CYTOSKELETON/MEMBRANE INTERACTIONS

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The actin-associated membrane skeleton is involved in cell adhesion, motility, and the regulation of cell survival. This application proposes the continued study of a new type of membrane skeleton characterized by the presence of supervillin, a recently-discovered actin-binding peripheral membrane protein. Supervillin is isolated from bovine neutrophil plasma membranes in association with stabilized actin filaments, fodrin, phosphatidyl-inositol-4-kinase alpha, Rho kinase, and several other proteins. Supervillin contains several F-actin binding sites, a focal adhesion targeting sequence, predicted nuclear localization signals, a potential nuclear export signal, and possible coiled-coil domains. Depending upon growth state and/or stage in the cell cycle, supervillin localizes within nuclei or with F-actin at sites of cell-cell and cell-substrate adhesion. Overexpression of supervillin sequences leads to disruption of focal adhesions and re-structuring of the actin cytoskeleton into bundles at the plasma membrane and/or in cell nuclei. Increased levels of supervillin message and/or protein are observed in many carcinoma cell lines, suggesting that supervillin overexpression may occur during tumorigenesis. Preliminary antisense experiments suggest that supervillin may be essential for cell survival. We propose that the supervillin-associated membrane skeleton contributes to the structural integrity of motile cells and that supervillin may be involved in the control of actin and myosin assembly at the plasma membrane. We further hypothesize that supervillin and associated proteins participate in aspects of nuclear architecture and/or function and that this type of membrane skeleton contributes to the regulation of cell adhesion, growth, survival, and/or motility. To test these hypotheses, we propose: (1) to complete the biochemical and morphological characterization of the supervillin-associated membrane skeleton in neutrophil plasma membranes and (2) to determine the role(s) of the supervillin-associated membrane skeleton during growth, adhesion, motility, and mechanical stress of epithelial and carcinoma cells. The ultimate goal of this project is to understand how the actin-associated membrane skeleton functions during cell motility, adhesion, and detachment from substrates and other cells. This information will increase our understanding of both normal cellular behaviors and pathological conditions, such as developmental abnormalities and cancer cell metastasis, and may even produce some useful diagnostic tools for tumor staging.