Molecular Motor Cargo Transport: Multidisciplinary Training in In Vitro and In Silico Model Systems

项目摘要/摘要 这项拟议中的研究的目标是确定支配 肌球蛋白-Va和动蛋白-1分子马达组合在三维中的细胞内转运 (3D)肌动蛋白细丝和微管网络(MT)。在目标1中，体外生物物理分析将 用于测量包被肌球蛋白-Va组的液状脂质体的转运 和Kinesin-1马达在肌动蛋白细丝和MT重新构建的3D网络中。脂质体 3D细丝网络中的轨迹将与底层细丝位置相关 和取向，以确定脂质体的运动模式(定向、扩散或 固定)与本地灯丝网络体系结构有关。为了实现这一目标，培训 将在马达蛋白表达(Trybus Lab)、3D超分辨率显微镜、 复杂系统重构和肌动球蛋白生物学(沃肖实验室)。在目标2中，机械论 数学建模将被用来理解决定生物物理参数 脂质体在AIM中的体外运动性观察1.马达、货物和 在硅胶细胞骨架网络中，轨迹影响脂质体的轨迹与映射的轨迹相同 体外实验将突出这一建模努力。建模方面的新专业知识将通过一款迷你 “休假”在沃尔科特实验室(WPI)。AIM 3将在体外货物运输之间架起一座桥梁 研究目标1和细胞内发生的情况。因此，脂质体的团队转运 Kinesin-1和Myosin-Va马达将在暴露的细胞骨架网络中被表征 通透性培养的COS7细胞。通过在图案化的衬底上培养细胞，这些“设计师” 细胞采用所需的几何形状(例如圆形或正方形)，从而组织它们的肌动蛋白 相应的微丝和MT细胞骨架。拟议的实验代表着新的培训 组织培养新技术(Howe Lab)。在AIMS 1和2中生成的知识库将 检验以下假设：脂质体体外转运原理对 在体外沿细胞骨架网络运输。这一提议代表了一种协同效应， 多学科培训经验，学员将在其中获得和利用新的专业知识 蛋白质表达、新的生物物理技术、数学建模和细胞生物学 技巧。

PROJECT SUMMARY/ABSTRACT The goal of the proposed research is to define the molecular mechanisms that govern intracellular transport by teams of myosin-Va and kinesin-1 molecular motors in a 3-dimensional (3D) network of actin filaments and microtubules (MTs). In Aim 1, in vitro biophysical assays will be used to measure the transport of fluid-like liposome cargoes coated with teams of myosin-Va and kinesin-1 motors in a reconstituted 3D network of actin filaments and MTs. Liposome trajectories in the 3D filament network will be correlated with the underlying filament position and orientation to determine how the liposome’s modes of motion (directed, diffusive, or stationary) are related to the local filament network architecture. To achieve this goal, training will be acquired in motor protein expression (Trybus Lab), 3D super-resolution microscopy, complex system reconstitution, and actomyosin biology (Warshaw Lab). In Aim 2, mechanistic mathematical modeling will be utilized to understand the biophysical parameters that govern liposome motility observed in vitro in Aim 1. How properties of the motors, the cargo, and the tracks influence liposome trajectories in in silico cytoskeletal networks identical to those mapped in vitro will highlight this modeling effort. New expertise in modeling will be gained by a mini “sabbatical” in the Walcott Lab (WPI). Aim 3 will bridge the gap between in vitro cargo transport studies in Aim 1 and that which occurs within a cell. Therefore, liposome transport by teams of kinesin-1 and myosin-Va motors will be characterized in exposed cytoskeletal networks of permeabilized cultured, COS7 cells. By growing cells on patterned substrates, these “designer” cells adopt to the desired geometry (e.g. circular or square) and thus organize their actin filament and MT cytoskeleton accordingly. The proposed experiments represent new training in novel tissue culture techniques (Howe Lab). The knowledgebase generated in Aims 1 and 2 will test the hypothesis that principles governing liposome transport in vitro are operative for transport along cellular cytoskeletal networks ex vivo. This proposal represents a synergistic, multidisciplinary training experience in which the trainee will gain and utilize new expertise in protein expression, new biophysical techniques, mathematical modeling, and cell biological techniques.