Microtubule regulation of actomyosin dynamics and force generation in T lymphocytes

由黏附和信号受体介导的细胞间相互作用是高度动态的，并受到 在界面上施加大量机械力的细胞骨架运动。细胞如何结合机械 而生化信号执行特定功能的机制还不是很清楚。免疫系统的细胞存在 研究力传递和机械传感的引人注目的背景，因为它们在结构上是动态的 是生化信息传递的场所。T细胞信号与细胞骨架密切相关，它是 显然，肌动蛋白细胞骨架施加在T细胞受体上的力被传递到生化信号 导致T细胞活化。然而，这些力被调节的分子机制以及如何 它们对T细胞功能的贡献仍不清楚。在这里，我们建议剖析 驻留在肌动蛋白和微管(MT)动力学交点的蛋白质，以促进我们对 T细胞中的力产生和机械传感。我们假设动态微管调节T细胞 细胞骨架和近端信号均通过1)调节片层中的肌动蛋白聚合动力学 以及板层结构的组装和2)调节RhoA的激活导致肌球蛋白收缩 和力量的产生。最终，我们假设MT/肌动蛋白相互作用有助于T细胞 调整它们的激活和效应器功能，以响应靶细胞的僵硬。我们的第一个目标将是 通过检测MT与肌动蛋白之间的特定相互作用来研究MT调节肌动蛋白动力学的机制 和肌动蛋白VIA+TIP蛋白。我们将结合光遗传技术和突变来探测特定的相互作用 MT和肌动蛋白之间调节T细胞激活。我们的第二个目标将是剖析 肌球蛋白的动态MTS驱动的收缩力量的产生。我们将结合光遗传控制RhoA 用定量成像和牵引力显微镜阐明激活和抑制的时空 T细胞活化过程中RhoA的活化特征。我们将使用新型传感器来检测全环基金-H1的活性，并 突变以确定其在MT/肌动蛋白偶联、力产生和T细胞信号转导中的作用。最后，我们将表演 用小鼠细胞进行功能研究，以检验肌动球蛋白动力学调节的假设 而收缩调节细胞毒性T淋巴细胞激活的机械协调及其在治疗中的作用 杀死癌细胞。我们提议的研究将阐明机械刺激和生化信号是如何 再加上在免疫反应中。此外，本提案中研究的具体路径与 一些免疫缺陷和淋巴瘤的进展，因此将有助于更好地理解 它们的功能障碍如何导致人类疾病，从而为免疫干预提供了新的靶点 心理治疗。

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 in immune therapy.