BIOPHYSICAL STUDIES OF CYTOSKELETAL FUNCTION

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The long range goal of the proposed work is to understand the mechanical functions and properties of animal cells. The cytoskeleton, a system of interacting filaments within the cell which governs its mechanical behavior, is at the center of attention. Three overlapping and mutually complementary areas of study are emphasized. The first concerns the role of the cytoskeleton in maintaining cell shape, contractile tension, and consistency or viscoelasticity. The roles of the three cytoskeletal filament systems, microfilaments, microtubules, and intermediate filaments in determining cellular viscoelasticity will be studied with measurements of cellular deformability using methods developed for specifically this purpose. The second area concerns the mechanical functions of dystrophin, the protein absent from patients afflicted with Duchenne muscular dystrophy. Evidence suggests that this protein provides a mechanical link between the surface membrane of the muscle cell and its actin filament system the purpose of which is the mechanical stabilization of the cell. This hypothesis will be tested with measurements of the deformability of muscle cells cultured from normal mice and mice lacking dystrophin which provide a model for the human disease. The possibility of an assay for dystrophin function based on these measurements will be explored. This assay would help guide efforts to restore dystrophin to cells which lack it using molecular genetic or cell biological methods. The third area of work concerns mechanical processes that contribute to locomotion of the amoeba Dictyostelium discoideum. This single cell organism is favorable for these studies because of the methods available to delete selected cytoskeletal proteins using molecular genetic methods and the extensive data about its mechanisms of locomotion and chemotactic responses. The deformabilities of different regions of cells at different times in their locomotory cycles, the role of adhesion to the substrate, and the transport of particles on the cell surface will measured to probe the mechanics of locomotion. The locomotion of Dictyostelium amoebae is similar in many ways to that of mammalian leukocytes, and so provides a model for a function of the latter cells which is an essential component of their contribution to immune responses.