A variety of diseases of the cardio-vasculature including, spherical elliptocytosis, atherosclerosis, clotting disorders, and hypertension have been linked to structural alteration and dysfunction of the actin cytoskeleton and acto-myosin activity. Relevant to this application are the platelet mediated responses during clotting where these cells are structurally transformed, undergo degranulation, and extensively aggregate to participate in clot formation. Essential to these events is the restructuring of the actin cytoskeleton and its participation in exocytosis of intracellular granules. In the present application, we employ sea urchin coelomocytes as a model for platelet activation since, like platelets, they function in clot formation, undergo structural transformation reliant upon the actin cytoskeleton, and exhibit degranulation. However, unlike platelets, these cells are excellent models for high resolution light microscopic studies and correlative biochemical analyses. The planned training program will focus on experiments designed to define the associated of a 110 kDa unconventional myosin with intracellular vesicles and to determine unconventional myosin's role in vesicle motility and cytoskeletal reorganization. Within the scope of the project, the participants will be trained in a variety of methodologies including, but not limited to, protein fractionation, light and electron microscopy, fluorescence analog cytochemistry, immunocytochemistry, biophysics, and molecular biology. Results of ,D studies will expand our knowledge of how extracellular signals lead to cytoskeletal transformation, vesicle motility, and degranulation. Since, structural and regulatory pathways within the actin cytoskeleton appear to be highly conserved across diverse species, defining the mechanisms at work in coelomocyte activation will without doubt impact our understanding of equivalent events in mammalian platelets.
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