The long term aims of this proposal are two fold: to understand the importance of actin filament networks and assembly in human platelets during activation and secretion, and to determine the extent of calcium regulation of filament and network formation. Using a fluorescent actin, NBD-actin, we have shown that cytoplasmic extracts from activated, but not unactivated platelets have a complex of actin and a 90 kd protein that strongly promotes assembly. The complex and the 90 kd protein have been purified to homogeneity and used to show that the complex will cap the fast growing end of a filament. The interactions between Ca++, actin and the 90 kd protein are partially understood. The complex can bind an additional actin, cap a filament or serve as a growth nucleus in the presence of Ca++ or EGTA. The binding of the first actin, however, is strongly dependent on Ca++. Monoclonal antibodies (MAbs) have been prepared and show that the 90 kd protein is related to gelsolin and is located on stress fibers in WI-38 and 3T3 cells. This result has been compared with the location of vinculin that we isolated from platelets. We now propose to use a variety of biochemical and biophysical methods, including fluorescence polarization and stopped flow techniques, to detail the interactions between Ca++, actin and gelsolin. One general objective is to evaluate the relative rates and extents of gelsolin binding to monomers, filament ends and also to filaments during Ca++ induced severing. The effects of profilin, vinculin and alpha-actinin on these reactions will be explored.
A second aim i s to do structural work on gelsolin by isolating and mapping the actin and MAb binding domains. The MAbs also will be used in microinjection experiments to study gelsolin function in living cells. The final objective is to explore the mechanism(s) that trigger actin and actin-associated proteins to form a contractile network in extracts from activated but not unactivated platelets.
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