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

项目概要/摘要 细胞内货物运输，如脂质结合的胰岛素颗粒和突触前囊泡，目的是 在质膜上的分泌，依赖于驱动蛋白-1（kin 1）和肌球蛋白Va（myoVa）的协同作用， 分子发动机。这些双头分子发动机通过步进式推进来携带它们共同的货物 沿着它们各自的细胞骨架轨迹，即用于kin 1和肌动蛋白的微管（MT 肌纤维为了成功运送货物，货物表面上的kin 1和myoVa发动机组， 必须克服由MT的3维（3D）复杂网络提出的物理挑战， 肌动蛋白丝构成了细胞的细胞骨架，也是这些马达的高速公路。到 确定如何有效的细胞内货物运输和交付完成，尽管物理 面对细胞复杂的细胞骨架高速公路所带来的挑战，我们已经开发出一种接近生理学的方法， kin 1和myoVa转运的体外模型系统，其由复杂但明确定义的3- 三维（3D）、MT和肌动蛋白丝网络。将形成生理学相关的脂质结合脂质体 具有包埋在脂质体膜中的Rab受体蛋白，使得分子马达可以连接到 Rab受体通过其各自的衔接蛋白，如在体内。一旦形成，马达包被的脂质体 被引入到3D网络中，并使用最先进的单个定义其传输轨迹 分子生物物理技术具有高的空间和时间分辨率。与MT的轨道结合蛋白 (MAP7，Tau）和肌动蛋白丝（原肌球蛋白）将被添加以指示脂质体转运的方向。 使用这个定义良好的模型系统要解决的关键问题是：1）电机如何加载到 货物表面？2)货物表面上的发动机是不是不从事运输，被动搭便车， 货物系链与异源轨道静电相互作用，以增强通过异源轨道的货物运输。 发动机积极？3)从MT到ACT运输的货物交接是一个协调事件，还是 是拔河的结果吗4)轨道结合蛋白是否通过增强或抑制运输来帮助货物分类？ 特异性MT和肌动蛋白丝？为了解释这些实验的结果，我们将开发一个机械的， 计算机传输模型，其结合了脂质体上运动组之间的机械相互作用 面我们认为，细胞骨架轨道，马达， 和货物，这决定了分子马达团队如何满足细胞对它们的需求， 3D细胞骨架高速公路。所获得的数据将为该领域提供丰富的力学空间知识库 并作为理解复杂细胞结构中分子马达运输的基础 细胞的环境以及如何设计有效的马达运输系统来递送和保留细胞内的蛋白质。 货物到达目的地。

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.