Mechanisms of dynein regulation

项目概要 微管相关运动蛋白将 ATP 水解产生的能量耦合到机械功，从而 然后用于为与细胞分裂、迁移和细胞运输相关的细胞内运动提供动力 货物。微管相关马达有两类：细胞质动力蛋白-1（dynein）和驱动蛋白。 这些马达在微管轨道上以相反的方向移动：动力蛋白朝微管负方向移动 端，通常靠近细胞核，驱动蛋白向微管正端移动。虽然有 四十多种驱动蛋白中，只有一种动力蛋白可以促进细胞质货物运输。动力蛋白高度 与调节其功能和活性的庞大蛋白质网络相互作用。然而，分子 有多少动力蛋白结合蛋白调节动力蛋白活性的机制在很大程度上是未知的。理解 如何调节动力蛋白是迫切需要的，因为动力蛋白或其调节因子的突变与许多因素相关 神经发育和神经退行性疾病。该提案旨在破译管理规则 使用跨学科方法结合蛋白质组学、生物化学、 结构生物学、单分子荧光显微镜以及固定细胞和活细胞成像。我们将重点关注 两个总体目标。第一个目标是确定一类新型动力蛋白调节蛋白的功能 我们的实验室已经发现了。这些新型调节剂与驱动蛋白、动力蛋白和已知的动力蛋白调节剂相关 并促进粘着斑的形成。通过确定这些新监管机构的职能，我们将 确定基于微管的运输在粘着斑组装和动力学中的作用。第二个进球 目的是确定动力蛋白调节蛋白 Nudel 发挥作用的分子机制。在这里，我们将 重点确定 Nudel 如何影响动力蛋白激活动力学以及动力蛋白结合和移动的能力 在微管轨道上。完成这一目标是迫切需要的，因为 Nudel 功能障碍与 小头畸形、癫痫和精神分裂症。总之，这项工作的结果将揭示新的机制 动力蛋白调节，对人类健康和疾病具有广泛影响。

PROJECT SUMMARY Microtubule-associated motor proteins couple the energy derived from ATP hydrolysis to mechanical work, which is then used to power intracellular movements associated with cell division, migration, and transport of cellular cargoes. There are two classes of microtubule-associated motors, cytoplasmic dynein-1 (dynein) and kinesins. These motors move in opposite directions on the microtubule track: dynein moves toward the microtubule minus- end, which is typically near the nucleus and kinesin moves toward the microtubule plus-end. While there are over forty kinesins, there is only one dynein that promotes cytoplasmic cargo trafficking. Dynein is highly regulated and interacts with a vast network of proteins that modulate its function and activity. However, molecular mechanisms of how many dynein binding proteins regulate dynein activity is largely unknown. Understanding how dynein is regulated is a pressing need as mutation in dynein or its regulators is associated with many neurodevelopmental and neurodegenerative diseases. This proposal seeks to decipher the rules that govern dynein-mediated cellular trafficking using an interdisciplinary approach combining proteomics, biochemistry, structural biology, single molecule fluorescence microscopy, and fixed and live cell imaging. We will focus on two overarching goals. The first goal is to determine the function of a new class of dynein regulatory proteins that our lab has discovered. These novel regulators associate with kinesin, dynein, and known dynein regulators and promote the formation of focal adhesions. By determining the function of these new regulators, we will determine the role of microtubule-based transport in focal adhesion assembly and dynamics. The second goal is to determine the molecular mechanism of how the dynein regulatory protein, Nudel functions. Here, we will focus on determining how Nudel effects the kinetics of dynein activation and dynein’s ability to bind to and move on the microtubule track. Completion of this goal is a pressing need, as Nudel dysfunction is associated with microcephaly, epilepsy, and schizophrenia. Together, the results of this work will reveal novel mechanisms of dynein regulation, which has broad implications in human health and disease.