Cytoskeletal force-sensing mechanisms

About This Grant

Cells perceive mechanical cues in their local environments, which must be converted into intracellular biochemical signals to modulate cellular physiology and control gene expression. This process of mechanical signal transduction (“mechanotransduction”) is critical for development and frequently dysfunctions in disease states such as cancer. Despite increasing appreciation of its importance for human physiology, the molecular mechanisms of mechanotransduction remain poorly understood, hampering efforts to define mechanistically distinct mechanical signaling pathways, delineate their specific biological functions, and target them therapeutically. The actin cytoskeleton, a network of dynamic actin filaments (F-actin), myosin motor proteins, and hundreds of associated factors, enables cells to mechanically interface with their surroundings. While the cytoskeleton is classically understood as a force generation and transmission apparatus that indirectly facilitates mechanotransduction, our research has provided evidence that actin filaments can also serve as direct molecular force transducers. By developing innovative cryo-electron microscopy (cryo-EM) sample preparation and machine-learning based computational analysis approaches, we have uncovered multiple classes of force- dependent structural transitions in F-actin (elicited by fluid flow and myosin molecular motor forces) that can be discriminated by force-sensitive actin-binding proteins. This work has provided a first direct glimpse at how forces alter protein structure to regulate function. Using biophysical reconstitution and cell biology studies, we have also shown how force-activated F-actin binding by proteins from the LIM (LIN-11, Isl-1 & Mec-3) domain superfamily can coordinate downstream mechanotransduction processes, including repair of physical damage to actin- myosin cables mediated by zyxin and extracellular matrix stiffness-dependent nuclear localization of Four-and- a-Half LIM domains (FHL) transcriptional control proteins. Additionally, we have developed cryo-EM and cryo- electron tomography (cryo-ET) approaches for visualizing crosslinking proteins bridging actin filaments, setting the stage for studying how forces alter the geometry and composition of higher-order cytoskeletal networks in atomistic detail. To build upon this progress, we will now study the structural mechanisms and cellular functions of a ubiquitous class of force-stabilized protein-protein interactions known as catch bonds, which are prominent in cytoskeletal crosslinkers and cell adhesion proteins implicated in mechanotransduction. We will also probe the structural basis of force-activated cytoskeletal engagement by LIM proteins, focusing on both individual actin filaments and cytoskeletal networks in vitro and in cells, as well as scrutinize the nuclear localization and gene regulatory mechanisms of FHL proteins. In the near term, our efforts will provide detailed mechanistic insights into mechanical signaling pathways, facilitating precise dissection of their functions in vivo. In the longer term, our work may guide the development of chemical probes and therapeutics which selectively target mechanotransduction.