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

项目总结/摘要 这项更新申请的目的是阐明脂质膜包裹细胞货物的影响， 基于微管的主要运动蛋白驱动蛋白1的功能。基于运动蛋白的运动性 许多生理上重要的过程，包括从一个亚细胞中运送囊泡物质， 在神经元中的位置。这种细胞内运动的功能障碍与许多疾病有关， 包括神经退化虽然运动蛋白的性质已被广泛研究，无论是在体内 在体外，许多重要的问题仍然存在，包括货物本身的性质如何影响马达 功能这一建议的中心假设是，货物膜的流体性质和货物膜的流体性质。 膜中微区的形成是基于运动蛋白的运动性的关键调节剂。在细胞中，电机 蛋白质通常以小组的形式移动膜结合的、含有货物的囊泡。使用传统的 在缺乏封闭膜的体外货物中，货物的运动性显示与 移动货物的发动机数量货物膜在确定 在一个团队中有几个马达的建议已经提出很久了。首先，膜流动性可以使再分配和 微管附近的马达蛋白聚集。第二，膜微区可以作为优先的 使马达聚集的结合位点这两种机制都可以增加可用于 一起搬运货物至关重要的是，目前体外测定中的大多数货物仍然缺乏生理学上的特异性。 膜的因此，这些提出的机制的定量研究受到缺乏 合适的体外实验系统。 为了弥补这一重大差距，在目前的资助期间，研究小组结合了以下方面的进展： 膜生物物理学与建立单分子光学捕获，以表征运动的 离体膜包被物。利用这种新的体外实验系统，研究小组发现， 第一个直接联系是流体膜的存在积极影响了主要的运动性， 基于微管的运动蛋白驱动蛋白。初步分析进一步表明， 运动性与移动货物的驱动蛋白数量的增加相关。总之，这项最新的工作奠定了 下一个资助期的基础，届时研究团队将直接测试中心假设， 货物膜流动性和微区形成是基于运动蛋白的运动的关键调节剂。 实现拟定目标将确立本提案中使用的体外系统为受控系统， 实验平台，用于询问运动蛋白的生理调节。拟议预算的结论 研究有可能揭示疾病的分子机制，包括 神经变性新的研究和新的治疗目标和战略，以减轻 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.