A major problem in cell biology is how cells regulate their shape and motility during the performance of such critical events as cytokinesis, membrane ruffling, pseudopod extension or microspike formation. Studies on the biochemistry, structure and function of the cytoskeleton in recent years has suggested that actin filaments, actin binding proteins, myosin, and various control proteins play a significant role in the maintenance of cell shape and the movement of cells. The intestinal epithelial cell brush border has been one of the most extensively studied model systems whereby we now know the major actin filament bundling proteins and the membrane linking proteins of the microvillus core and the structure of the terminal web. Recent experiments suggest that the brush border is motile via a terminal web contractile ring. We propose to continue our studies on the structure and function of the brush border with a series of experiments designed to provide new information about the regulation of motility of the brush border and about the ways in which the length and stability of microvilli are regulated. Intact and skinned intestinal epithelial cells will be used in a series of experiments to determine if changes in free calcium regulate brush border motility and myosin phosphorylation. Biochemical studies on brush border myosin are planned to determine if levels of phosphorylation correlate with filament assembly and enzyme activity. Myosin light chain kinase will be localized in the brush border to determine if it is in the contractile ring. Microvillus length will be studied by experiments to detemine the actin monomer/polymer pool size, turnover of cytoskeletal proteins during growth of microvilli, and the presence of profilin in intestinal cells. Reconstitution experiments will be done to determine if microvillus rootlets and cores can be put together from their known component parts which may give insights into the stability of actin filament bundles within other cell types. These studies will provide new information about the functioning of the normal cytoskeleton which will assist in the interpretation of certain diseased states where cells move abnormally.
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