Project Abstract/Summary Cells utilize an array of actin binding proteins with diverse and complementary properties to assemble, maintain and disassemble a range of distinct F-actin networks to facilitate different fundamental functions including motility, polarization and division. To serve its function, each of these networks has a unique architecture defined by the number, lengths and connectivity of its filaments, which is maintained by a continuous dynamical balance of actin filament assembly, remodeling and disassembly. A fundamental challenge is to understand how functional network architectures are formed and maintained by the continuous coupling of architecture and assembly dynamics. Here we are focusing on the cell cortex, a dynamic network of actin filaments, crosslinkers and myosin motors, lying just beneath the plasma membrane, that undergoes rapid deformation and flow to drive cell movement, polarization, division and tissue morphogenesis. How ensembles of actin regulatory factors work in concert to simultaneously regulate actin filament network architecture assembly and dynamics at the cortex of living cells is poorly understood. The one cell C. elegans embryo provides a uniquely powerful opportunity to address these questions in a single large cell, that is directly accessible to high resolution microscopy, with powerful genetics, transgenics, CRISPR and RNAi. The C. elegans cortex is primarily composed of an F-actin network of linear filaments and small filament bundles that are assembled by formin CYK-1-mediated polymerization of profilin-actin, and decorated by actin filament crosslinkers plastin PLST-1, anillin ANI-1, and by mini-filaments of non-muscle myosin II NMY-2. Conversely, cofilin UNC-60A and capping protein CAP-1/CAP-2 appear to be key regulators of filament disassembly and filament length, respectively. We are addressing how the C. elegans cortical F-actin network architecture is formed and maintained through the dynamic integration of formin-dependent filament assembly, filament crosslinking, filament capping, and cofilin-dependent filament disassembly, all while the network experiences continuous myosin- driven deformation and flow. We are combining the complementary state-of-the-art in vivo expertise (quantitative single molecule imaging and particle analysis) and in vitro expertise (reconstitution and biophysical analysis) of the Ed Munro and David Kovar lab’s to address two major questions concerning the architecture and dynamics of the C. elegans F-actin cortex. First, we will characterize the fundamental dynamics, regulation and feedback control of formin- mediated actin filament and bundle assembly (Aim 1). Second, we will investigate the fundamental dynamics, regulation and feedback control of cofilin-mediated actin filament disassembly of the formin actin filament networks (Aim 2).