This research is developing and applying biophysical assays for cytoskeletal function. The project uses three different mutually complementary approaches. One assay, recognizing that a principal function of the cytoskeleton is to maintain or change cell shape, uses a new method for measuring cellular deformability. This provides a quantitative index of the physical state of the cytoskeleton, measured in terms of the force needed slightly to indent the exposed surface of a cell adherent to a solid substratum. Biophysical analysis of this measurement using continuum mechanical theory and model experiments comprises one portion of the project. Its goal is to extract quantitative estimates for cellular viscosities and elasticity moduli. Concurrently, the dependence of deformability on the shape, physiological state and cytoskeletal structure of a cell and the participation of the cytoskeleton in physiological functions such as capping and secretion will be characterized empirically. A second portion of the project carries this work to the molecular level by determining the function in living cells of proteins which modulate the extent of polymerization and interactions in microfilaments. Proteins such as gelsolin and alpha-actinin, which have been well characterized in vitro, will be introduced into the cytoplasm of cells. An operational assay of cytoskeletal state which includes morphology, cellular viscoelasticity, and intracellular transport will gauge the effects of the modulating proteins on the cytoskeleton. A third portion of the project extends Fluorescence Correlation Spectroscopy and Fluorescence Photobleaching Recovery to cytoskeletal polymerization reactions. The former will be used to measure the distribution of degrees of polymerization; the latter, to measure polymerization reaction kinetics in cells. These methods will provide information not readily available by more conventional techniques.
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