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.

项目概要： 肌动蛋白丝和微管是细胞骨架聚合物，对于细胞分裂、运动和细胞内活动至关重要 这些蛋白质的缺乏与癌症、心脏病和其他疾病有关。 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， 例如驱动蛋白和肌球蛋白以及相关的结合蛋白这种通讯的分子基础。 通过张力和压力以及这些信号如何通过细胞骨架传播尚不清楚 传统上，研究此类细胞骨架现象的方法要么是单一的。 分子水平或全细胞水平，一般评估肌动蛋白和微管细胞骨架 作为体外的独立系统，而使用光学捕获等方法进行的单分子实验已经取得了进展。 在破译单个电机的机械原理方面具有无价的价值，这是一种完全简化的方法，只需一个 丝和一种运动蛋白并不能准确地代表交联的结构层次 另一方面，细胞水平的研究是在相当复杂的环境中进行的。 在这项研究计划中，我们将通过工程新颖的、生理相关的来弥补规模和测定控制方面的差距 细胞骨架环境或纳米细胞，可在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，我们可以选择将哪些细胞骨架元素纳入纳米细胞的架构中， 相应地调整构建模块，以了解分子水平的变化如何传播到系统水平 使用这种创新方法，我们的首要目标是提供一种 对马达、交联剂、细丝和信号因子如何沟通的基本分子理解 利用光学捕获、石英晶体在整体中相互结合以及与局部细胞骨架环境 具体来说，我们将研究肌球蛋白的工作原理。 肌动蛋白组装体中的整体以及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元素组成的混合纳米细胞将被探测 了解不同硬度的聚合物、不同柔韧性的交联蛋白质和运动蛋白质如何 不同的持续性和力产生能力会影响细胞骨架串扰，因为 E-hooks 是其中的关键。 微管蛋白的多样性位点并对不同驱动蛋白家族的运动产生独特的影响，我们将探究 E- 钩结构影响纳米细胞中整体驱动蛋白力的产生，这项研究将为这一研究铺平道路。 我们的长期目标不仅是了解维持生命的基本机制，而且最终 能够在体外重建细胞过程的生理学真实模型，提供巨大的 开发细胞骨架疾病诊断和治疗策略的潜力。