Biophysical Mechanisms of Force Transmission in Cytoskeletal Ensembles

细胞骨架中力传递的生物物理机制

• 批准号: 10672426
• 负责人: Dana Nicole Reinemann
• 金额: $ 33.45万
• 依托单位: UNIVERSITY OF MISSISSIPPI
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-01 至 2027-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10672426
• 关键词: Actins; Affect; Architecture; Binding Proteins; Biological Assay; Biophysical Process; Cell division; Cell model; Cell physiology; Cells; Communication; Complex; Crosslinker; Cytoskeleton; Disease; Disparate; Elements; Engineering; Environment; Equilibrium; Family; Filament; Foundations; Generations; Goals; Heart Diseases; Hybrids; In Vitro; Individual; Intracellular Transport; Kinesin; Lead; Life; Malignant Neoplasms; Mechanics; Methods; Microfilaments; Microtubules; Molecular; Molecular Motors; Motor; Myosin ATPase; Physiological; Pliability; Polymers; Productivity; Proteins; Quartz; Research; Screening procedure; Signal Transduction; Site; Structure; System; Techniques; Tubulin; Work; biophysical tools; cell motility; crosslink; diagnostic strategy; diagnostic tool; experimental study; innovation; interdisciplinary approach; novel; optic trap; optical traps; protein crosslink; protein function; reconstitution; single molecule; transmission process; treatment strategy

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.

项目摘要： 肌动蛋白丝和微管是细胞分裂，运动性和细胞内必不可少的细胞骨架聚合物 这些蛋白质中的运输和缺乏在癌症，心脏病和其他疾病中隐含。 为了促进跨越整个细胞的重要任务，这些细丝通过运动蛋白相互协调， 例如动力素和肌球蛋白，以及相关的结合蛋白。这种交流的分子基础 通过张力和压缩力以及这些信号如何通过细胞骨架传播不好 理解齿。传统上，研究这种细胞骨架现象的方法是 分子水平或整个细胞水平以及肌动蛋白和微管细胞骨架通常已评估 作为单独的体外系统。而单分子实验（使用光学诱捕）的方法具有 我们在解读单个电动机的机制方面是无价的，这是一种完全还原主义的方法 细丝和一种运动蛋白不能准确代表交联的结构层次结构 电机和蛋白质功能。另一方面，细胞水平研究发生在非常复杂的环境中。在 本研究计划，我们将通过工程小说，身体相关的规模和测定控制差距弥合差距 细胞骨架环境或纳米细胞，其中探测运动蛋白力学和细胞骨架串扰。 就像乐高积木一样，我们可以选择哪些细胞骨架元素纳入我们的纳米壁架构和 调整构件块相应地了解分子水平的变化如何传播到系统水平 力产生和网络刚度。使用这种创新的方法，我们的总体目标是提供 基本的分子理解电动机，交联，细丝和信号因素如何交流 在合奏中彼此之间以及利用光学诱捕的局部细胞骨架环境，石英晶体 耗散和光谱技术的微生物。具体来说，我们将研究肌球蛋白的工作方式 在肌动蛋白组件中的合奏中，哪些分子成分决定生产力的产生。 由肌动蛋白和微管细胞骨架的元素组成的混合纳米核素将被探测到 了解不同刚度的聚合物如何使蛋白质具有不同的柔韧性和运动蛋白 随着加工性和力的产生能力的变化影响细胞骨架串扰。因为电子钩是 微管蛋白的多样性部位和唯一影响不同动力素家族的运动性，我们将询问如何电子 钩结构会影响纳米球中的集合运动蛋白力的产生。拟议的研究将铺平道路 符合我们的长期目标，这不仅是了解维持生命的基本机制，而且最终 能够在体外重新建立细胞过程的物理现实模型，从而提供了巨大的 开发细胞骨架疾病的诊断和治疗策略的潜力。