Rearrangements of the actin cytoskeleton are critical to numerous cellular processes and are defective in many genetic and infectious diseases. Members of the Wiskott-Aldrich Syndrome Protein (WASP) family play key roles in controlling actin dynamics throughout biology. In the previous period we discovered a new mechanism of WASP family regulation that unified a disparate body of unexplained data under a common framework. We also reconstituted two pentameric assemblies, the 400 kDa WRC and the 550 kDa SHRC, that contain and control the WASP proteins WAVE and WASH, respectively. Our WRC crystal structure explained inhibition of WAVE within the assembly. Our biochemical analyses resolved a long-standing dispute regarding WRC activity. Here, we will exploit our unique access to recombinant WRC and SHRC to understand structurally and biochemically how these assemblies respond in complex fashion to upstream stimuli to promote cell migration, neuronal adhesion and vesicle trafficking. We will determine the crystal structure of the WRC bound to the Rac GTPase. The structure, plus complementary biochemical and collaborative cell biological studies, will explain how the WRC is cooperatively activated by GTPases, phospholipids and kinases. We will characterize a novel WRC-binding motif (WIPS) that we have discovered in 30 neuronal adhesion receptors, including many members of the enigmatic protocadherin family. We will determine the structure of a WRC-WIPS complex and learn how various WIPS-containing receptors cooperate with Rac to activate the WRC in vitro and in cells. Finally, we will learn how the SHRC is recruited to membranes through multivalent binding to the retromer coat complex, and the functional consequences of these interactions on actin assembly. Our work will allow the first physical comparisons between WASP proteins that function as single chains (WASP/N-WASP) and those that function within multi-component assemblies (all other family members), revealing new and general principles of signal integration that span the family. We will learn how protocadherins and other neuronal receptors communicate to actin and are coordinated with other signaling inputs as part of their poorly understood adhesive functions. Our findings will provide new reagents and concepts to guide neuroscientists in understanding these receptors in cels and organisms. We wil gain new insights into diseases including autism, epilepsy and deafness, which can be caused by protocadherin mutations. Finally, our studies of the SHRC will address a broadly significant problem in cell biology--how actin assembly and vesicle coat formation are cordinated during endocytic traficking-through a new hypothesis, that multivalency provides a mechanism for SHRC recruitment to respond to retromer density on membranes. This work will suggest general mechanisms by which multivalent interactions can be used to control the specificity and timing of membrane interactions of soluble species. PUBLIC HEALTH RELEVANCE: Our research focuses on understanding control of actin dynamics by members of the Wiskott-Aldrich Syndrome Protein (WASP) family. These molecules are critically involved in many normal biological processes and in numerous diseases, including metastatic cancer, immune disorders, infection, and as suggested by our recent findings, neuronal diseases including autism, epilepsy, deafness and blindness. An understanding of how WASP proteins function will reveal new, general principles in basic biology and could lead to new agents for the diagnosis and treatment of many diseases.