Biophysical Mechanisms of Force Transmission in Cytoskeletal Ensembles

项目总结： 肌动蛋白细丝和微管是细胞分裂、运动和细胞内必不可少的细胞骨架聚合物。 运输，而这些蛋白质的缺乏与癌症、心脏病和其他疾病有关。按顺序 为了促进跨越整个细胞的重要任务，这些细丝通过马达蛋白相互协调， 例如肌动蛋白和肌球蛋白，以及相关的结合蛋白。这种交流的分子基础 以及这些信号是如何通过细胞骨架传播的还不是很好。 明白了。研究这种细胞骨架现象的方法传统上要么是单一的 分子水平或全细胞水平，以及肌动蛋白和微管细胞骨架通常已被评估 在体外作为独立的系统。而单分子实验，通过光学捕获等方法，已经 在破译单个马达的机械方面是无价的，一种完全简化论的方法 细丝和一种马达蛋白不能准确地代表交联物的结构层次 马达和蛋白质起作用。另一方面，细胞水平的研究是在一个相当复杂的环境中进行的。在……里面 这项研究计划，我们将通过工程学上新颖的、与生理相关的，弥合规模和检测控制方面的差距 细胞骨架环境，或纳米细胞，在其中探索运动蛋白质力学和细胞骨架串扰。 就像乐高积木一样，我们可以选择将哪些细胞骨架元素整合到我们的纳米细胞的架构中，并 相应地调整构建块，以了解分子级别的变化如何传播到系统级别 力生成和网络刚度。使用这种创新的方法，我们的首要目标是提供 对马达、交联剂、细丝和信号因子如何进行通讯的基本分子理解 利用光学捕捉，石英晶体，对局部细胞骨架环境 具有耗散功能的微天平和光谱技术。具体地说，我们将研究肌球蛋白是如何工作的 在肌动蛋白组装和什么分子成分决定生产力产生的整体中。 由肌动蛋白和微管细胞骨架元素组成的杂交纳米细胞将被探索到 了解不同硬度的聚合物、不同柔韧性的交联蛋白和马达蛋白 不同的加工能力和力产生能力会影响细胞骨架的串扰。因为E-Hook是 微管蛋白的多样性位置并独特地影响不同的运动家族的运动性，我们将询问E- 钩子结构影响纳米细胞内力产生的系综运动。这项拟议的研究将为 我们的长期目标，不仅是为了了解维持生命的基本机制，而且最终 能够在体外重建生理上真实的细胞过程模型，提供了巨大的 制定细胞骨架疾病诊断和治疗策略的潜力。

Project Summary: Actin filaments and microtubules are cytoskeletal polymers essential for cell division, motility, and intracellular transport, and deficiencies in these proteins are implicated in cancer, heart disease, and other disorders. In order to facilitate vital tasks that span the entire cell, these filaments coordinate with each other through motor proteins, such as kinesin and myosin, and associated binding proteins. The molecular basis for this communication through tension and compression forces and how these signals propagate through the cytoskeleton is not well understood. Approaches to study such cytoskeletal phenomena have traditionally been either at the single molecule level or whole cell level, and the actin and microtubule cytoskeletons have generally been evaluated as separate systems in vitro. While single molecule experiments, with methods such as optical trapping, have been invaluable in deciphering the mechanics of individual motors, a completely reductionist approach with one filament and one motor protein does not accurately represent the structural hierarchy in which crosslinking motors and proteins function. On the other hand, cell level studies take place in a quite complex environment. In this research plan, we will bridge the gap in scale and assay control by engineering novel, physiologically relevant cytoskeletal environments, or nanocells, in which to probe motor protein mechanics and cytoskeletal crosstalk. Much like LEGOs, we can choose which cytoskeletal elements to incorporate in our nanocell’s architecture and tune the building blocks accordingly to understand how changes at the molecular level propagate to system level force generation and network stiffness. Using this innovative approach, our overarching goal is to provide a fundamental molecular understanding of how motors, crosslinkers, filaments, and signaling factors communicate with each other in ensembles and to the local cytoskeletal environment utilizing optical trapping, quartz crystal microbalance with dissipation, and spectroscopic techniques. Specifically, we will investigate how myosins work together in ensembles in actin assemblies and what molecular components dictate productive force generation. Hybrid nanocells that consist of elements from both the actin and microtubule cytoskeleton will be probed to understand how polymers of different stiffnesses, crosslinking proteins with different pliability, and motor proteins with varying processivity and force generation capability affect cytoskeletal crosstalk. As E-hooks are the diversity site of tubulin and uniquely influence motility in disparate kinesin families, we will interrogate how E- hook structure affects ensemble kinesin force generation in nanocells. The proposed research will pave the way to our long-term goal, which is not only to understand fundamental mechanisms that sustain life, but ultimately be able to reconstitute physiologically realistic models of cellular processes in vitro, providing an enormous potential for developing diagnostic and treatment strategies for cytoskeletal diseases.