Unbiased Discovery and Functional Analysis of Mechanosensitive Protein Interactions

Abstract

Mechanical cues play fundamental roles in development and normal physiology, guiding processes such as morphogenesis, differentiation, tissue organization, and wound healing. When these mechanical cues become dysregulated, however, they can drive pathogenesis. For instance, atherosclerosis forms at sites of disturbed fluid flow, fibrosis is categorized by pathological stiffness, and metastatic phenotypes are promoted by stiff tumor environments. Underlying each of these processes are mechanosensitive signaling pathways. These pathways are responsible for transducing the mechanical forces into biochemical signals that the cell can respond to. At mechanosensitive adhesion complexes, such as focal adhesions, this mechanotransduction is thought to occur by applied load leading to a conformational change across a specific mechanosensitive protein. These conformational changes can then alter the protein-protein interactions formed and initiate biochemical signaling. These mechanosensitive protein interactions are, therefore, the critical step in mechanosensitive signaling pathways and their characterization is needed to accurately control and treat mechanosensitive processes and diseases.While conceptually straightforward, the identification of such force-sensitive protein interactions has proven more difficult. This is largely due to limitations in our current experimental techniques. Common methods either lack the needed molecular specificity or are low-throughput, candidate-driven approaches. Additionally, our progress may be hindered by an incomplete knowledge of the underlying regulatory mechanisms. For instance, it is unknown whether another regulatory mechanism works in tandem with cryptic binding or how the mechanosensitive protein interactions themselves feedback to impact molecular loading. This gap in knowledge may exacerbate the technical limitations we observe. The overall motivation of this dissertation, therefore, is to improve our understanding of mechanosensitive protein interactions and advance the experimental tools we use to investigate them. In this work, we used the linker protein vinculin as a model mechanosensitive protein, due to its ability to bear load in cellulo, its core function in focal adhesions, and its multitude of protein interactions. Using vinculin, we aimed to 1) investigate how molecular tension affects protein complex formation and 2) determine how protein interactions affect molecular tension.To investigate the effect of force on mechanosensitive protein interactions, we developed a novel technique that leveraged proximity-dependent biotinylation and mass spectrometry to compare interactions formed by vinculin when it can or cannot bear load. Using this technique, we observed vinculin-tension-enhanced, -repressed, and -insensitive proximal interactions. The tension-sensitive populations included previously unrecognized binding partners, and combinations of proteins whose opposing tension sensitivities represent potential biomechanical switches. Bioinformatic analyses illustrated that these force-sensitive interactions are involved in distinct cellular processes and may be regulated by a new class of mechanosensitive LIM domains. Furthermore, super-resolution imaging was used to reveal tension-dependent extension of vinculin that altered its spatial stratification. This stratification was mirrored in our tension-sensitive interactions, suggesting a novel regulatory mechanism termed tension-sensitive compartmentalization.To determine how protein interactions affect molecular tension, we used a combination of biochemical binding assays and FRET-based biosensor studies. Specifically, fluorescence polarization was used to both confirm residual binding of vinculin A50I and validate a true talin-binding null mutant, I12K-A50I. Vinculin molecular tension sensors harboring this mutation revealed talin-binding as an important regulator of both vinculin recruitment to, and tension distribution within, focal adhesions. However, vinculin-talin binding is not solely responsible for vinculin loading, suggesting a novel force transmission pathway within focal adhesions. Moreover, a vinculin conformation sensor harboring the point mutation revealed vinculin-talin binding as dispensable for vinculin activation.In summary, this dissertation elucidated key aspects of mechanosensitive protein interactions. We accomplished this by developing a novel technique for their unbiased discovery, that successfully identified vinculin-tension-sensitive interactions and helped reveal a new regulatory mechanism that may underpin mechanotransduction. Additionally, we used a combination of biochemical and imaging techniques to functionally analyze the vinculin-talin interaction, revealing key aspects of vinculin function that are regulated by talin-binding and illustrating a novel force transmission pathway within focal adhesions. Together, these studies provide critical insight into the interplay between vinculin tension and its mechanosensitive interactions. Overall, this work informs our understanding of mechanosensitive signaling pathways and will aid in our ability to control and treat mechanosensitive diseases.