848 6209

Mr SCHIAVO Giampietro giampietro.schiavo@cancer.org.ukCancer Research UK,

London Research Institute44 Lincoln's Inn

FieldsLondon WC2A

3LYUK 44-207 269 3300

Mr WAY Michael michael.way@cancer.org.ukCell Motility Laboratory,

Cancer Research UK,London Research Institute

44 Lincoln's Inn Fields

London WC2A 3LY

UK 44-207 269 3733

Mr WEIGERT Roberto weigertr@mail.nih.gov

Intracellular Membrane Trafficking Unit,

Oral and Pharyngeal Cancer Branch,

National Institute of Dental and Craniofacial Research,

NIH

30 Convent Dr. Room 303A

Bethesda,MD 20892-4340

USA

Ms K'OUAS Guylène guylene.kouas@pasteur.frCentre d'Enseignement,

Institut Pasteur25/28 rue du Dr

Roux75724 Paris Cedex 15

France 33-(0)1 40 61 30 02

Mr WUNDER Christian christian.wunder@curie.fr

Laboratoire Trafic, Signalisation & Criblage

Intracellulaire,UMR 144,

Institut Curie

26, rue d'Ulm75248 Paris Cedex 05

France

Ms COURMARCEL Fabienne fabienne.courmarcel@pasteur.frCentre d'Enseignement,

Institut Pasteur25/28 rue du Dr

Roux75724 Paris Cedex 15

France 33-(0)1 40 61 38 79

PRACTICAL WORK

SECRETARY