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

细胞内转运和细胞骨架组装中分子马达活性的协调 建筑。 私家侦探-理查德·J·麦肯尼 研究总结 在真核生物中，细胞内转运是细胞内平衡所必需的。这一过程的大部分都是 由分子马达执行，将三磷酸腺苷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。几十年的研究发现了结构性的 以及解释这些马达中有多少沿着它们的丝状轨迹移动的分子细节 与世隔绝。在蜂窝环境中，大多数这些马达与复杂的调节机制协同工作。 这将它们与各自的货物联系起来，调节它们的运动属性，并指示 时空活动。这台机器是如何控制单个电机输出的，目前还不清楚 在复杂的细胞环境中清晰而难以剖析。此外，许多货物都是 在一种称为双向传输的过程中，由相反极性的电机同时运动。多么 单独的马达被招募到货物上，被激活，并与其他类别的马达集成 对这一领域提出了巨大的挑战。此外，不同电机的活动被利用来 建立和维护关键的细胞骨架结构，如有丝分裂纺锤体、纤毛和卵裂 犁沟。如何协调马达和监管活动，以推动这些 结构是目前理解正常和疾病细胞生理学的一个重要障碍。 这项应用旨在开发新的分析方法和工具来研究运动招募的复杂性 和调节、货物的双向运输和细胞骨架结构的自组装 由马达和相关分子驱动。我们将生物化学和单一- 对测试分子功能的体外重组进行分子分析，并翻译我们的 对体内系统测试这些重组产生的假说的发现将会打开 卓有成效的长期研究途径。我们建议：1)重新构建和研究招聘， 细胞质动力蛋白和运动蛋白马达与膜性货物结合的调节和运动 通过内源性Rab GTPase机制，已知将这些马达与内吞联系起来 细胞中的囊泡和线粒体；2)动力蛋白和动力蛋白功能的重建和研究 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 在剖析细胞质动力蛋白马达的调节机制方面的专业知识和先前的工作， 旨在为解剖复杂的运动功能提供强有力的新工具。我们的工作 将阐明推动细胞动态平衡的基本分子和细胞生物学原理 深入了解分子马达故障引起的病理机制。

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.