Most cells in the human body are functionally polarized as reflected by different regions of the plasma membrane having distinct morphologies and protein compositions. The goal of the current research is to investigate mechanism that cells use to achieve this polarity. Specifically, this application focuses on the microfilament-based cytoskeleton of the apical aspect of polarized epithelial cells and how it contributes to the assembly, dynamics and composition of the abundant microvilli that characterize this domain. The membrane-cytoskeletal linker ezrin, the ezrin-binding scaffolding protein EBP50 and its ligand EPI64 are key proteins that control microvilli on the apical aspect of cells, and this proposal is to understand their individual and collective functions. Our experimental system utilizes the JEG3 cell line, derived from human placenta, as these cells express robust and dynamic microvilli. Ezrin is a very abundant, conformational regulated essential protein in microvilli, so we propose that its role in maintaining these structures is regulated in part by sampling the plasma membrane for appropriate binding partners. Therefore, the first aim seeks to establish a sensitive system to identify proteins that associate with the active conformation of ezrin, and then explore their role in microvilli maintenance.
The second aim has two parts investigating EBP50, also essential for microvillar structure. Recent advances have shown that each of EBP50's two PDZ domains contribute to microvillar dynamics. First, proteins that bind EBP50's PDZ domains to regulate microvilli will be identified and characterized. Second, since microvilli are dynamic structures, and EBP50 is a highly dynamic component of them, we will use live cell imaging of wild type cells and those expressing characterized ezrin and EBP50 mutations to understand the relationship between the dynamics of components and the dynamics of a structure.
The third aim concerns EPI64, an Arf6-GTP binding RabGAP protein that binds EBP50 and is now implicated in both an endocytic cycle and in regulating microvilli. The role of EPI64 and its interacting partners in endocytosis will be explored, as well as their impact on microvillus structure and dynamics.
The fourth aim i s to continue structural analyses of ezrin, EBP50 and EPI64 to gain insight into how these proteins function together. Overall, this proposal is to explore mechanisms involved in placing the right proteins at the right place and in the right abundance on the cell surface. These studies have relevance to many diseases. For example, the EBP50 ligands, CFTR and EGFR, both have to be present on the cell surface in the right abundance, and defects in their presence of over-abundance can cause cystic fibrosis or cancer, respectively.

Public Health Relevance

All non-infectious diseases are caused by cellular defects that translate into dysfunction of organ(s). The proposed work seeks to understand how cells organize their plasma membrane, both in terms of morphology and abundance of specific proteins. This is of crucial importance as the regulated presence of specific proteins, like growth factor receptors, transporters like the cystic fibrosis transmembrane conductance regulator, and adhesion molecules in the membrane determine the functions of cells, and defects in these processes contribute too many diseases, including cancer.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM036652-27
Application #
8423697
Study Section
Nuclear and Cytoplasmic Structure/Function and Dynamics Study Section (NCSD)
Program Officer
Nie, Zhongzhen
Project Start
1986-04-01
Project End
2015-11-30
Budget Start
2012-12-01
Budget End
2013-11-30
Support Year
27
Fiscal Year
2013
Total Cost
$468,732
Indirect Cost
$167,514
Name
Cornell University
Department
None
Type
Organized Research Units
DUNS #
872612445
City
Ithaca
State
NY
Country
United States
Zip Code
14850
Widemann, Brigitte C; Acosta, Maria T; Ammoun, Sylvia et al. (2014) CTF meeting 2012: Translation of the basic understanding of the biology and genetics of NF1, NF2, and schwannomatosis toward the development of effective therapies. Am J Med Genet A 164A:563-78
Viswanatha, Raghuvir; Bretscher, Anthony; Garbett, Damien (2014) Dynamics of ezrin and EBP50 in regulating microvilli on the apical aspect of epithelial cells. Biochem Soc Trans 42:189-94
Wayt, Jessica; Bretscher, Anthony (2014) Cordon Bleu serves as a platform at the basal region of microvilli, where it regulates microvillar length through its WH2 domains. Mol Biol Cell 25:2817-27
Garbett, Damien; Bretscher, Anthony (2014) The surprising dynamics of scaffolding proteins. Mol Biol Cell 25:2315-9
Garbett, Damien; Sauvanet, Cecile; Viswanatha, Raghuvir et al. (2013) The tails of apical scaffolding proteins EBP50 and E3KARP regulate their localization and dynamics. Mol Biol Cell 24:3381-92
Bretscher, Anthony (2013) Magazine or journal--what is the difference? The role of the monitoring editor. Mol Biol Cell 24:887-9
Hokanson, David E; Bretscher, Anthony P (2012) EPI64 interacts with Slp1/JFC1 to coordinate Rab8a and Arf6 membrane trafficking. Mol Biol Cell 23:701-15
LaLonde, David P; Bretscher, Anthony (2011) The UBX protein SAKS1 negatively regulates endoplasmic reticulum-associated degradation and p97-dependent degradation. J Biol Chem 286:4892-901
LaLonde, David P; Garbett, Damien; Bretscher, Anthony (2010) A regulated complex of the scaffolding proteins PDZK1 and EBP50 with ezrin contribute to microvillar organization. Mol Biol Cell 21:1519-29
Garbett, Damien; LaLonde, David P; Bretscher, Anthony (2010) The scaffolding protein EBP50 regulates microvillar assembly in a phosphorylation-dependent manner. J Cell Biol 191:397-413

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