Force-sensitive Linker Proteins as Mediators of Cellular Mechanosensitivity - ABSTRACT: Virtually every fundamentally important cellular process, including cell migration, division, death, differentiation, and metabolism, has been found to be dependent on mechanical cues. Similarly, many disease states impacting human health, but lacking effective treatments, are at least partially caused or exacerbated by perturbed mechanical cues. Examples include cancer and fibrotic disease, which are associated with increased stiffness of tissues, and atherosclerosis, which is associated with perturbed hemodynamics. Despite the clear importance, the underlying molecular mechanisms through which cells sense and respond to mechanical cues, commonly referred to as mechanosensitivity, are poorly understood. In particular, the lack of knowledge regarding both the key mediators and the pertinent regulatory mechanisms highlights a substantial knowledge gap associated with the inability to translate the current understanding of mechanosensitivity into the detailed mechanistic knowledge required for manipulating cell behavior and developing novel therapeutics. These deficits are driven by a significant technological gap. The current challenge is that most existing techniques in cell biology assume a purely biochemical basis for biological function and either destroy or cannot distinguish these mechanical interactions. In particular, there are relatively few technologies to assess how the application of force affects protein functions in cellulo. Our work is guided by a new conceptualization of how the specificity of mechanosensitivity is determined by the mechanical state, defined by localization, ability to form interactions with other proteins, degree of mechanical loading, and amount of phosphorylation of the pertinent linker protein. The proposal will address three goals. First, leveraging our expertise in protein engineering, we will create and use a suite of biosensors to elucidate how phosphorylation affects other aspects of the mechanical state of linker proteins, rigidity sensing, and force transmission between the cell and the ECM (Goal 1). Second, building on previous work studying force-sensitive protein interactions, we will develop a novel approach for unbiasedly identifying the key mediators of mechanosensitivity for a given mechanical linker protein (Goal 2). Third, we will evaluate the role of these mediators in stiffness sensing and various forms of cell migration, potentially identifying proteins that drive mechanosensitive cellular processes and disease states (Goal 3). This R35 will give the lab the flexibility and power to advance the molecular understanding of mechanosensitivity and open new avenues for the study of mechanosensitive cellular processes. Findings from the proposed studies will also lay the foundation for advances in the diagnosis, treatment, and prevention of mechanosensitive diseases.
