Cargo Transport by Myosin Va and Kinesin-1 Molecular Motors: In Vitro Model Systems that Build Complexity in 3-Dimensions.

Myosin Va 和 Kinesin-1 分子马达的货物运输：构建 3 维复杂性的体外模型系统。

• 批准号: 10204620
• 负责人: David M Warshaw
• 金额: $ 43.57万
• 依托单位: UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-05-01 至 2026-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10204620
• 关键词: 3-Dimensional; Actins; Active Biological Transport; Adaptor Signaling Protein; Address; Binding Proteins; Biological Models; Cell membrane; Cell physiology; Cells; Complex; Cytoskeleton; Data; Destinations; Electrostatics; Environment; Event; Foundations; Hand; Human; In Vitro; Intracellular Transport; Kinesin; Life; Link; Lipid Binding; Liposomes; MYO5A gene; Mechanics; Membrane; Microfilaments; Microtubules; Modeling; Molecular; Molecular Motors; Mothers; Motor; Myosin ATPase; Nature; Physiological; Property; Research Personnel; Surface; System; Transport Process; Tropomyosin; Vesicle; War; base; biophysical techniques; design; experimental study; in silico; in vitro Model; in vivo; innovation; insulin granule; knowledge base; presynaptic; receptor; single molecule; tau Proteins; temporal measurement; vesicle transport; 3-Dimensional; Actins; Active Biological Transport; Adaptor Signaling Protein; Address; Binding Proteins; Biological Models; Cell membrane; Cell physiology; Cells; Complex; Cytoskeleton; Data; Destinations; Electrostatics; Environment; Event; Foundations; Hand; Human; In Vitro; Intracellular Transport; Kinesin; Life; Link; Lipid Binding; Liposomes; MYO5A gene; Mechanics; Membrane; Microfilaments; Microtubules; Modeling; Molecular; Molecular Motors; Mothers; Motor; Myosin ATPase; Nature; Physiological; Property; Research Personnel; Surface; System; Transport Process; Tropomyosin; Vesicle; War; base; biophysical techniques; design; experimental study; in silico; in vitro Model; in vivo; innovation; insulin granule; knowledge base; presynaptic; receptor; single molecule; tau Proteins; temporal measurement; vesicle transport

Project Summary/Abstract Intracellular cargo transport, such as lipid-bound insulin granules and presynaptic vesicles that are destined for secretion at the plasma membrane, rely on the concerted effort of kinesin-1 (kin1) and myosin Va (myoVa) molecular motors. These double-headed molecular motors carry their common cargo by stepping processively for considerable distances along their respective cytoskeletal tracks, i.e. microtubules (MTs) for kin1 and actin filaments for myoVa. To successfully deliver cargo, teams of kin1 and myoVa motors on the cargo surface, must overcome the physical challenges presented by the 3-dimensional (3D) complex network of MTs and actin filaments that comprise the cell’s cytoskeleton, which also serves as these motors’ highways. To determine how efficient intracellular cargo transport and delivery are accomplished despite the physical challenges presented by the cell’s complex cytoskeletal highway, we have developed a near-physiological in vitro model system of kin1 and myoVa transport that is composed of a complex, but well-defined, 3- dimensional (3D), MT and actin filament network. Physiologically-relevant lipid-bound liposomes will be formed having Rab receptor proteins embedded in the liposome membranes so that molecular motors can be linked to the Rab receptors through their respective adapter proteins as in vivo. Once formed, motor-coated liposomes are introduced into the 3D networks and their transport trajectories defined using state-of-the-art single molecule biophysical techniques with high spatial and temporal resolution. Track-binding proteins to MTs (MAP7, Tau) and actin filaments (tropomyosins) will be added to dictate the direction of liposome transport. Key questions to be addressed using this well-defined model system are: 1) How are motors loaded onto the cargo surface? 2) Are motors on the cargo surface that are not engaged in transport, passive hitchhikers or are they cargo tethers that electrostatically interact with the heterologous track to enhance cargo transport by the actively engaged motors? 3) Is cargo hand-off from MT- to actin-based transport a coordinated event or the result of a tug of war? 4) Do track-binding proteins help to sort cargo by enhancing or inhibiting transport on specific MT and actin filaments? To interpret the results of these experiments, we will develop a mechanistic in silico transport model that incorporates the mechanical interactions between motor teams on the liposome surface. We propose that a functional interplay exists between the properties of the cytoskeletal tracks, motors, and cargos, which determines how teams of molecular motors meet the cellular demands placed on them by 3D cytoskeletal highways. The data obtained will provide a rich, mechano-spatial knowledgebase for the field and serve as a foundation for understanding molecular motor transport in the complex cytoarchitectural environment of the cell and how efficient motor transport systems are designed for delivery and retention of cargo at its destination.

项目摘要/摘要 细胞内的货物运输，例如脂质结合的胰岛素颗粒和注定为 质膜的分泌，依靠jinesin-1（kin1）和肌球蛋白VA（MYOVA）的一致努力 分子电动机。这些双头分子电动机通过逐步阶梯踏上了共同的货物 为了考虑沿各自的细胞骨架轨道的考虑距离，即Kin1和肌动蛋白的微管（MTS） Myova的细丝。为了成功交付货物，Kin1和Myova Motors的货物货物， 必须克服3维（3D）MTS和 肌动蛋白丝包含细胞的细胞骨架，也用作这些电动机的高速公路。到 确定有效的细胞内货物运输和交付如何完成dospite 该细胞复杂的细胞骨架高速公路带来的挑战，我们在 KIN1和MYOVA转运的体外模型系统，由复杂但定义明确的3--组成 尺寸（3D），MT和肌动蛋白细丝网络。将形成与生理上相关的脂质结合脂质体 将Rab受体蛋白嵌入脂质体机理中，以便将分子电机链接到 Rab受体通过其各自的衔接蛋白作为体内。一旦形成，运动涂层的脂质体 被引入3D网络及其使用最新单曲定义的传输轨迹 具有高空间和临时分辨率的分子生物物理技术。轨道结合蛋白到MTS （MAP7，TAU）和肌动蛋白丝（tropomyosins）将添加以决定脂质体转运的方向。 使用此明确定义的模型系统要解决的关键问题是：1）如何将电动机加载到 货物表面？ 2）是货物表面上不从事运输，被动搭便车者或 他们载有与异源轨道静电相互作用的货物，以增强通过 积极参与电动机？ 3）是从MT-到基于Actin的运输事件的货物交接或 拖船的结果？ 4）做轨道结合蛋白通过增强或抑制运输的轨道结合蛋白有助于对货物进行分类 特定的MT和肌动蛋白丝？为了解释这些实验的结果，我们将在 在脂质体上合并电动机之间的机械相互作用的硅运输模型 表面。我们建议在细胞骨架轨道，电动机，，， 和Cargos，它决定了分子电动机团队如何满足通过 3D细胞骨架高速公路。获得的数据将为该领域提供丰富的机械空间知识库 并作为理解复合物细胞结构中分子运动传输的基础 电池的环境以及如何设计用于交付和保留的高效电机运输系统 货物在目的地。