Developing Approaches to Identify Mechanosensitive Protein Recruitment and Interactions

Abstract

Important physiological processes, including migration, morphogenesis, and differentiation, and pathophysiological processes, including cancer and fibrosis, have been increasingly tied to cell’s abilities to sense mechanical stimuli from the extracellular matrix (ECM) and either generate or respond to mechanical loads in turn. Mechanical stimuli from the ECM is integrated at focal adhesions (FAs), a subcellular structure consisting of hundreds of interacting proteins that mediate physical connections between the ECM and force-generating actin cytoskeleton. At the molecular level, underlying this integration process is mechanotransduction, where the mechanical deformation of load-bearing proteins alters protein function to regulate signaling. This process is thought to expose cryptic protein binding domains that lead to the downstream recruitment and formation of mechanosensitive protein complexes. However, an incomplete understanding of mechanotransduction, and the relevant molecular players involved, prevents a mechanistic understanding of all mechanosensitive processes. In turn, this has hindered advancements in the development of therapies to combat mechanosensitive diseases, as well as efforts to manipulate cell response through the design of bio-instructive scaffolds in tissue engineering and regenerative medicine. To address this issue, the central goal of this dissertation is to develop and utilize molecular-scale tools to probe the role of molecular-scale forces on protein function and elucidate relevant molecular players in mechanotransduction. To date, available techniques for studying the role of molecular-scale forces on protein function remain technically challenging and low throughout. Thus, we sought to develop novel imaging- and biochemical-based assays that were capable of probing protein response to molecular tension within cellular contexts where both spatiotemporal control of cellular force generation and signaling networks were maintained. More specifically, we developed two separate assays that work in concert to first, characterize the specificity of the protein’s molecular tension-sensitive recruitment to FAs, and then unbiasedly uncover all molecular tension-sensitive protein interactions. We developed an imaging-based assay, termed Fluorescence Tension Co-localization (FTC), that integrates immunofluorescence labeling, molecular tension sensors, and machine learning to determine the sensitivity, specificity, and context-dependence of molecular tension sensitive protein recruitment mechanisms. When we applied FTC to study the mechanical linker protein, vinculin, we found constitutive and context specific molecular tension-sensitive protein recruitment mechanisms that varied with adhesion maturation. More specifically, we found that in immature FAs, vinculin tension specifically recruits integrin-associated proteins while in mature FAs, vinculin tension specifically recruits actin-associated proteins. We also developed a separate biochemical based assay, that integrates proximity-dependent biotin labeling techniques with biophysical knowledge of key residues required for protein loading to determine the mechanosensitive binding interactions of key FA proteins. Using streptavidin pulldown assays to isolate the interacting proteins, we found that vinculin forms proximal protein interactions with an FA protein, migfilin, that has not been previously identified as a vinculin binding partner. In summary, this dissertation focuses on developing novel molecular-scale assays for studying mechanosensitive protein recruitment and interaction mechanisms. Using these tools, we identified multifaceted tension sensitive protein recruitment mechanisms associated with vinculin during adhesion maturation, as well as identified a novel proximal binding partner, migfilin. Overall, this establishes the importance of molecular loads across single proteins in regulating other protein activity. Widespread use of these developed assays will help elucidate a more mechanistic understanding of mechanotransduction through the identification and study of relevant molecular players.