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

项目摘要/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 质膜的分泌，依赖于Kinesin-1(Kin1)和myosin Va(MyoVa)的协同作用 分子马达。这些双头分子马达通过连续的步进来携带它们共同的货物。 沿各自的细胞骨架轨迹有相当长的距离，即Kin1和Actin的微管(MT) MyoVa的细丝。为了成功运送货物，货物表面上的kin1和myoVa马达团队， 必须克服MTS的三维(3D)复杂网络带来的物理挑战 肌动蛋白细丝，构成细胞的细胞骨架，也是这些马达的高速公路。至 确定在不考虑物理环境的情况下如何实现细胞内货物运输和交付的效率 细胞复杂的细胞骨架公路带来的挑战，我们已经开发出一种近生理学的 Kin1和myoVa转运的体外模型系统由一个复杂的、但定义明确的3- 维度(3D)、MT和肌动蛋白细丝网络。将形成与生理相关的脂质结合脂质体 将Rab受体蛋白嵌入到脂质体膜中，以便分子马达可以连接到 与体内一样，Rab受体通过它们各自的接头蛋白进行调节。一旦形成，马达包衣脂质体 被引入到3D网络中，并且它们的传输轨迹使用最先进的单个 具有高时空分辨率的分子生物物理技术。与MTS结合的轨迹蛋白 (MAP7，Tau)和肌动蛋白细丝(原肌球蛋白)将被加入以决定脂质体的运输方向。 使用这个定义明确的模型系统要解决的关键问题是：1)如何将电机加载到 货物表面？2)货物表面上不从事运输的马达、被动搭便车者或 它们的货物系绳与异种轨道静电相互作用，以增强货物运输 主动参与的发动机？3)从MT到基于肌动蛋白的运输的货物交接是协调事件还是 拉锯战的结果？4)轨道结合蛋白是通过促进还是抑制运输来帮助分拣货物？ 特定的MT和肌动蛋白细丝？为了解释这些实验的结果，我们将在 结合脂质体上马达团队之间的机械相互作用的硅胶传输模型 浮出水面。我们认为在细胞骨架轨道，马达， 以及货物，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机械空间知识库 为理解复杂细胞结构中的分子马达运输奠定了基础。 细胞的环境，以及如何设计有效的机动运输系统来交付和保留 到达目的地的货物。

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.