Force-sensitive macromolecular cytoskeletal assembly

描述（由申请人提供）：细胞在广泛的机械环境下进行不同的过程，如细胞分裂、生长、运动、粘连形成和组织形态发生。这些过程的核心是机械力，它可能来自细胞外部，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，包括肌动蛋白为基础的肌球蛋白II马达和肌动蛋白交联蛋白，组装，功能，然后分解，以响应这些力量和信号通路。这种动态力响应组件提供了机械的自调谐，导致自然的正反馈和负反馈，并进一步允许机械输入被转换（转换）成信号输出。我们的集体研究涵盖了从单个分子到整个细胞水平的功能，重点是收缩系统如何运作，以驱动胞质分裂和运动，并为细胞提供机械感觉功能。在这个应用中，我们提出了两个主要的模型蛋白质系统，捕捉力敏大分子组装的关键方面的研究。大量已发表和未发表的数据，包括定量细胞成像结合机械和遗传扰动，再加上计算建模激发了这个建议中的问题。特别是，我们的目标是确定力依赖性组装的肌球蛋白II双极粗丝（BTF）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并使其磷酸化以促进BTF分解。定量成像将测试这些机制如何允许体内力依赖性BTF组装。在目的2中，我们将研究肌动蛋白交联剂α-辅肌动蛋白的不同亚型，根据它们在体外测量的动力学性质，预测显示不同程度的机械敏感性亚细胞积累。我们将比较每种α-辅肌动蛋白亚型（人ACTN 1和ACTN 4以及变形虫ACTN）的机械敏感性蓄积。由于计算建模支持捕捉-滑动行为和/或结构协同性作为机械敏感性累积的物理基础，因此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则，指导细胞过程，如细胞分裂，细胞运动，干细胞分裂，组织形态发生和稳态。

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 Substantial published and unpublished data, including quantitative cell imaging combined with mechanical and genetic perturbations and coupled with computational modeling motivate the questions in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.