Measuring Actin Dynamics

The actin cytoskeleton is a highly dynamic system responsible for maintaining cell shape and rigidity. In endothelial cells, the actin cytoskeleton undergoes a remarkable reorganization in response to shear at the cell surface. This rearrangement likely occurs through regulation of cytoskeletal dynamics. This thesis presents a viable technology for the measurement of two fundamental dynamic properties of the actin cytoskeleton, actin monomer diffusion coefficients, and actin filament turnover. The experimental method employed is photoactivation of fluorescence (PAF). In these experiments, actin monomer is labeled with a non-fluorescent fluorophore derivative (caged-actin) and microinjected into living cells. After incorporation of the injected actin into the endogenous cytoskeleton, a cell is flashed with a narrow band of UV light converting the fluorophore in the band back to its fluorescent parent. The subsequent decay of fluorescence is monitored to infer information about monomer diffusion and filament turnover. The thesis proceeded through three developmental steps, 1) the synthesis of caged-actin, 2) the design and assembly of the necessary hardware for PAF experiments, and 3) the development of a model of the steady-state dynamics of the actin cytoskeleton from which the first comprehensive protocol for interpreting cytoskeletal dynamics from PAF data records was prescribed. A limited number of experiments on non-sheared endothelial cells have been conducted. Results agree qualitatively with model predictions and previous studies. Estimates of monomer diffusion coefficients and filament turnover times agree with some estimates available in the literature, but disagree with others. More experiments may resolve these discrepancies. Thesis Supervisor: Dr. C. Forbes Dewey, Jr. Title: Professor of Mechanical Engineering To my parents, Jim and Gerry McGrath, whose steady and unconditional love has always seen me through.