PROJECT SUMMARY Recently, it has become apparent that mechanical cues in the cellular microenvironment drive cell migration, stem cell differentiation into distinct cell types and even how a surveilling T-cells is triggered by its correct antigen, solidifying tension-sensing as a key regulatory switch in cellular function. Not surprisingly, alteration of mechanical forces is an emerging factor in diseases like cancer, which makes intuitive sense given that diagnosis often involves detecting a lump that feels harder and stiffer than the surrounding tissue. Indeed, distinct and quantifiable “mechanical phenotypes” of normal and diseased cells/tissues have been measured. Underlying these cellular “mechanical phenotypes” characteristic of normal and diseased cellular microenvironments are mechanosensing proteins that convert sensed physical perturbations into biochemical signals in a process known as mechanotransduction. These signaling pathways are putative targets of emerging “mechano- therapeutic” strategies aimed to correct aberrant mechanical phenotypes. The overall vision of the Gordon lab is to innovate technology to identify the molecular players underlying disease-relevant mechanical-phenotypes, and dissect their tension-sensing mechanisms to cure disease. The greatest challenge to determining the molecular basis of force sensing is that the technology to measure picoNewton (pN) forces sensed by an individual protein in the context of the cell emerged only ten years ago, and is still under constant development. This has crippled identification of new mechanosensing proteins involved in a given cellular or disease process and also left a huge gap in testable hypotheses regarding how force alters the conformation of receptors to trigger a biological response. Our lab has established three major areas to tackle this problem that blend technology development and hypothesis driven questions. Program I. In combination with cellular imaging, we develop and use molecular tension sensors (MTS) to measure forces sensed by hypothesized mechanosensing proteins in the cellular context. We plan to combine MTS and CRISPR screens to identify mechanosensors involved in glioblastoma and T-cell migration. Program II. Second, we aim to test the hypothesis that proteolysis of receptors is a mechanism to convey mechanical stimuli. We will use structural biophysics to study newly identified Notch-like proteolytic switches and use CRISPR-tagging and mass spectrometry to study global receptor proteolysis in response to applied force. Program III. Finally, our lab has expanded into a third area- function and application of HUH-endonucleases as “HUH-tags” to covalently link proteins and DNA. We plan to engineer sequence specificity and RNA-binding of HUH-tags. We are poised to use HUH-tags to improve DNA- based MTS and to link mechanosensing-domains to DNA-nanostructures to coax proteins into mechanically activated conformations. The interleaving of new protein-DNA co...