PROJECT SUMMARY/ABSTRACT The actin cytoskeleton is a major cellular component with key functions in virtually every aspect of cell physiology including cell motility, shape and mechanics, cell and tissue morphogenesis, cell-cell and cell-matrix interactions, and dynamics of membrane organelle. Aberrations in actin cytoskeleton structure, functions and/or dynamics contribute significantly to human pathologies, especially to cancer and neurodegenerative, immune and cardiovascular diseases. The actin cytoskeleton plays indispensable roles in cells due to its ability to generate large pushing, pulling and resistance forces in many different combinations. To perform these diverse functions, actin filaments are organized into diverse structural arrays by multiple accessory proteins. Despite extensive research, the exact organization of these actin-based molecular machineries is frequently unknown. The main barrier toward this goal is the difficulty of resolving actin cytoskeleton architecture at a single-filament level. Without knowledge of the structure, functional understanding of the machinery is incomplete. My lab uses a distinctive approach to overcome this problem. We take advantage of platinum replica electron microscopy (PREM), which is uniquely able to combine high resolution imaging of the cytoskeleton with full coverage of the whole cell and to efficiently correlate the cytoskeleton structure with live cell dynamics. With help of PREM, my lab has made multiple fundamental contributions toward understanding of cytoskeleton functions in a range of generic and specialized cell types. In this application, we propose in the course of the next five years to address the following questions representing each of the four major categories of actin cytoskeleton functions: (1) Protrusion – How microtubules regulate protrusive activity of the actin cytoskeleton for directional migration; (2) Contraction – How an interplay between nonmuscle myosin II paralogs regulates polarized subcellular distribution of contractile forces; (3) Cell mechanics – How differences in the molecular architecture of the actin cortex are linked to different mechanical properties of normal and cancer cells; (4) Membrane dynamics – How branched actin networks promote invagination of clathrin-coated membrane domains. Our expertise in PREM in addition to a broad range of other cell biological, imaging, functional, biochemical, and molecular biological methods puts us in unique position to significantly advance our understanding of actin cytoskeleton functions. In turn, this knowledge may provide important new insights into how to combat human diseases associated with actin cytoskeleton malfunctions.