Thursday, November 21, 2024
11/21/2024
Project Summary/Abstract Leukocyte migration out of the vasculature into peripheral tissue is crucial for their role in fighting pathogens, promoting tissue repair, and attacking solid tumors. This process is a key control point in the inflammatory response and relies heavily on signaling events downstream of leukocyte integrins and their endothelial ligands. Manipulating integrin signaling could serve as untapped avenue to control immune cell functions in inflammatory diseases and cancer, but hindering efforts on this front is a lack of mechanistic understanding of integrin signaling events. Importantly, integrins are known to be central players in mechanotransduction (converting mechanical information into a biochemical response), yet very little is known about how immune cells use integrins to sense and respond to the mechanical cues on the molecular level. This is surprising, since there is strong evidence that inflammation causes endothelial cells to change their mechanical properties, driving immune cell infiltration. Our preliminary data show that, indeed, T cells can respond to the mechanical properties of their substrate through the integrin LFA-1, kicking off cytoskeletal changes that drive T cell migration. Importantly, we found a signaling scaffold, named CasL, that is mechanically activated and required for T cell migration downstream of LFA-1. Surprisingly, T cells lacking CasL fail to form an actin-rich leading edge, but instead display numerous membrane blebs, suggesting CasL may control cytoskeletal responses and/or cortical integrity. Based on our preliminary data, we hypothesize that CasL is a crucial mechanosensitive signaling hub governing cytoskeletal responses during T cell migration. Specifically, we hypothesize CasL controls cytoskeletal organization by regulating the activity of one or more of the Rho family GTPases, thus coordinating proper cytoskeletal responses and optimum motility. In Aim 1, we will take a systematic approach to determine how CasL regulates cytoskeletal dynamics and cortex-membrane integrity, focusing on the Rho GTPases and their downstream effector proteins, and then test the hypothesis that CasL function is crucial to maintain normal cortex-membrane stiffness. In Aim 2, we will leverage our ability to introduce mutants into primary T cells lacking CasL to undertake a structure/function analysis of CasL in T cell migration. We will define how CasL phosphorylation, as well as how each individual domain, contributes to actin responses, migration, and membrane blebbing. Lastly, in Aim 3, we ask how loss of CasL affects T cell migration into inflamed tissue in vivo. Taken together, these studies will provide fundamental information on the mechanisms of T cell migration and mechanotransduction and lay the foundation for future work aimed and manipulating these pathways in inflammatory diseases and cancer.
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