Biophysical Mechanisms of Force Transmission in Cytoskeletal Ensembles

项目摘要： 肌动蛋白丝和微管是细胞分裂、运动和细胞内分泌所必需的细胞骨架聚合物。 这些蛋白质的缺乏与癌症、心脏病和其他疾病有关。为了 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， 如驱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 以及这些信号如何在细胞骨架中传播 明白研究这种细胞骨架现象的方法传统上要么是在单一的细胞骨架上， 分子水平或全细胞水平，以及肌动蛋白和微管细胞骨架一般已被评估 作为单独的体外系统。虽然单分子实验，如光学捕获， 在破译单个电机的力学方面是非常宝贵的，这是一种完全简化的方法， 丝和一个马达蛋白不能准确地代表交联的结构层次， 马达和蛋白质的功能。另一方面，细胞水平的研究发生在一个相当复杂的环境。在 在这项研究计划中，我们将通过工程新颖，生理相关， 细胞骨架环境或纳米细胞，在其中探测运动蛋白力学和细胞骨架串扰。 就像LEDGE一样，我们可以选择将哪些细胞骨架元素纳入纳米细胞的结构中， 相应地调整构建块，以了解分子级的变化如何传播到系统级 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一个 对马达、交联剂、细丝和信号因子如何交流的基本分子理解 利用光学捕获、石英晶体 微天平与耗散和光谱技术。具体来说，我们将研究肌球蛋白如何工作， 在肌动蛋白装配中的集合体以及哪些分子成分决定了生产力的产生。 由肌动蛋白和微管细胞骨架元素组成的混合纳米细胞将被探测到， 了解不同硬度的聚合物，具有不同柔韧性的交联蛋白质和马达蛋白质 具有不同的持续合成能力和力产生能力的细胞会影响细胞骨架串扰。由于E钩是 微管蛋白的多样性位点，并在不同的驱动蛋白家族中独特地影响运动，我们将询问E- 钩状结构影响纳米细胞中整体驱动力的产生。拟议中的研究将为 我们的长期目标不仅是了解维持生命的基本机制， 能够在体外重建细胞过程的生理现实模型，提供巨大的 开发细胞骨架疾病的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.