Microfilaments are cytoskeletal elements involved in the determination of cell shape as well as in many aspects of cell motility. We have chosen two projects to contribute to an understanding of microfilament organization and function. The first is a functional analysis of the components of the isolated intestinal microvillus cytoskeleton, and an investigation of how these findings relate to microfilament arrangements in other cells. We propose studies on two microvillus proteins. One is the 110K-calmodulin complex that links the microvillus core bundle to the plasma membrane. The complex can be specifically solubilized from the microvillus by a nucleoside triphosphate. The complex has been purified and shown to have a nucleoside triphosphatase activity. We propose to characterize the complex and explore the relationship between these properties, and its interaction with F-actin. We also propose to identify and characterize membrane components with which the complex associates. The other microvillus cytoskeletal protein we will study is ezrin, whose function is not known. In cultured cells, ezrin is a major substrate for the inducible tyrosine kinase activity of the epidermal growth factor receptor and certain kinases of tumor viruses. We propose to determine the function of ezrin in the micrivillus and explore the effects of in vitro tyrosine phosphorylation on its properties. The second project involves characterization of caldesmon from smooth and non-muscle cells. Current evidence indicates that it is a key component of a thin-filament Ca2+-regulatory system in smooth muscle. It is a potent F-actin bundling protein whose F-actin binding activity can be inhibited by the presence of Ca2+-calmodulin. Caldesmon will be characterized in detail and the molecular mechanism that gives it these properties explored. Proteins immunologically and functionally related to smooth muscle caldesmon are present in other cells and show considerable molecular weight heterogeneity. We propose to purify and characterize these proteins and compare them to smooth muscle caldesmon. Since cloned cultured cell lines contain more than one caldesmon species, we propose to explore the functional differences between classes of caldesmon species. The work set out in this study focusses on three proteins that play key roles in microfilament organizations. The results should provide some important basic information about the regulation and molecular organization of microfilaments.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM036652-06
Application #
3291045
Study Section
Cellular Biology and Physiology Subcommittee 1 (CBY)
Project Start
1986-04-01
Project End
1995-03-31
Budget Start
1991-04-01
Budget End
1992-03-31
Support Year
6
Fiscal Year
1991
Total Cost
Indirect Cost
Name
Cornell University
Department
Type
Schools of Arts and Sciences
DUNS #
City
Ithaca
State
NY
Country
United States
Zip Code
14850
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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