The long term goals of our work are to understand how epithelial cells organize into monolayers through specialized cadherin-mediated cell-cell contacts, and localize proteins to functionally different plasma membrane domains. We integrate innovative experimental approaches to address these problems: structural analysis of proteins and protein complexes, high resolution live cell imaging of proteins and biosensors, biochemical analysis of protein complex assembly and function in cells, and novel in vitro reconstitution assays. During this funding period, we defined stages in cell-cell adhesion and the mechanism involved, and determined how plasma membrane proteins are targeted to and organized in the forming basolateral membrane domain upon cell-cell adhesion. We will build upon these results in proposed studies. We will define roles of alpha-catenin and p120 in regulating cell-cell adhesion and membrane dynamics. We will examine the effects of sequestering endogenous alpha-catenin and p120 to the plasma membrane independently of E-cadherin, and to mitochondria using location-specific tags on cell migration, actin dynamics and cell-cell adhesion using live cell imaging, FRET, biosensors and small molecule inhibitors. We will use affinity purification and MALDI/TOF Mass Spectrometry to define proteins that regulate alpha-catenin and p120 functions and association with the actin cytoskeleton. We will dissect mechanisms regulating sorting of vesicles at the basolateral plasma membrane targeting patch. We will use innovative reconstitution assays on membrane patches bound to an E-cadherin substrate to visualize how transport vesicles are delivered to the targeting patch, and how each of the componentsof the targetign patch function. These studies will be extended to functions of Lgl, Discs Large, Scribble and Par proteins in the development of cell surface polarity. We will investigate the role of microtubule (MT) and septin cytoskeleton in cell-cell adhesion and vesicle delivery to the basolateral plasma membrane targeting patch. We will define roles of septins in cell-cell adhesion and in specifying Glu-MT organization and vesicle trafficking towards the plasma membrane, and investigate protein-protein interactions between septins/MTs and the targeting patch. Together, these studies will provide a comprehensive molecular picture of the signaling and structural protein networks involved in cell-cell adhesion and the development of cell surface polarity.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37GM035527-28
Application #
8245091
Study Section
Special Emphasis Panel (NSS)
Program Officer
Flicker, Paula F
Project Start
1990-07-01
Project End
2013-03-31
Budget Start
2012-04-01
Budget End
2013-03-31
Support Year
28
Fiscal Year
2012
Total Cost
$641,287
Indirect Cost
$234,030
Name
Stanford University
Department
Biophysics
Type
Schools of Medicine
DUNS #
009214214
City
Stanford
State
CA
Country
United States
Zip Code
94305
Toret, Christopher P; D'Ambrosio, Michael V; Vale, Ronald D et al. (2014) A genome-wide screen identifies conserved protein hubs required for cadherin-mediated cell-cell adhesion. J Cell Biol 204:265-79
Buckley, Craig D; Tan, Jiongyi; Anderson, Karen L et al. (2014) Cell adhesion. The minimal cadherin-catenin complex binds to actin filaments under force. Science 346:1254211
Mbom, Bertrade C; Siemers, Kathleen A; Ostrowski, Maggie A et al. (2014) Nek2 phosphorylates and stabilizes ?-catenin at mitotic centrosomes downstream of Plk1. Mol Biol Cell 25:977-91
Dash, Surjya Narayan; Lehtonen, Eero; Wasik, Anita A et al. (2014) Sept7b is essential for pronephric function and development of left-right asymmetry in zebrafish embryogenesis. J Cell Sci 127:1476-86
Lowndes, Molly; Rakshit, Sabyasachi; Shafraz, Omer et al. (2014) Different roles of cadherins in the assembly and structural integrity of the desmosome complex. J Cell Sci 127:2339-50
Miller, Phillip W; Clarke, Donald N; Weis, William I et al. (2013) The evolutionary origin of epithelial cell-cell adhesion mechanisms. Curr Top Membr 72:267-311
Lowndes, Molly; Nelson, W James (2013) Fabricating surfaces with distinct geometries and different combinations of cell adhesion proteins. Methods Mol Biol 1046:219-30
Miller, Phillip W; Pokutta, Sabine; Ghosh, Agnidipta et al. (2013) Danio rerio *E-catenin is a monomeric F-actin binding protein with distinct properties from Mus musculus *E-catenin. J Biol Chem 288:22324-32
Ghossoub, Rania; Hu, Qicong; Failler, Marion et al. (2013) Septins 2, 7 and 9 and MAP4 colocalize along the axoneme in the primary cilium and control ciliary length. J Cell Sci 126:2583-94
Nelson, W James; Dickinson, Daniel J; Weis, William I (2013) Roles of cadherins and catenins in cell-cell adhesion and epithelial cell polarity. Prog Mol Biol Transl Sci 116:3-23

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