Mechanisms controlling cellular actin dynamics and cytoskeletal crosstalk - The goal of this research is to understand the mechanisms controlling the assembly and turnover of cellular actin structures, and crosstalk among the actin, microtubule, and septin cytoskeletons. Proper regulation of cellular actin dynamics and crosstalk is critical for cell motility, organelle transport, cell adhesion, cell contractility, cell polarity, cell and tissue morphogenesis, among many other processes. While many of the key actin regulatory proteins have now been identified and characterized individually for their biochemical effects on actin, in vivo these proteins work in groups, which often leads to emergent functional effects on actin that cannot be identified by studying each protein alone. To tackle this problem, we are using multi-wavelength single molecule TIRF microscopy to directly observe groups of actin binding proteins functioning together in real time to control actin filament dynamics and organization. We then dissect these processes at a molecular level and use this information to develop highly specific tools to test these mechanisms in vivo (in yeast and mammalian cells) through genetic manipulation and live imaging. During the recent funding period, we used these approaches to gain fundamental new insights into how key groups of actin regulators work in concert. For example, we discovered that Srv2/CAP (yeast and human) synergizes with cofilin to accelerate pointed end depolymerization by >300-fold. We found that yeast coronin, GMF, and Arp2/3 form a high-affinity ternary complex that robustly catalyzes actin filament debranching. We discovered that human APC, formin (Dia1), and G-actin associate to form a stable, inactive ‘prenucleation’ complex, which is then activated by a ligand of APC and Dia1. We learned that yeast septins are organized at the bud neck into evenly-spaced ‘pillar’ structures that align with actin cables, and proper alignment and function of actin cables requires multiple factors linking septins and actin (e.g., Hof1, Syp1, Iqg1, and Myo1). This work has also raised new questions, which we will address in the next cycle, including: how is the turnover of branched Arp2/3-actin filament networks in mammalian cells precisely tuned by branch stabilizers and destabilizers to regulate motility? How are the potent activities of yeast formins (Bni1 and Bnr1) spatially and temporally controlled in cells to produce actin cables of a precise length, shape, and architecture? How do bud neck-associated septin structures scaffold and regulate multiple actin-binding proteins to ensure proper actin cable formation? How do human APC, IQGAP1, and Dia1 collaborate and interact with Cdc42 and its GEF (ASEF) to control actin assembly at the leading edge of mammalian cells? As part of our planned research, we have introduced new technologies, new collaborators, and new directions, including cryo-ET to study actin network ultrastructure at the leading edge, studying actin rearrangements in yeast cell quiescence, and identification of ‘FEED’ motif proteins that link formins to diverse cellular processes (in yeast and humans), which includes new leads into actin-microtubule and actin-septin crosstalk.
