The long range goal of this work is to study the molecular basis of membrane-cytoskeleton associations and their role in the directed intracellular movements of membranes and organelles. Specifically, it is planned to investigate the idea that actin filament-membrane attachments are instrumental in the translocation of secretory vesicles to the plasma membrane during exocytosis. The approach taken here will be to demonstrate specific and regulated associations of actin with purified membrane components. The catecholamine-containing secretory vesicles (chromaffin granules) are an experimentally accessible system because they can be easily prepared in large quantities and high purity. Also, actin filaments can bind to a protein site on purified chromaffin granule membranes and these interactions are regulated by free calcium ion concentrations thought to mediate exocytosis in vivo (Fowler, V.M. and H.B. Pollared. 1982. Nature 205:336-339). However, using available methods of measuring actin binding to membranes, it was not possible to distinguish a small number of high affinity sites on the membranes from a large number of low affinity sites, nor to clearly distinguish effects of regulatory factors on actin binding from effects on actin polymerization or on filament self-association. Therefore, a primary goal of this proposal will be to quantitatively characterize actin binding to membranes, using define, radiolabelled actin species (e.g., monomer, convalently cross-linked dimer) at subcritical concentrations so that actin polymerization will not interfere with analysis of binding data. Subsequently, it is planned to identify and purify the actin-binding protein(s) from the chromaffin granule membrane, to characterize its interaction with monomeric and filamentous actin, and to determine its relationship to known actin-binding proteins, as well as its subcellular location in the cell. Regulatory features of actin-membrane association (e.g., [Ca++] free, phosphorylation) will be investigated at three levels: actin binding to membranes, interactions of the purified protein with actin, and during exocytosis from intact cells. It is also planned to determine ultrastructurally how actin filaments are attached to the chromaffin granule membrane in vitro, and in situ in the cell, and to determine whether immunologically cross-reactive proteins might also be present on membranes of other subcellular organelles.