Microtubule regulation of actomyosin dynamics and force generation in T lymphocytes

T 淋巴细胞中肌动球蛋白动力学和力产生的微管调节

基本信息

批准号：
9889158
负责人：
Arpita Upadhyaya
金额：
$ 30.78万
依托单位：
UNIV OF MARYLAND, COLLEGE PARK
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-03-15 至 2024-02-29
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9889158
关键词：
Actins Actomyosin Adaptor Signaling Protein Adenomatous Polyposis Coli Protein Adhesions Adhesives Antigens Binding Biochemical Biological Biomechanics CD8B1 gene Cell Communication Cell physiology Cells Characteristics Clonal Expansion Coupled Coupling Cues Cytoskeleton Cytotoxic T-Lymphocytes Development Environment Family Filament Functional disorder Future Gel Generations Goals Guanine Guanine Nucleotide Exchange Factors Human Immune response Immune system Immunologic Deficiency Syndromes Immunotherapy In Vitro Infection Inflammatory Interleukin-12 Interleukin-2 Intervention Lead Link Lymphocyte Activation Lymphoma Maintenance Mechanics Mediating Microtubules Molecular Movement Mus Mutation Myosin ATPase Optics Pathway interactions Play Plus End of the Microtubule Process Proteins Receptor Signaling Regulation Rho-associated kinase Role Signal Pathway Signal Transduction Site Small Interfering RNA Stimulus Structure Surface System T-Cell Activation T-Cell Receptor T-Lymphocyte Techniques Testing Therapeutic Traction Force Microscopy Translating Work adaptive immune response base cancer cell cell killing cytokine cytotoxic CD8 T cells genetic regulatory protein human disease immunological synapse in vivo knock-down mechanical force mechanical properties mechanotransduction mutant neoplastic cell novel novel strategies optogenetics physical property polymerization quantitative imaging response sensor spatiotemporal transmission process tumor

项目摘要

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.

由粘附和信号传导受体介导的细胞间相互作用是高度动态的并且受到细胞骨架运动在界面处施加大量机械力。细胞如何结合机械执行特定功能的生化信号尚不清楚。存在免疫系统细胞为研究力传递和机械传感提供了令人信服的背景，因为它们在结构上是动态的是生化信息传递的场所。 T 细胞信号传导与细胞骨架密切相关，显然，肌动蛋白细胞骨架对 T 细胞受体施加的力被转化为生化信号传导导致T细胞活化。然而，调节这些力的分子机制以及如何调节它们对 T 细胞功能的贡献仍不清楚。在这里，我们建议剖析以下方面的相互作用和活动：位于肌动蛋白和微管 (MT) 动力学交叉点的蛋白质，以增进我们对 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传导均通过 1) 调节片状足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 激活导致肌球蛋白收缩性和力的产生。最终，我们假设 MT/肌动蛋白相互作用有助于 T 细胞的能力调整它们的激活和效应器功能以响应靶细胞的硬度。我们的首要目标是通过探究 MT 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通过+TIP蛋白。我们将结合光遗传学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 细胞活化。我们的第二个目标是剖析联系机制动态 MT 到肌球蛋白驱动的收缩力产生。我们将结合RhoA的光遗传学控制通过定量成像和牵引力显微镜的激活和抑制来阐明时空 T细胞激活过程中RhoA激活的特征。我们将使用新颖的传感器来检测 GEF-H1 的活性突变以确定其在 MT/肌动蛋白偶联、力产生和 T 细胞信号传导中的作用。最后我们将表演在功能环境中对小鼠细胞进行研究，以检验肌动球蛋白动力学调节的假设和收缩性调节细胞毒性 T 淋巴细胞激活的机械协调及其在杀死癌细胞。我们提出的研究将阐明机械刺激和生化信号如何发挥作用在免疫反应期间耦合。此外，该提案中研究的具体途径与一些免疫缺陷和淋巴瘤进展，因此将有助于更好地了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新的目标治疗。