The overall goal of the NIGMS-funded research in my lab is to define the molecular and cellular mechanisms underlying dynamic rearrangements of the actin cytoskeleton, and to explore how these mechanisms are harnessed in vivo (in yeast and animal cells) to control diverse actin-based processes such as cell motility, endocytosis, intracellular transport, and cell morphogenesis. Genetic and biochemical research has been rapidly producing a ?molecular parts list? for the actin cytoskeleton, and many of the components have been characterized individually for their biochemical effects on actin filaments and their genetic effects on cellular actin organization and function. However, it is becoming clear that most of these proteins do not function alone, but rather in groups to perform their biological roles, and thus, new approaches are needed to define how they work in concert to perform their cellular functions. Our lab is tackling this problem using advanced single molecule TIRF microscopy to directly observe multi-component actin regulatory mechanisms in real time, and testing these mechanisms using genetic, cell biological, biochemical, and structural approaches. Through this approach, we have made fundamental new insights into actin regulation. For instance, we defined the first collaborative actin nucleation mechanisms of formins (with Bud6 & APC). We discovered that formins and Capping Protein can bind simultaneously at filament ends to accelerate each other?s dissociation. We showed that Cofilin, AIP1, and Coronin work together via an ordered mechanism to sever and disassemble F-actin. We discovered that Srv2/CAP works in conjunction with Cofilin and Twinfilin to depolymerize filament ends. In parallel, we have combined genetics, cellular imaging, and separation-of-function mutants to dissect the contributions of these mechanisms to actin-based processes in yeast and mammalian cells. Moving forward, we will ask the following questions: what are the complete regulatory cycles of the two yeast formins (Bni1 and Bnr1)? How is Arp2/3 complex-mediated actin nucleation balanced by its inhibitors (Coronin and GMF) and activators (Las17/WASP and Abp1)? How is actin nucleation at the leading edge of motile cells controlled by interactions among IQGAP1, APC and formins? How do interactions at filament ends between Capping Protein and formins (and their in vivo binding partners) control actin network growth? How do the filament severing and depolymerization mechanisms (Cofilin, AIP1, Coronin, Twinfilin, and Srv2/CAP) drive net disassembly of actin under the assembly-promoting conditions of the cytosol? Are there actin-associated proteins that accelerate the nucleotide state transition on F-actin to promote disassembly? In addition, we will introduce new technologies and directions to our research, including in vitro reconstitution of cellular actin structures, cryo-EM to study protein structure, cell-free extracts to genetically-biochemically dissect actin mechanisms, and a systems-level approach to determine how genetic disruptions in individual actin regulators affect the cellular levels, localization, and functions of the remaining actin-associated proteins.
Dynamic rearrangements of the actin cytoskeleton are essential for countless biological processes, including cell motility, endocytosis, cell division, intracellular transport, and cell and tissue morphogenesis. Furthermore, defects in actin regulation lead to a range of disorders, including Alzheimer?s disease, Huntington?s disease, Parkinson?s diseases, Amyotrophic Lateral Sclerosis (ALS), cardiovascular diseases, developmental disorders (e.g., limb deformities, infertility, and hearing impairment), and cancer. This proposal focuses on the molecular and cellular mechanisms underlying actin dynamics and organization. It takes aim at major gaps in our knowledge of how groups of actin regulatory proteins work together in multi-component mechanisms to govern actin dynamics in living systems. The proposed research will resolve these mechanisms, and in doing so provide new insights into the molecular basis of human disease.
