Actin filament length regulation mediated by formin’s polymerization activities – nucleation and processive elongation

Abstract

Actin cytoskeletal organization governs essential cellular processes like cell motility, adhesion, cytokinesis, and morphogenesis. The assembly of actin filaments into higher order structures is regulated by a plethora of actin-binding proteins, which specialize the structures for specific functions. A significant fraction of these structures are assembled using unbranched actin filaments. The formin family of actin polymerases are uniquely responsible for polymerizing the majority of these unbranched filaments in cells. Formins function by binding the barbed end of filaments using their dimerized FH2 domains. They enhance the nucleation of filaments and facilitate monomer additions at the barbed end. The FH2 dimers step processively onto incoming actin monomers and stay bound to the barbed end to allow sustained filament growth. By remaining processively associated to the barbed end through thousands of cycles of subunit addition, formins protect filaments from capping and assemble filaments with physiologically relevant lengths. Despite its fundamental importance in actin cytoskeletal architecture, a clear understanding of formin’s processive elongation property remains elusive. Along with processive elongation, another way formins influence filament length is by nucleation of filaments. To understand how formins assemble filaments that attain precise lengths, we need an understanding of how both processive elongation and nucleation impact polymerization. To investigate how formin’s polymerization properties regulate filament length, we used in vitro actin reconstitution experiments, TIRF-microscopy based single molecule imaging, and stochastic modeling. We studied the budding yeast formin Bni1p to understand the regulation of formin processivity and found that the mechanism by which subunits are added to the filament barbed end has a vital role in dictating formin’s dwell time at the barbed end. Additionally, we uncovered a kinetic balance between formin-mediated nucleation and filament elongation, which is central to filament length regulation. Altogether, these findings provide novel insights into the workings of formins and advance the field in understanding their role in actin filament length regulation. This thesis provides a framework for future investigations to comprehend formin function and the roles played by other cellular factors in formin-mediated filament length regulation and actin cytoskeletal organization.