The cortical cytoskeleton supports the plasma membrane, the cell's barrier through which all small molecules pass, all signals are sensed, and all communication and attachments between cells are made. Critical to an understanding is how the cell surface is organized, especially how cells regulate attachment of the membrane to the cell cortex. This project investigates the ERM (ezrin/radixin/moesin) proteins, whose founding member, ezrin, provides a regulated linkage between the actin cytoskeleton and membrane-associated proteins in apical microvilli on polarized epithelial cells. Activated ezrin binds EBP50, a scaffolding protein with two PDZ domains that bind to many different transmembrane proteins, and a C-terminal ezrin binding domain. In addition, a cytosolic protein, EPI64 binds the first PDZ domain of EBP50. Recent work shows that EPI64 regulates the presence of microvilli on the cell surface, which is investigated in the first two aims. In the first, specific EPI64, EBP50 and ezrin mutants will be generated and their role in microvilli regulation assessed. Additional factors involved in this regulatory process will be identified and characterized. In the second, in vivo imaging of microvilli dynamics will be documented, and the role of the regulatory factors on microvilli life cycle parameters determined. In this aim, the basis for the recent finding that microvilli have subdomains will also be explored.
The third aim i nvestigates the coordination between the presence of microvilli on the cell surface and receptor-mediated endocytosis. Several studies have implicated EBP50 in endocytosis, and recent results suggest an involvement of EPI64. Analysis of endocytosis of various ligands will be determined in cells perturbed for microvillar structure.
The final aim concerns the role of ezrin and moesin in the formation of the immunological synapse between a T-cell and an antigen presenting cell. Recent studies have revealed that ezrin and moesin perform distinct roles, and this aim investigates these at the molecular level. Several proteins from T cells that bind specifically to ezrin or moesin have been identified, and their role in immunological synapse formation is to be investigated. The proposed basic research has relevance to the molecular basis of disease. Many diseases can be traced to defects in plasma membrane organization and endocytic trafficking (e.g. CFTR, growth factor receptors, etc), some of which are regulated by the system under study. Moreover, ezrin is elevated in many tumors, and an elevated level of ezrin has been shown to be necessary for metastasis in some tumor cell lines.
|Pelaseyed, Thaher; Viswanatha, Raghuvir; Sauvanet, Cécile et al. (2017) Ezrin activation by LOK phosphorylation involves a PIP2-dependent wedge mechanism. Elife 6:|
|Sauvanet, Cécile; Wayt, Jessica; Pelaseyed, Thaher et al. (2015) Structure, regulation, and functional diversity of microvilli on the apical domain of epithelial cells. Annu Rev Cell Dev Biol 31:593-621|
|Sauvanet, Cécile; Garbett, Damien; Bretscher, Anthony (2015) The function and dynamics of the apical scaffolding protein E3KARP are regulated by cell-cycle phosphorylation. Mol Biol Cell 26:3615-27|
|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|
|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|
|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|
|Garbett, Damien; Sauvanet, Cécile; 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|
|Viswanatha, Raghuvir; Wayt, Jessica; Ohouo, Patrice Y et al. (2013) Interactome analysis reveals ezrin can adopt multiple conformational states. J Biol Chem 288:35437-51|
|Bretscher, Anthony (2013) Magazine or journal--what is the difference? The role of the monitoring editor. Mol Biol Cell 24:887-9|
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