The formation and contraction of actin gels in vitro by actin associated proteins give a means to study the mechanism(s) of regulating changes in cytoplasmic consistency which are associated with non-muscle motility. using sea urchin eggs and coelomocytes we have defined the physiological role of one actin bundling protein, fascin, and demonstrated its role in the formation of microvilli. We have shown that a second 220,000 Dalton protein will crosslink actin-fascin bundles into a three dimensional gel in vitro, but have not shown its role in situ. We are proposing to use microinjection methods and well defined antibodies to study the role of both proteins in early development. Sea urchin myosin is relatively sollble in extracts at low ionic strength in the absence of ATP. The addition of ATP results in bipolar filament formation which produces a myosin precipitate. The characteristics of this reaction suggest a calcium independent phosphorylation mechanism. We propose to test this idea by looking for incorporation of phosphate into myosin heavy and/or light chains after incubation with [32P]-AlphaATP. If successful we will look for a phosphorylation-dephosphorylation cycle during cytokinesis. We will purify and characterize the myosin specific kinases and phosphatases, produce immunological probes and test their effects in situ by antibody injection. Using human platelets we have identified and purified a 90,000 Dalton calcium dependent capping protein which blocks actin monomer addition at the barbed end od a filament. This protein also will promote bundle formation in the presence of EGTA. Our preliminary evidence suggest a similar activity is present in the eggs. We propose to purify and charactrize this activity, study its effects on actin and actin filaments and define its physiological role using antibody injection. Finally we will pursue to ongoing projects: Using a newly developed gel overlay technique with iodinated actin we will search for new acxtin binding proteins including membrane bound actin binding proteins which are poteintial filament nucleation and anchoring sites. We also will continue to characterize our monoclonal antibodies aginst muscle action in an effort to fine specific probes will be used to localize these ends and to study monomer addition in situ.
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