Movement is a ubiquitous feature of all cells and includes such processes as mitosis, cytokinesis, locomotion of single cells through ameboid motion, axonal transport, and membrane trafficking. One of the cellular systems involved in many of these movements is the actin-based cytoskeleton which composed of actin, myosin, and actin binding proteins. The actin cytoskeleton is remarkable for its stability as well as its dynamic nature. We have been investigating the intestinal enterocyte brush border actin cytoskeleton which forms one of the most stereotyped actin cytoskeletons making it a useful model for investigation. The brush border is composed of over one thousand finger-like microvilli supported by a core bundle of actin filaments penetrating into the underlying terminal web of anastomosing filaments. Distinct actin associated proteins are found in each domain. The brush boarder is involved in nutrient transport and therefore an understanding of how it forms and is maintained is of paramount importance. Three lines of investigation are proposed. First, we will use brush border and muscle proteins in an effort to understand how this cell or a muscle makes extremely stable actin filaments which do not disassemble off the preferred end for disassembly. We hypothesize that different tropomyosins, alone or in association with other binding proteins, stabilize the actin pointed end from disassembly. Actin depolymerization assays using spectrofluorometric methods will be used. Second, we will investigate two classes of actin binding proteins using the microvillar proteins, villin and fibrin, as representative examples. We will determine, through competition assays with intact proteins or with purified proteolytic fragments, whether they bind to similar or distinct sites on actin. Third, we will investigate further the development of the brush border cytoskeleton. We have recently detected phosphotyrosine-containing proteins in the epithelium which are restricted to the undifferentiated mitotic crypt cells. We will study the protein tyrosine kinases in order to determine if they are specific growth hormone receptors or proto-oncogenes. We will analyze the phosphotyrosine-containing proteins to evaluate their function and to determine if they have a regulatory role in controlling development of the brush border.
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