Project Summary A hallmark of eukaryotic cells is their ability to migrate, divide, adhere and respond to environmental cues. Nonmuscle myosin-2 (NM2) motors play an essential role in many aspects of these fundamental cellular processes by forming short bipolar filaments that interact with actin filaments. NM2 motors are binary switches that alter between inactive and active states depending on the cellular context. The precise control of NM2 motor activity is critical for its cellular function as master regulator of the actin cytoskeleton. Aberrant regulation due to mutations in NM2 paralogs contribute to a whole host of diseases including blood and neurological disorders, heart diseases, deafness, nephritis, and cancers. NM2-specific therapies are thus needed, yet the lack of basic knowledge about the structure and regulation of NM2 paralogs is a bottleneck to their development. We aim to develop a detailed structural and mechanistic understanding of how force generation by NM2 motors drive various cellular functions. Using innovative and interdisciplinary techniques including the state-of-the-art cryo- electron microscopy, X-ray crystallography, steady-state kinetics, in vitro motility assays and high-resolution fluorescence microscopy, we will systematically dissect the mechanisms of activation and regulation of NM2. To achieve this, in Aim 1, we will determine the major structural states in the ATPase cycle of NM2 motors to explain enzyme function. In Aim 2, we will determine a high-resolution cryo-EM structure of the inactive state of full- length NM2 to explain its molecular architecture. In Aim 3, we will study the consequences of abolishing the ability to form an inactive state on the dynamics of NM2 filaments in cells. Collectively, our studies will provide a deeper understanding of the structure, function and regulation of NM2. Importantly, this knowledge will advance our understanding of emergent NM2 functions in cells and thus, lay the foundation for future development of NM2-specific therapeutics.