BIOPHYSICAL STUDIES OF CYTOSKELETAL FUNCTION

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The long range goal of the proposed work is to understand generally how cells function mechanically and, more specifically, what forces and structures determine their shapes and deformabilities. The approach is based on the hypothesis that cell shape and deformability are governed by the cytoskeleton. Since the cytoskeleton is a system interacting polymer filaments, both its and the cell's viscoelasticity should be understandable in terms of polymer chemistry. To achieve this level of understanding it is necessary to measure quantitatively cellular, and therefore cytoskeletal, viscoelasticity. Toward this end a nev method for measuring cellular viscoelasticity based on determining the resistance of the cell to localized indentation has been developed. This method will be used to determine the relative contributions of the three cytoskeletal systems to the control of cellular deformability by interfering separately with the structure of each of the systems. In addition the mechanical function of the nucleus and of cell-substrate and cell-cell interactions will be examined. The mechanical functions of myosin will be examined in mutant Dictyostelium cells which entirely lack this protein. The mechanical changes resulting from physiological activation of secretory cells and leukocytes will be determined and interpreted in terms of cytoskeletal processes. Supporting this work on cells there will also be studies of the interactions of purified cytoskeletal proteins in simplified reconstituted systems. For example, fluorescence photobleaching recovery will be used to characterize the interactions of alpha-actinin with actin filaments.