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And this is what we are working on at the UofC…

Identifying components of an archaeal pathway for the insertion of tail-anchored membrane proteins

In eukaryotes, tail-anchored (TA) proteins are targeted to and inserted into the endoplasmic

reticulum (ER) membrane by a conserved post-translational pathway, involving the transmembrane

domain recognition complex (TRC). Targeting occurs when TRC40 (or its yeast homolog Get3)

binds a TA substrate and targets it to the ER membrane by an interaction with the TRC receptor

(called Get1 and Get2 in yeast). No similar targeting pathway has yet been identified in archaea or

bacteria.

Our recent data suggest that archaea have a TRC40 homolog that is structurally and functionally

similar to its eukaryotic protein. Now, we are interested in knowing whether there are other protein

components required in the archaeal TA protein targeting pathway. We identified a sequence

homolog of the putative mammalian TRC receptor (called WRB) in multiple archaeal strains. BLAST

analysis shows that the archaeal homolog shares only ~18% identity to WRB, but possesses key

predicted sequence features, including three transmembrane domains and a functionally important,

cytosolic facing coiled-coil domain.

We have expressed and purified coiled-coil fragment and full-length membrane protein from two

archaebacteria: Methanobacterium thermoautotrophicum Delta H and Methanocaldococcus

jannaschii DSM2661. Here we present initial functional analysis of their interactions with the

archaeal TRC40 protein.

Methods used:

Cloning, expression, detergent, purification screening

Western blot, Size Exclusion Chromatography, Mass Spectrometry, Multi Angle Light Scattering, Single molecule

Fluorescence Photobleaching, Native PAGE, In vitro translation, Fluorescence Resonance Energy Transfer, Crystallography

Functional analysis of Moesin interacting proteins in cytoskeletal organization

Katarzyna Lepeta, Fehon lab

• Moesin is known to organize the actin cytoskeleton andregulate the small GTPase RhoA in the apical domain ofepithelial cells. Using a truncated form of Moesin thatstabilizes interactions with its binding partners andmass spectrometry, we have been identifying Moesininteracting proteins to better understand how Moesinis regulated and its role in regulating Rho1. Usingtechniques listed below, we are currently trying todetermine the role of these putative Moesin interactingproteins in cytoskeletal organization and epithelialintegrity.

Confocal microscopy was used to visualize the localization of a putative Moesin interactor (CG7112, in red) in S2 cells

Wing disc of a transgenic fly, expressing RNAi for a putative Moesin interactors (CG1677).

Techniques:

- Fly genetics

- Fluorescence/confocal microscopy

- Cell culture

- In vitro and in vivo protein expression assays

- Co-immunoprecipitation

- Immunohistochemistry

- In vitro and in vivo RNA interference

- Molecular biology

- Mass spectrometry

Lab website: http://fehonlab.bsd.uchicago.edu/Contact: klepeta@uchicago.edu

Innovative tools for investigating filopodia-dependent cell migrationEwa Warchol, Rock Lab

Project 1: Synthetic Antigen Binders as imaging reagents for fascin

Fascin is an actin cross-linker that functions in formation offilopodia. Those specific cellular protrusions found at the leadingedge of the cell act as environment sensors during migration. Byutilizing phage display technique we generated synthetic AntigenBinders (sABs) for fascin. Fluorescently labeled sABs delivered tothe cell can act as novel imaging reagents enabling us to fullyunderstand mechanisms of fascin in filopodia formation.

Project 2: Misdirecting Myosin X’s cargo

Myosin X is a molecular motor that participates in cargo delivery tothe cell membrane by choosing fascin-actin bundles. Maincomponents of that cargo – integrins and cadherins are important forfilopodia attachment to the substrate and cell-cell adhesions,respectively. I created a chimeric construct that transports Myosin X’scargo to the interior of the cell. We hypotesize it will disturb filopodiaformation, cell-cell interactions and therefore also cell motility.

a b

a: wild type U2OS cells; b: U2OS cell expressing chimeric protein (GFP-tagged); actin stained with TMR-phalloidin, nucleus with Hoechst dye.

Methods:

- Molecular cloning- Cell culture- Recombinant protein expression and purification (affinity chromatography, protein A ion exchange chromatography)- Phage display, mutagenesis, protein engineering- Competitive phage ELISA- TIRF microscopy- Surface plasmon resonance- Actin spin down assays- Coimmunoprecipitation

The role of the transmembrane domain in signal

transduction of human growth hormone receptor

Katarzyna Soczek, Anthony A. Kossiakoff

The mechanism of signal transduction in cytokine transmembrane receptors is poorly understood. It was recently shown that in the

absence of ligands, receptors are predimerized through the transmembrane domains (TM). Here, we investigate the role of thetransmembrane domain of human growth hormone receptor (hGHR) in signaling. We show that introduction of point mutations into the

TM that disrupt dimerization surprisingly leads to an increase in signaling. Additionally, we want to apply structural studies to determineexact interaction interfaces of TM helises in on and off conformations. To do this, we apply two different models. First, is based on NMR

studies of TM domain only. Second, involves extracellular domain (ECD) and TM complex biophysical studies. Combining of the cellbased assays with NMR data will provide new insights into the mechanism of signal transduction in hGHR.

Fig.1. In the classical model receptors

are dimerized after hormone binding.

In the new model, the receptor is

predimerized in the absence of ligand

Fig.2. Molecular modeling indicates that there can be 5 equally possible TMconformations. This should be verified experimentally.

Fig.3. ECD with TM and boundhormone, incorporated into nanodisc

which mimics cell membrane. Modeldesigned for structural studies.

Methods:Toxcat assay

Luciferase assay

Cell and tissue culture

Protein expression and purification

Cloning

Faculty of the Biological Sciences Division

Research Summary / Selected Publications(i) One of our research interests centers around studying at atomic resolution the structural and functional properties that define

molecular recognition systems that activate and regulate biological properties. In particular, we study the energetics of hormone-induced receptor activation and regulation of growth hormone and its receptor using X-ray crystallography, site-directed mutagenesis, phage display mutagenesis and biophysical analysis.

(ii) Synthetic Antibodies- We use novel phage display libraries and screening procedures to produce a new class of synthetic affinity binders (sABs) based on Fab antibody scaffolds. These synthetic antibodies are much more versatile than traditional monoclonal antibodies and can be tuned to bind to multi-protein complexes and specific conformational states of their protein targets. These attributes make them the reagents of choice to study complex processes like cell signaling and cytokinesis.

(iii) Drug delivery- We have developed a unique drug delivery method that utilizes ligand-induced receptor-mediated endocytosis pathways. We call this method Receptor-Mediated Delivery (RMD) and have shown that we can deliver functionally active proteins and RNA/DNA cargoes into live cells for functional and imaging experiments. 4) Synthetic biology- We use a combination of peptide synthesis and phage display (biosynthetic phage display) to produce proteins with novel properties.

Tony KossiakoffChair & Ortho S.A. Sprague Professor, Department of Biochemistry & Molecular Biology

B.S. (Chemistry and Mathematics) 1968, Davis and Elkins CollegePh.D. (Physical Chemistry) 1972,University of Delaware, Newark

Horn, J.R., Sosnick, T.R., Kossiakoff, A. A. (2009) " Principal determinants leading to transition state formation of a protein-protein complex, orientation trumps side chain interactions" Proc. Natl. Aca. Sci. (USA) 106(8):2559-64.Rizk, S.S., Luchniak, A., Uysal, S., Brawley, C., Rock, R.S., Kossiakoff, A.A. (2009) “An engineered substance P variant for receptor-mediated delivery of synthetic antibodies into tumor cells”. Proc. Natl. Acad. Sci. U.S.A 106(27): 11011-5.Uysal, S., Vasquez, V., Tereshko, V., Esaki, K., Fellouse, F.A., Sidhu, S.S., Koide, S., Perozo, E., Kossiakoff, A. (2009) ”Crystal structure of full-length KcsA in its closed conformation”. Proc. Natl. Aca. Sci. (USA) 106(16):6644-9.

Research Summary / Selected Publications(i) An important property of living organisms is their ability to move when needed. All such directed movements, including

intracellular trafficking, cell division, and muscle contraction, are driven by a set of molecular machines that are only a few nanometers in diameter. We would like to understand how one of these motors, myosin, couples ATP hydrolysis into motility along actin filaments, and how it has been tuned for a wide variety of tasks in the cell.

(ii) While stepping, myosin travels through a specific sequence of tightly coupled biochemical and mechanical states. Withoutsuch coordination, structural transitions would occur at improper times and the motor would not function. Our challenge is to unravel the coordination mechanisms in these motors. We focus on the unconventional myosins, including myosin V, VI, and X. These motors drive several forms of cargo transport and play key roles in the organization of actin-based structures. Unlike myosin II, which operates in large ensembles to drive high-speed motility in muscle, these unconventional myosins operate in smaller numbers and thus have different mechanical and kinetic properties.

(iii) We primarily use single-molecule techniques to study motility, including optical tweezers to measure forces and single fluorophore imaging to follow biochemical events. These methods allow us to probe the protein motions in a manner that is unobscured by other motor molecules, which may or may not be acting in concert.

Ronald S RockAssistant Professor, Department of Biochemistry & Molecular Biology

B.S., Chemistry, University of Chicago, 1992

Ph.D., Chemistry, California Institute of Technology, 1999

Nagy, S., Ricca, B.L., Norstrom, M., Courson, D.S., Brawley, C.M., Smithback, P., Rock, R.S. (2008) "A myosin motor that

selects bundled actin for motility." Proc. Natl. Acad. Sci. USA 105: 9616-20. PubMed

Rizvi, S.A.; Courson, D.S.; Keller, V.A.; Rock, R.S.; Kozmin, S.A. (2008) The dual mode of action of bistramide A entails

severing of filamentous actin and covalent protein modification. Proc. Natl. Acad. Sci. USA 105: 4088-92. PubMed

Rock, R. S., Ramamurthy, B., Dunn, A. R., Beccafico, S., Rami, B. R., Morris, C., Spink, B. J., Franzini-Armstrong, C.,

Spudich, J. A. and Sweeney, H. L. (2005). "A Flexible Domain Is Essential for the Large Step Size and Processivity of Myosin

VI." Mol Cell 17: 603-9. PubMed

Research Summary / Selected Publications(i) The major goals of our research are to understand the relationship between sequence, structure and function in biological

systems, and also to leverage this knowledge to generate proteins with new or optimized function. We use two fundamental and complementary approaches: 1) directed evolution and 2) structural analysis.

(ii) The success of directed evolution depends critically on library quality and the ability to rapidly and effectively interrogate libraries using an appropriate assay. Thus, we combine structure- and homology-based mutagenesis strategies to generate functionally rich libraries that allow us to efficiently access sequence space. Similarly, we develop high- (low information content) and low-throughput (high information content) screening strategies to facilitate the identification of proteins with desirable properties.

(iii) We use these tools in a variety of ways. Current projects in the lab include: 1) optimization of fluorescent reporters for biological imaging; 2) detailed analysis of sequence-structure-function relationships in systematically varied populations of enzymes; 3) generation of novel biosensors and 4) application of directed evolution to overcome limiting factors in macromolecular crystallography.

Robert J KeenanAssistant Professor, Department of Biochemistry & Molecular Biology

B.S., Biology & Chemistry, Bates College, 1990

Ph.D., Biochemistry & Biophysics, UCSF, 1998

Siehl, D.L., Castle, L.A., Gorton, R. and Keenan, R.J. (2007). "The molecular basis of glyphosate resistance by an optimized

microbial acetyltransferase." J. Biol. Chem., accepted for publication.

Keenan, R.J., Siehl, D.L., Gorton, R. and Castle, L.A. (2005). "DNA shuffling as a tool for protein crystallization." Proc Natl

Acad Sci USA, 102:8887-8892.

Siehl, D.L., Castle, L.A., Gorton, R., Chen, Y.H., Bertain, S., Cho, H.J., Keenan, R.J., Liu, D. and Lassner, M.W. (2005).

"Evolution of a microbial acetyltransferase for modification of glyphosate: a novel tolerance strategy." Pest Manag. Sci.,

61:235-240.

Research Summary / Selected PublicationsThe goals of our research are to elucidate factors governing molecular recognition events underlying protein function and to produce novel

function by exploiting such knowledge. Current research topics include: (i) Minimalist interaction interfaces. Protein-protein interactions are central to biological regulation. Natural protein interaction interfaces are

large and complex. We aim to define the "minimalist" requirements for tight and specific interfaces (e.g. how large does an interface needs to be?; how much chemical and structural diversity is required for affinity and specificity?). Our research focuses on interactions mediated by surface loops, ubiquitously seen in antibodies and cytokine receptors. We employ iterative processes of engineering synthetic binding proteins by altering loops of a small protein and analyzing the structure and function of binding proteins. Our research has helped establish the concept of "molecular scaffolds" and the field of "antibody mimics."

(ii) Peptide self-assembly. Self-assembly of peptides into water-insoluble, beta-sheet-rich fibrils is implicated in protein misfolding diseases (e.g. Alzheimer's) and it is also a process leading to novel nanomaterials. We aim to understand contributions of various factors governing peptide self-assembly and design novel nanostructures. We have developed a unique model system called "peptide self-assembly mimic", which captures the essence of peptide self-assembly within a water-soluble protein and enables us to investigate atomic structures and energetics of otherwise recalcitrant materials.

(iii) Structural biology. We use solution NMR spectroscopy, x-ray crystallography and the antibody mimic technology to characterize the atomic structure and dynamics of proteins involved in signal transduction. As a member of the Structural Genomics Initiative, we are developing powerful technologies to facilitate protein structure determination.

Shohei KoideAssociate Professor, Biochemistry & Molecular Biology

B.Sc. University of Tokyo, 1986;Ph.D. University of Tokyo, 1991;Postdoctoral, The Scripps Research Institute

Gilbreth RN, Esaki K, Koide A, Sidhu SS & Koide S. (2008). A dominant conformational role for amino acid diversity in minimalist protein-protein interfaces. J Mol Biol, 381, 407-418. LinkHuang J, Koide A, Makabe K & Koide S (2008) Design of protein function leaps by directed domain interface evolution. Proc Natl Acad Sci U S A, 105, 6578-6583. LinkBiancalana M, Makabe K, Koide A & Koide S. (2008) Aromatic cross-strand ladders control the structure and stability of β-rich peptide self-assembly mimics. J Mol Biol, 383, 205-213. Link

Research Summary / Selected PublicationsCells regulate actin filament assembly to drive a wide range of fundamental cellular processes such as division and motility. The

focus of our research group is to determine the biochemical mechanisms that govern how fission yeast and the nematode worm coordinate actin assembly. We utilize interdisciplinary approaches in and out of live cells including genetics, fluorescence microscopy, biochemistry, biophysics and innovative single actin filament imaging assays.

David KovarAssistant Professor, Molecular Genetics & Cell Biology, Biochemistry & Molecular Biology, Committee on Developmental Biology, Committee on GeneticsB.A.-Ohio Wesleyan University 1995; Ph.D.-Purdue University 2001;

Postdoctoral fellow-Yale University 2001-2005

Neidt, E.M., Scott, B.J. and D.R. Kovar. 2009. Formin differentially utilizes profilin isoforms to rapidly assemble actin

filaments. J. Biol. Chem. 284, 673-84.

Neidt, E.M., Skau, C.T. and D.R. Kovar. 2008. The cytokinesis formins from the nematode worm and fission yeast

differentially mediated actin filament assembly. J. Biol. Chem. 283, 23872-83.

Kovar, D.R., Harris, E.S., Mahaffy, R., Higgs, H.N. and T.D. Pollard. 2006. Control of the assembly of ATP- and ADP-actin by

formins and profilin. Cell. 124, 423-435.

Kovar, D.R. Molecular details of formin-mediated actin assembly. 2006. Curr. Opin. Cell Biol. 18, 11-17.

Kovar, D.R., Wu, J.-Q., and T.D. Pollard. 2005. Profilin-mediated competition between capping protein and formin Cdc12p

during cytokinesis in fission yeast. Mol. Biol. Cell. 16, 2313-2324.

Kovar, D.R. and T.D. Pollard. Insertional assembly of actin in association with formins produces piconewton forces. 2004.

Proc. Natl. Acad. Sci. USA. 41, 14725-14730.

Kovar, D.R., Kuhn, J.R., Tichy, A.L., and T.D. Pollard. 2003. The fission yeast cytokinesis formin Cdc12p is a barbed end actin

filament capping protein gated by profilin. J. Cell Biol. 161, 875-887.

Research Summary / Selected Publications(i) Interaction between microtubules and target sites (e.g. kinetochores) is critical for cellular processes such as mitosis, development, and

stem cell maintenance. To function in these diverse roles, the dynamic behavior of microtubules must be properly regulated. For example, disruption of microtubule function/organization has been linked to neurodegenerative disease. Alternately, inhibiting microtubule dynamics is among the most effective strategies for cancer therapeutics. Thus, understanding these processes represents a major challenge for cell biology with potential to have significant impact on issues of human health.

(ii) Microtubules are regulated by a large and diverse group of proteins. However, due to the transient and dynamic nature of the interactions, the mechanisms involved have been elusive. My lab uses the model organism S. cerevisiae to address fundamental questions about the mechanisms that regulate microtubule function and microtubule interactions within the cell. We utilize various approaches; high-resolution and quantitative microscopy, cell biological approaches in living cells, molecular biology, protein biochemistry, and in-vitro reconstitution assays.

(iii) Kinesin motor proteins generally power movement along microtubules. We recently discovered that the important, but poorly understood Kinesin-8 family represents a ‘hybrid’ motor that combines walking and depolymerase activity in the same molecule. Furthermore, we demonstrated that Kinesin-8 operates at the interface between dynamic microtubules and their interaction sites.

(iv) Currently, we are working to elucidate the molecular mechanisms and regulation of Kinesin-8 in the context of microtubule interactions. Kinesin-8s are highly conserved and function in critical processes such as spindle positioning, chromosome segregation, and spindle morphogenesis. Thus, Kinesin-8 is an ideal ‘molecular handle’ to leverage against understanding the mechanisms that govern dynamic microtubule interactions.

Mohan GuptaAssistant Professor, Molecular Genetics & Cell Biology,

B.S., Biochemistry, University of Kansas, 1992

Ph.D., Biochemistry (Honors), University of Kansas, 2001

Austin, K. M., Gupta, M. L., Jr., Coats, S., Tulpule, A., Mostoslavsky, G., Balazs, A. B., Mulligan, R. C., Daley, G., Pellman, D., and

Shimamura, A. (2008). Mitotic spindle destabilization and genomic instability in Shwachman-Diamond syndrome. J Clin Invest.,

118:1511-8. (PubMed)

Gupta, M. L., Jr., Carvalho, P., Roof. D. M., and Pellman, D. (2006). Plus end-specific depolymerase activity of Kip3, a kinesin-8 protein,

explains its role in positioning the yeast mitotic spindle. Nat Cell Biol. 8:913-23. (PubMed)

Carvalho, P., Gupta, M. L., Jr., Hoyt, M. A., and Pellman, D. (2004). Cell cycle control of kinesin-mediated transport of Bik1 (CLIP-170)

regulates microtubule stability and dynein activation. Dev Cell, 6:815-29. (PubMed)

Techniques

Drosophila genetics

molecular cloning

antibody staining

confocal microscopy

live-cell imaging

in situ hybridization

Sally Horne-Badovinac

Assistant Professor, Molecular Genetics and Cell Biology

Committee on Developmental Biology

Committee on Genetics, Genomics and Systems Biology

http://www.shblab.org

Research Summary

The proper function of organs like the heart, kidneys, liver or lungs depends on these organs acquiring their unique

shapes during embryonic development. If the dynamic tissue movements that create these complex three

dimensional structures go awry, birth defects and metabolic diseases result. My lab is using the superb genetic and

cell biological tools of the fruit fly, Drosophila melanogaster, to elucidate the molecular and cellular mechanisms that

determine the shape of a simple fly organ known as an egg chamber. Fruit flies provide tremendous experimental

advantages for studying organ morphogenesis, because they allow us to quickly identify the underlying genetic

networks and then test our hypotheses about gene function in an animal where we have an unrivaled ability to

temporally and spatially manipulate gene expression. Given the high degree of conservation in developmental

signaling mechanisms throughout evolution, the discoveries we make in flies are likely to be directly relevant to

human organ formation, as well as to the abnormalities and/or diseases that occur when these developmental

processes are perturbed.

The specific question my lab is addressing is, how does the initially spherical egg chamber take

on an elliptical shape as it grows? Each egg chamber consists of an internal germ cell cluster,

surrounded by a somatic epithelium. The cellular processes that drive egg chamber elongation

are poorly understood, but we know that a precise planar arrangement of actin filaments and

ECM fibrils in and around the epithelium is required. Planar polarity is a mechanism used many

times during development to effect changes in tissue shape. Interestingly, the egg chamber uses

a different molecular framework to establish this type of polarity than the classic planar polarity

systems. We expect, therefore, that the study of egg chamber elongation will reveal novel

molecular and cellular mechanisms controlling planar morphogenesis and organ shape. Through

a genetic screen, we have identified a large collection of mutations that disrupt egg chamber

elongation, which now provide an unprecedented opportunity to gain molecular insight into this

fascinating morphogenetic event.

The prospective master’s student will employ a variety of techniques to

phenotypically characterize one of the mutants and determine the

disrupted protein’s normal function in

egg chamber elongation.