Informational flow from mechanosensing to signaling for extracellular matrix stiffness sensing

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Project Summary Tissue stiffness changes during development, aging and diseases. Sensing this stiffness by cells determines differentiation, proliferation, migration, and survival, which are all important for development and tissue regeneration. Local tissue stiffening is a hallmark of cancer, sensing of which by cancer cells causes further tumor progression and metastasis. Understanding stiffness sensing mechanism is thus essential for designing appropriate treatment strategy against developmental disorders and cancer. A prominent sensor for the tissue stiffness is a molecular complex located between the cell and the environment, referred to as a focal adhesion. The first step in stiffness sensing involves transmission of increased level of a force across molecules in the focal adhesion against higher tissue stiffness. From the on-going funding, we discovered that this differential force transmission is independent of myosin contractility, the main force generator within cytoskeleton, and strongly depends on actin polymerization assisted by its nucleators by creating a backward flow against the cell membrane. The remaining question is how this differential force in response to the tissue stiffness can be translated to different signals that ultimately regulate cells’ developmental functions. The goal of the proposal is to understand whether a key focal adhesion-based signaling is caused by sensing activity of a key structural sensor protein and the force running through it. To achieve this goal, we have developed a set of experimental, microscopic, computational, and statistical frameworks that allow us to draw a conclusion about causality between the two time-dependent signals at individual focal adhesions captured from a live-cell imaging and computer vision. Specifically, we focus on focal adhesion kinase (FAK), which is a signaling hub for focal adhesion-based signaling but not well known for its coupling to the mechanical sensor, talin. Talin can be stretched under force and expose binding sites for other molecules like a vinculin, another mechanical linker protein. The overall objective of this renewal proposal is to use these multi-disciplinary pipelines to test a novel conceptual model of stiffness sensing in which the stiffness-dependent FAK activation is induced by talin’s mechanical sensing and the differential force in a manner that is dependent on focal adhesions’ dynamic state. We will determine 1) if FAK recruitment and activation are caused by talin recruitment and mechanosensitivity, 2) if FAK activation is caused by the mechanical force, 3) how FAK activation promotes RhoA signaling for stiffness sensing. An enhanced mechanistic understanding of these processes would increase our fundamental knowledge of how cells sense and respond to tissue mechanics. Thus, the proposed studies are relevant to the NIH's mission, as they will lead to new insights in physiology and pathophysiology including tissue development, regeneration and cancer progression.