Force-sensitive macromolecular cytoskeletal assembly

力敏感大分子细胞骨架组装

• 批准号: 9242654
• 负责人: DOUGLAS N ROBINSON
• 金额: $ 26.14万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-07-01 至 2019-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9242654
• 关键词: Actinin; Actins; Adhesions; Affect; Behavior; Binding; Biological Models; Cell Proliferation; Cell division; Cell physiology; Cells; Computer Simulation; Contractile System; Coupled; Crosslinker; Cytokinesis; Cytoskeletal Proteins; Cytoskeleton; Data; Development; Dictyostelium; Elements; Environment; Enzymes; Feedback; Genetic; Goals; Growth; Homeostasis; Human; Hypertension; In Vitro; Kinetics; Lead; Learning; Location; Maintenance; Measurement; Measures; Mechanical Stress; Mechanics; Methods; Modeling; Molecular; Morphogenesis; Motion; Motor; Muscular Dystrophies; Myosin ATPase; Myosin Heavy Chains; Myosin Type II; Operating System; Organism; Output; Pathologic; Pattern; Phosphotransferases; Play; Process; Property; Protein Isoforms; Proteins; Published Comment; Publishing; Research; Role; Shapes; Signal Pathway; Signal Transduction; Site; Stimulus; Structure; Sum; System; Testing; Thick Filament; Tissues; alpha Actinin; base; cell motility; cellular imaging; crosslink; in vivo; laser tweezer; macromolecular assembly; mechanical force; mechanotransduction; myosin-heavy-chain kinase; optical traps; programs; protein crosslink; public health relevance; quantitative imaging; reconstitution; response; sensor; single molecule; stem cell division

DESCRIPTION (provided by applicant): Cells perform diverse processes, such as cell division, growth, motility, formation of adhesions, and tissue morphogenesis, under a wide range of mechanical environments. Central to these processes are mechanical forces, which may come from outside the cell or may be generated internally and which are integrated with signaling pathways to guide the cellular process. The cell's macromolecular cytoskeletal machinery, including the actin-based myosin II motors and actin crosslinking proteins, assemble, function and then disassemble in response to these forces and signaling pathways. This dynamic force-responsive assembly provides self-tuning of the machinery, leading to natural positive and negative feedback and further allows mechanical inputs to be converted (transduced) into signaling outputs. Our collective research spans from single molecule to whole cell level functions with an emphasis on how contractile systems operate to drive cytokinesis and motility and to provide mechanosensory functions for the cell. In this application, we propose studies of two major model protein systems that capture key aspects of force-sensitive macromolecular assembly. Substantial published and unpublished data, including quantitative cell imaging combined with mechanical and genetic perturbations and coupled with computational modeling motivate the questions in this proposal. In particular, we aim to determine the molecular basis for force-dependent assembly of the myosin II bipolar thick filament (BTF). In Aim 1, we will determine the compliance within the BTF and then determine how this compliance restricts the activity of the myosin heavy chain kinase, which tracks the assembled BTF and phosphorylates it to promote BTF disassembly. Quantitative imaging will test how these mechanisms allow for force-dependent BTF assembly in vivo. In Aim 2, we will examine different isoforms of the actin crosslinker alpha-actinin which, based on their in vitro measured kinetic properties, are predicted to display different degrees of mechanosensitive sub-cellular accumulation. We will compare the mechanosensitive accumulation of each alpha-actinin isoform (human ACTN1 and ACTN4 as well as amoeboid ACTN). Because computational modeling supports a catch-slip behavior and/or structural cooperativity as the physical basis of mechanosensitive accumulation, we will determine the force-dependent binding lifetimes for each isoform using single molecule methods. In sum, this research effort will decipher key principles of force-dependent cytoskeletal assembly, which guide cellular processes such as cell division, cell motility, stem cell divisions, and tissue morphogenesis and homeostasis.

描述（由申请人提供）：细胞在各种机械环境下执行不同的过程，例如细胞分裂、生长、运动、粘附形成和组织形态发生。这些过程的核心是机械力，机械力可能来自细胞外部，也可能在内部产生，并与信号通路整合以指导细胞过程。细胞的大分子细胞骨架机制，包括基于肌动蛋白的肌球蛋白 II 马达和肌动蛋白交联蛋白，根据这些力和信号传导途径进行组装、发挥作用，然后分解。这种动态力响应组件提供机械的自调节，从而产生自然的正反馈和负反馈，并进一步允许机械输入转换（转换）为信号输出。我们的集体研究涵盖从单分子到全细胞水平的功能，重点是收缩系统如何驱动胞质分裂和运动，并为细胞提供机械感觉功能。在此应用中，我们提出了对两种主要模型蛋白质系统的研究，这些模型蛋白质系统捕获了力敏感大分子组装的关键方面。大量已发表和未发表的数据，包括定量细胞成像与机械和遗传扰动相结合，并与计算模型相结合，激发了本提案中的问题。特别是，我们的目标是确定肌球蛋白 II 双极粗丝 (BTF) 力依赖性组装的分子基础。在目标 1 中，我们将确定 BTF 内的顺应性，然后确定这种顺应性如何限制肌球蛋白重链激酶的活性，肌球蛋白重链激酶跟踪组装的 BTF 并将其磷酸化以促进 BTF 解体。定量成像将测试这些机制如何实现体内力依赖性 BTF 组装。在目标 2 中，我们将检查肌动蛋白交联剂 α-肌动蛋白的不同亚型，根据其体外测量的动力学特性，预计它们会表现出不同程度的机械敏感亚细胞积累。我们将比较每种 α-肌动蛋白亚型（人类 ACTN1 和 ACTN4 以及变形虫 ACTN）的机械敏感性积累。因为计算模型支持捕获滑动行为和/或结构协同性作为机械敏感积累的物理基础，所以我们将使用单分子方法确定每个亚型的力依赖性结合寿命。总之，这项研究工作将破译力依赖性细胞骨架组装的关键原理，这些原理指导细胞分裂、细胞运动、干细胞分裂以及组织形态发生和稳态等细胞过程。