Cell-cell interactions, mediated by adhesion and signaling receptors, are highly dynamic and subject to cytoskeletal movements that impart substantial mechanical force at the interface. How cells combine mechanical and biochemical signals to carry out specific functions is not well understood. Cells of the immune system present a compelling context for studying force transmission and mechanosensing because they are structurally dynamic and are sites of biochemical information transfer. T cell signaling is closely linked to the cytoskeleton, and it is evident that forces applied by the actin cytoskeleton at the T cell receptor are transduced to biochemical signaling leading to T cell activation. However, the molecular mechanisms by which these forces are regulated and how they contribute to T cell function remain obscure. Here, we propose to dissect the interactions and activities of proteins that reside at the intersection of actin and microtubule (MT) dynamics to advance our understanding of force generation and mechanosensing in T cells. We hypothesize that dynamic microtubules modulate the T cell cytoskeleton and proximal signaling both by 1) regulating actin polymerization dynamics in the lamellipodium and the assembly of structures in the lamella and 2) regulating RhoA activation leading to myosin contractility and force generation. Ultimately, we hypothesize that MT/actin interactions contribute to the ability of T cells to adapt their activation and effector function in response to the stiffness of target cells. Our first goal will be to examine the mechanisms by which MT regulate actin dynamics by probing the specific interactions between MT and actin via +TIP proteins. We will combine optogenetic techniques with mutations to probe specific interactions between MT and actin that regulate T cell activation. Our second goal will be to dissect the mechanisms that link dynamic MTs to myosin driven contractile force generation. We will combine optogenetic control of RhoA activation and inhibition with quantitative imaging and traction force microscopy to elucidate the spatiotemporal characteristics of RhoA activation during T cell activation. We will use novel sensors for GEF-H1 activity and mutations to establish its role in MT/actin coupling, force generation and T cell signaling. Finally, we will perform studies with mouse cells in a functional context to test the hypothesis that regulation of actomyosin dynamics and contractility tunes the mechanical coordination of cytotoxic T lymphocyte activation and their efficacy in killing cancer cells. Our proposed studies will clarify how mechanical stimuli and biochemical signaling are coupled during the immune response. Furthermore, the specific pathways studied in this proposal are linked to a number of immunodeficiencies and lymphoma progression and thus will help lead to a better understanding of how their dysfunction can contribute to human disease, thus providing new targets for intervention...