Coordination of molecular motor activity in intracellular transport and assembly of cytoskeletal architecture.

细胞内运输和细胞骨架结构组装中分子运动活动的协调。

• 批准号: 9382131
• 负责人: Richard James McKenney
• 金额: $ 37.6万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-30 至 2022-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9382131
• 关键词: ATP Hydrolysis; Actins; Architecture; Behavior; Biochemistry; Biological; Biological Assay; Cell physiology; Cells; Chemicals; Cilia; Complex; Disease; Dynein ATPase; Endocytic Vesicle; Environment; Eukaryota; Filament; Goals; Health; Homeostasis; Huntington Disease; Impairment; In Vitro; Individual; Intracellular Transport; Kinesin; Link; Malignant Neoplasms; Microtubules; Mitochondria; Mitotic spindle; Molecular; Molecular Machines; Molecular Motors; Motion; Motor; Motor Activity; Motor output; Pathologic; Process; Property; Proteins; Recruitment Activity; Regulation; Research; Structure; System; Testing; Translating; Work; cell motility; in vivo; insight; novel; rab GTP-Binding Proteins; reconstitution; self assembly; single molecule; spatiotemporal; tool

Title: Coordination of molecular motor activity in intracellular transport and assembly of cytoskeletal architecture. P.I. – Richard J. McKenney Research Summary Intracellular transport is essential for cellular homeostasis in eukaryotes. Much of this process is carried out by molecular motors that convert the chemical energy from ATP hydrolysis into motion along the actin and microtubule cytoskeletal networks. Decades of research has uncovered structural and molecular details that explain how many of these motors move along their filament tracks in isolation. In the cellular milieu, most of these motors act in concert with complex regulatory machinery that links them to their respective cargos, modulates their motile properties, and dictates spatiotemporal activity. How individual motor output is controlled by this machinery is currently not clear and difficult to dissect in the complex environment of the cell. In addition, many cargos are moved simultaneously by motors of opposite polarity, in a process called bidirectional transport. How individual motors are recruited to cargo, activated, and integrated with other classes of motors presents a large challenge to the field. Further, the activities of disparate motors are harnessed to build and maintain critical cytoskeletal structures such as the mitotic spindle, cilium, and cleavage furrow. How motor and regulatory activities are coordinated to drive the self-assembly of such structures is currently a significant barrier to understanding normal and diseased cellular physiology. This application seeks to develop novel assays and tools to study the complexity of motor recruitment and regulation, bidirectional transport of cargos, and the self-assembly of cytoskeletal structures driven by motors and associated molecules. Our approach to combine biochemistry and single- molecule analysis towards in vitro reconstitutions that test molecular function, and translate our findings into in vivo systems that test hypotheses generated by these reconstitutions, will open up fruitful long-term avenues of research. We propose to: 1) Reconstitute and study the recruitment, regulation, and motility of cytoplasmic dynein and kinesin motors bound to membranous cargo through the endogenous Rab GTPase machinery that is known to link these motors to endocytic vesicles and mitochondria in cells, and 2) Reconstitute and study functions of dynein and kinesin motors that drive the self-assembly of the mitotic spindle. These broad goals build and expand on our expertise and previous work in dissecting the regulatory mechanisms of the cytoplasmic dynein motor, and aim to provide powerful new tools useful towards dissecting complex motor function. Our work will illuminate basic molecular and cell biological principles that drive cellular homeostasis and provide insight into the pathological mechanisms that arise from molecular motor malfunction.

标题：细胞内转运和细胞骨架组装中分子马达活性的协调 架构 P.I. - 理查德·J·麦肯尼 研究综述 细胞内转运是真核生物细胞内环境稳定所必需的。这个过程的大部分是 由分子发动机将ATP水解产生的化学能转化为运动 沿着肌动蛋白和微管细胞骨架网络。几十年的研究揭示了 以及分子细节，解释了有多少马达沿着它们的细丝轨道运动， 隔离在细胞环境中，这些马达大多与复杂的调节机制协同作用 将它们与各自的货物联系起来，调节它们的运动特性， 时空活动目前还不清楚这种机器如何控制单个电机输出 在细胞的复杂环境中清晰且难以解剖。此外，许多货物 通过相反极性的马达同时移动，这个过程称为双向运输。如何 单个发动机被招募到货物上，激活，并与其他类别的发动机集成在一起 这对该领域提出了很大的挑战。此外，不同马达的活动被利用以 建立和维持关键的细胞骨架结构，如有丝分裂纺锤体、纤毛和卵裂 皱纹。运动和调节活动如何协调，以驱动这种自我组装 结构是目前理解正常和患病细胞生理学的重要障碍。 本申请旨在开发新的测定和工具来研究运动募集的复杂性 和调节，货物的双向运输，以及细胞骨架结构的自组装 由发动机和相关分子驱动。我们的方法是联合收割机生物化学和单一的- 体外重组的分子分析，测试分子功能，并将我们的 体内系统的发现，测试这些重建产生的假设，将打开 富有成效的长期研究途径。我们建议：1）重组和研究招聘， 细胞质动力蛋白和驱动蛋白马达与膜货物结合的调节和运动 通过已知将这些马达与内吞作用相联系的内源性Rab GT3机制， 2）动力蛋白和驱动蛋白的重组和功能研究 驱动有丝分裂纺锤体自组装的马达。这些广泛的目标建立并扩展了我们的 专业知识和以前的工作在解剖细胞质动力蛋白马达的调节机制， 并旨在提供对解剖复杂运动功能有用的强大的新工具。我们的工作 将阐明驱动细胞稳态的基本分子和细胞生物学原理， 提供对分子运动功能障碍引起的病理机制的深入了解。