Force-sensitive macromolecular cytoskeletal assembly

描述(申请人提供)：细胞在广泛的机械环境中执行不同的过程，如细胞分裂、生长、运动、形成粘连和组织形态形成。这些过程的中心是机械力，它可能来自细胞外，也可能来自内部，并与引导细胞过程的信号通路整合在一起。细胞的大分子细胞骨架机制，包括以肌动蛋白为基础的肌球蛋白II马达和肌动蛋白交联蛋白，在这些力和信号通路的响应下组装、发挥功能，然后分解。这种动态力响应型组件提供机械的自我调节，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。我们的集体研究跨越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来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的研究，这两个系统捕捉到了力敏感的大分子组装的关键方面。大量已发表和未发表的数据，包括与机械和遗传扰动相结合的定量细胞成像，以及与计算建模相结合的数据，激发了这一提案中的问题。特别是，我们的目标是确定肌球蛋白II双极粗丝(BTF)依赖于力的组装的分子基础。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的分解。定量成像将测试这些机制如何允许在体内进行依赖于力的BTF组装。在目标2中，我们将研究肌动蛋白交联剂α-肌动蛋白的不同亚型，根据它们在体外测量的动力学特性，预测它们将显示不同程度的机械敏感性亚细胞积聚。我们将比较每种α-肌动蛋白亚型(人ACTN1和ACTN4以及阿米巴ACTN)的机械敏感性积累。由于计算模型支持接滑行为和/或结构协同性作为机械敏感积累的物理基础，我们将使用单分子方法确定每个异构体的力相关结合寿命。总之，这项研究工作将破译依赖于力的细胞骨架组装的关键原则，这些原则指导细胞过程，如细胞分裂、细胞运动、干细胞分裂、组织形态发生和动态平衡。

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 Substantial published and unpublished data, including quantitative cell imaging combined with mechanical and genetic perturbations and coupled with computational modeling motivate the questions in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