Structural Mechanisms of Cytoskeletal Force-Sensing

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PROJECT SUMMARY Cells in the body perceive cues from their local environment, which control cellular behavior through a coordinated series of molecular events known as signaling. Signaling is critically important for telling a cell if it should grow and divide, migrate to a different part of the body, or commit suicide if it has completed its function or been irreparably damaged. Frequently, signaling processes are found to be working incorrectly in diseased cells. For instance, cancer cells divide and migrate out of control and ignore cues which should keep them in check. Signals come in multiple forms. Specific molecules bind and activate cognate receptor proteins in the cell, known as “chemical signaling”, which is broadly well-understood. Physical forces and the rigidity of a cell’s environment also elicit specific cell behaviors, but we have a comparatively poor understanding of how proteins transmit these “mechanical signals”. A significant fraction of successful drugs target protein molecules which operate in chemical signaling. The development of many such treatments was stimulated by determining the detailed three-dimensional chemical structures of the interactions between receptor proteins and the molecules which activate them, facilitating the design of drugs which precisely intervene in these processes. Despite its importance, efforts to therapeutically target mechanical signaling have been limited. The long-term goal of this research project is to visualize how forces modulate the three-dimensional structure of mechanical signaling proteins to activate them, in order to facilitate the development of drugs that block these changes. This proposal is specifically focused on understanding how cellular polymers (“filaments”) composed of the protein actin coordinate mechanical signaling. The cell contains many networks composed of actin filaments, myosin molecular motor proteins, and hundreds of other binding partners, which collectively generate and transmit diverse forces. We hypothesize that specific types of forces cause distinct physical rearrangements in actin filaments, which can be detected by other proteins in the cell through direct binding interactions. We will identify proteins which bind actin in a force-sensitive manner (Aim 1), focusing specifically on delineating the precise regions of the proteins which confer force-sensitivity. We will next visualize how side-wise bending forces (Aim 2) and length-wise tensile and compressive forces generated by myosin motor proteins (Aim 3) impact actin filament structure, hypothesizing these force regimes produce distinct rearrangements which can be discriminated by binding partners. In pursuit of these Aims, we are developing sample preparation and computational image analysis approaches to visualize the three-dimensional structure of actin polymers in the presence of mechanical forces with cryo-electron microscopy (cryo-EM). In addition to providing basic insights into how forces are perceived by cells through changes in protein structure, our studies will guide the development of precise molecular interventions into mechanical signaling processes governed by actin.