Project Summary Mechanotransduction is the process by which cells sense and transduce extracellular mechanical stimuli into intracellular signaling and gene expression. Mechanotransduction is ubiquitous across diverse organisms and has significant influences on cell function and behavior, in parallel with chemical and genetic signal transductions. The mechanics of microenvironments mediate mechanotransduction through cell-microenvironment interactions and its mis-regulation is at the heart of various pathologies. A major knowledge gap in the field is how mechanical stimuli from microenvironments are transduced into cellular signaling and what the relationships are between microenvironmental mechanics, cell signaling, gene expression, and cell functions. We recently discovered that multiple electrically non-excitable epithelial cell lines can initiate and propagate spontaneous long-distance intercellular calcium waves (ICWs), when cells are cultured in 2D/3D soft microenvironments, but not in stiff ones. Because calcium regulates a broad range of essential cell functions, our findings uncover an unprecedented mechanotransduction nexus between microenvironmental mechanics and diverse cellular signals. We hypothesize that the cellular actomyosin contractility that is regulated by soft microenvironments (10s kPa) promotes the initiation and propagation of the long-distance ICWs. In this R35 grant, we propose a cross-disciplinary research program that systematically elucidates this novel mechanotransduction process and establishes a framework to bridge the knowledge gap. This will be accomplished by leveraging innovative approaches such as genetically encoded fluorescent calcium sensors, CRISPR imaging, high-throughput cell selection, and algorithms to pursue two interrelated research themes. The first theme is the identification of regulatory mechanisms that initiate the calcium waves by active modulations and live-cell imaging of contractility- associated proteins. The expected results will provide important molecular insights of the link between mechanotransduction and the ICWs. The second theme is the dissection of mechanisms through which calcium waves enhance cell migration and achieve biological functions by manipulating ICWs and investigating the full transcriptome profiles. The results will advance our understanding of the relationships between microenvironmental mechanics, cell signaling, gene expression, and cell functions. We envision that the fundamental principles uncovered in this project will apply broadly to various cell systems and physiological functions. The long-term objective of our research program is to establish a mechanistic foundation of mechanobiology and promote the creation of therapeutic strategies which leverage these principles. The success of this proposal will enable my group to embark in a long-term research direction to tackle a variety of critical and challenging questions regarding the interplay between mechanotr...