Regulation of cytoplasmic dynein in vivo

项目摘要/摘要 我们研究一种叫做细胞质动力蛋白-1的微管马达(简写为“动力蛋白”)。在……里面 真核细胞，微管作为动力蛋白和运动蛋白等运动蛋白的轨迹 往前走。这些马达蛋白运送货物，包括细胞器、小泡、蛋白质和 MRNA，到不同的细胞位置以发挥作用。微管有两个不同的末端：加号 末端朝向细胞外围，负端靠近细胞中心。动力蛋白是一个负数结尾- 定向马达，它将货物从细胞外围输送到细胞中心。此外 生理性物质包括早期的内小体和其他细胞器/小泡，动力蛋白 病毒感染后将病毒颗粒向内输送。我们的实验室使用了一种丝状真菌 作为研究活细胞动力蛋白活性的遗传系统的尼杜拉曲霉 由其他蛋白质控制。我们和其他科学家发现动力蛋白通过 在微管+端的一种动蛋白，它通过适配器与早期内体相互作用 钩状复合体和动态肌动蛋白复合体等蛋白质。在活细胞中，这些适配器 蛋白质是激活动力蛋白向微管负端移动所必需的。然而， 这一过程还需要其他蛋白质，如Liis1(Lissephy-1)和veza(一种维他丁-1)。 如蛋白质)。Lis1促进动力蛋白的“开放”构象以促进其激活，但 否决的机制尚不清楚。我们的初步数据也表明，负面监管机构 需要防止dynein过早离开正数端而不携带其 货物，但负面监管机构仍有待确定。此外，目前还不清楚是什么因素 调节微管负端的货物释放。在未来五年，我们将结合 经典遗传学，活细胞成像和全基因组测序，以识别阳性和 动力蛋白活性的负调节因子。我们还将使用活细胞成像、蛋白质组学和 结构分析，以解决VEA的机制和新确定的调节器。这个 曲霉遗传系统最适合于这些研究。而体外实验则是 我们的基因系统是研究已知蛋白质的极佳工具，它有能力发现 未知的监管者。发现这些监管机构将为通向新的领域铺平道路 该领域的研究，这将刺激进一步的工作，涉及合作，以获得 对动力蛋白马达错综复杂的调节的机械洞察。 。

Project Summary/Abstract We study a microtubule motor called cytoplasmic dynein-1 (or “dynein” for simplicity). In eukaryotic cells, microtubules serve as tracks for motor proteins such as dynein and kinesins to move on. These motor proteins deliver cargoes, including organelles, vesicles, proteins, and mRNAs, to different cellular locations for function. A microtubule has two different ends: the plus end facing cell periphery and the minus end close to the cell center. Dynein is a minus-end- directed motor, and it transports cargoes from the cell periphery toward the cell center. Besides the physiological cargoes including early endosomes and other organelles/vesicles, dynein also transports virus particles inward after viral infection. Our lab uses a filamentous fungus called Aspergillus nidulans as a genetic system to study how dynein activities in live cells are controlled by other proteins. We and other scientists have found that dynein gets transported by a kinesin to the microtubule plus end where it interacts with early endosome via adapter proteins such as the HookA complex and the dynactin complex. In live cells, these adapter proteins are required for activating dynein to move toward the microtubule minus end. However, this process also requires other proteins such as LIS1 (Lissencephaly-1) and VezA (a vezatin- like protein). LIS1 promotes an “open” conformation of dynein to facilitate its activation, but the mechanism of VezA is unclear. Our preliminary data also suggest that negative regulators are needed to prevent dynein from moving away from the plus end prematurely without carrying its cargo, but the negative regulators remain to be identified. Moreover, it is unclear what factors regulate cargo release at the microtubule minus end. In the next five years, we will combine classical genetics, live cell imaging with whole genome sequencing to identify both positive and negative regulators of dynein activities. We will also use live cell imaging, proteomics and structural analysis to solve the mechanisms of VezA and newly identified regulators. The Aspergillus genetic system is best suited for these studies. While in vitro experiments are excellent for studying known proteins, our genetic system has the power for discovering unknown regulators. Discovering these regulators will pave the ways leading to new areas of research in the field, which will stimulate further work involving collaborations to gain mechanistic insights into the intricate regulation of the dynein motor. .