My laboratory has a long-term interest in understanding the basic mechanisms underlying actin cytoskeleton regulation and how these mechanisms drive fundamental processes such as cell migration, vesicle trafficking, immune cell activation, and neuron morphogenesis. We are particularly interested in solving the regulation mechanisms of the Wiskott-Aldrich Syndrome Protein (WASP) family proteins. These proteins play a central role in linking membrane signals to the Arp2/3-mediated actin polymerization throughout eukaryotic cells. Ge- netic mutation or misregulation of these proteins is heavily involved in human diseases, including various neu- rodevelopmental disorders, immune syndromes, pathogen infections, and cancer. Despite their importance, the regulation mechanisms of most WASP-family proteins are poorly understood, posing a large barrier to un- derstanding their roles in biology and human pathology and identifying new avenues of treating related dis- eases. Our recent studies have made a series of pivotal contributions to the understanding of the WASP-family protein WAVE and its role in disease. WAVE is incorporated in a large protein assembly named the WAVE Regulatory Complex (WRC), which is essential to polymerizing actin at the plasma membrane. We have deter- mined how WRC is activated by two distinct small GTPases, Rac1 and Arf, how disease-related mutations dis- rupt WRC activation, and how WRC interacts with various ligands, including the neuronal receptor HPO-30, the redox regulator p47phox, and the actin regulator Ena/VASP/UNC-34. In the next five years, we will continue to move the field vertically by addressing a series of new, important questions about WAVE regulation. In parallel, we will start exploring the mechanisms of two other WASP-family proteins, WHAMM and JMY, for which very little is known mechanistically despite their essential roles in regulating actin assembly at endomembranes. We will combine protein engineering, quantitative biochemistry and mass spectrometry, single molecule fluores- cence microscopy, and single particle cryogenic electron microscopy (cryo-EM) to determine how WRC is acti- vated by Arf binding, how distinct Arf and Rac1 binding sites establish cooperativity, and how WRC interacts with several novel ligands important to neuron morphogenesis, axon guidance, and parasite infection. In addi- tion to dissecting mechanisms of individual ligands, we will establish membrane-based single-molecule fluores- cence experiments to determine the mechanisms by which multiple ligands, including inositol phospholipids, GTPases, and membrane proteins, cooperatively activate the WRC in a context closely resembling cell mem- branes. Furthermore, we will determine how WHAMM and JMY are regulated by binding to various ligands. Our work will provide a comprehensive mechanistic framework for understanding WASP family protein regula- tion. This knowledge will be broadly useful to different fields studying actin-related proc...