Do Cargo Membrane Fluidity and Microdomain Formation Impact Motor Protein-Based Motility?

项目摘要/摘要 这种更新应用的目的是阐明包裹细胞货物的脂膜的影响。 主要基于微管的运动蛋白Kinesin-1的功能。基于运动蛋白的运动性是基础 许多生理上重要的过程，包括从一个亚细胞运送囊泡货物 神经元中的另一个位置。这种细胞内运动功能障碍与许多疾病有关， 包括神经退行性变。虽然马达蛋白的性质已经在体内得到了广泛的研究 在体外，许多重要的问题仍然存在，包括货物本身的特性如何影响马达 功能。这一建议的中心假设是货物膜的流体性质和 膜上微区的形成是运动蛋白运动的关键调节因素。在细胞中，马达 蛋白质经常以小团队的形式工作，移动结合在膜上的、含有货物的囊泡。使用传统输入 没有包膜的体外货物，货物的运动性被证明与 活跃移动货物的马达的数量。货膜在测定水合物中的重要性 长期以来，人们一直在提议团队中的马达数量。首先，膜的流动性可以使膜的再分布和 微管附近的马达蛋白聚集。第二，膜微域可以作为优先选择 聚集马达的结合部位。这两种机制都可以增加可用于 把货物作为一个团队搬运。最重要的是，目前的体外检测中的大多数货物仍然缺乏生理活性。 薄膜。因此，对这些拟议机制的定量调查因缺乏 适宜的体外实验系统。 为了缩小这一重大差距，在当前的资助期间，研究小组结合了以下方面的进展 膜生物物理学与已建立的单分子光学捕获来表征细胞的运动性 膜封闭的体外货物。使用这种新的体外实验系统，研究小组发现 第一个直接联系是液膜的存在对大动脉的运动有积极的影响 基于微管的运动蛋白激动素。初步分析进一步表明，货物数量的增加 运动性与移动货物的动蛋白数量的增加有关。总而言之，这项最近的工作奠定了 为下一个资助期奠定基础，届时研究团队将直接测试中心假设 货膜流动性和微区的形成是基于运动蛋白的运动的关键调节因素。 实现建议的目标将建立本提案中使用的体外系统作为受控系统 探索运动蛋白生理调控的实验平台。建议的调查结果 研究有可能阐明疾病的分子机制，包括 神经退行性变。新的研究和新的治疗目标和战略 神经病理学和促进细胞健康将从这里提出的研究中产生。

Project Summary/Abstract The goal of this renewal application is to elucidate the impact of the lipid membrane enclosing cellular cargos on the function of the major microtubule-based motor protein kinesin-1. Motor protein-based motility underlies many physiologically important processes, including the delivery of vesicular cargos from one subcellular location to another in neurons. Dysfunctions in this intracellular motility are implicated in many diseases, including neurodegeneration. While the properties of motor proteins have been extensively studied both in vivo and in vitro, many important questions remain, including how the properties of the cargo itself impact motor function. The central hypothesis of this proposal is that the fluid nature of the cargo membrane and the formation of microdomains in the membrane are key regulators of motor protein-based motility. In cells, motor proteins often work in small teams to move membrane-bound, cargo-containing vesicles. Using traditional in vitro cargos that lack an enclosing membrane, the motility of the cargo are shown to correlate positively with the number of motors actively moving that cargo. The importance of the cargo membrane in determining the number of motors in a team has long been proposed. First, membrane fluidity can enable the redistribution and clustering of motor proteins near the microtubule. Second, membrane microdomains can serve as preferential binding sites that cluster motors. Both mechanisms can increase the number of motors that are available to move the cargo as a team. Crucially, most cargos in current in vitro assays still lack the physiological membrane. Thus, quantitative investigations of these proposed mechanisms are limited by a lack of appropriate in vitro experimental systems. To close this major gap, during the current funding period, the research team combined advances in membrane biophysics with established single-molecule optical trapping to characterize the motility of membrane-enclosed cargos in vitro. Using this new in vitro experimental system, the research team uncovered the first direct link that the presence of a fluid membrane positively impacts the motility of the major microtubule-based motor protein kinesin. Preliminary analyses further indicate that the increase in cargo motility correlates with an increase in the number of kinesins moving the cargo. Together, this recent work lays the foundation for the next funding period, when the research team will directly test the central hypothesis that cargo-membrane fluidity and microdomain formation are key regulators of motor protein-based motility. Accomplishing the proposed Aims will establish the in vitro system used in this proposal as a controlled experimental platform for interrogating the physiological regulation of motor proteins. Findings of the proposed studies have the potential to shed light on the molecular mechanisms underlying diseases, including neurodegeneration. Both new investigations and novel therapeutic targets and strategies to mitigate neurological pathology and to promote cellular health will arise from the studies proposed here.