9723273 Wadsworth MT dynamic behavior has been shown to be regulated in living cells, and the parameters of MT turnover varies with cell type and stage of the cell cycle. Recently, it has been shown that microtubule turnover in epithelial cells is greatly suppressed when cells establish apical/basolateral polarity and is stimulated following treatment with a mitogenic growth factor (hepatocyte growth factor, or HGF) (which disestablishes polarity). In the first Aim of this project, the behavior of individual microtubules will be measured in the leading edge and non-leading lamellar regions of motile (growth factor-treated) epithelial cells to test the hypothesis that MT dynamic turnover is regionally regulated within the cell. These measurements will directly show if MT behavior varies among different specific regions of the cell, and if so, which parameters are altered. In addition, the correlation of MT dynamics to the establishment of cell polarity will be determined by measuring dynamics before and after polarization of motile cells. In the second Aim, the molecular basis for the stimulation of MT turnover in HGF-stimulated cells will be determined. Tubulin will be immunoprecipitated from extracts of control and HGF-treated cells using anti-tubulin antibodies, and any molecules that co-immunoprecipitate with tubulin dimers will be identified and characterized. In addition, MTs will be assembled in vitro from extracts of control and HGF-treated cells, using taxol to stimulate net polymerization, and MT-associated proteins (MAPs) will be released from the MTs using salt and/or ATP. The composition, phosphorylation and quantity of MAPs in control and treated cells will be compared. In the third Aim, molecules that suppress MT turnover in polarized epithelial cells will be identified. To accomplish this goal, a rapid, quantitative, in vitro polymerization assay will be developed to screen fractions of an epithelial cell lysate for factors that suppress net microtubule growth. The effect of the novel proteins thus identified will be directly demonstrated by microinjection into living cells and measurement of the parameters of dynamic instability. The results of these experiments will provide new information regarding the molecular mechanisms that regulate microtubule turnover as cells respond to growth factors and as they differentiate. Microtubules (MTs) are polymers of the subunit protein tubulin, and are one of the three major classes of cytoskeletal structures in eukaryotic cells. Their function is critical to a variety of fundamental cellular processes, such as cell division, flagellar and ciliary motility, intracellular trafficking of subcellular particles, and cell shape regulation. Individual MTs are inherently dynamic, alternating stochastically between periods of growth (polymerization) and rapid shortening (depolymerization). MT dynamic behavior has been shown to be regulated in living cells, and the parameters of MT turnover varies with cell type and stage of the cell cycle. This project will elucidate some of the biological consequences of MT dynamics, and will elucidate the biochemical mechanisms by which cells regulate the dynamic instability of MTs. ***
