In vivo Regulation of Cytoplasmic Dynein

描述（由申请人提供）：细胞质动力蛋白是一种多亚基复合物，是一种负端定向微管马达。细胞质动力蛋白与其附属复合物动力蛋白一起执行多种细胞功能，包括逆行囊泡运输和细胞器分布。调控胞质动力蛋白活性的分子机制尚不清楚。我们的长期目标是通过使用丝状真菌细粒曲霉作为遗传模型系统，了解胞内靶向和胞质动力蛋白的运动活性是如何在体内调节的。我们通过对中性芽孢杆菌核分布缺陷突变体（nud）的遗传分析，确定了在细胞质动力蛋白途径中起作用的多个基因。虽然一些nud基因编码细胞质动力蛋白和动力蛋白复合物的成分，但也发现了新的调节因子。例如，nudF基因与Lis1是同源的，Lis1是一种涉及神经元迁移的人类无脑畸形（平滑脑）疾病基因。我们最近发现GFP标记的细胞质动力蛋白、动力蛋白和NUDF都在体内微管的正端积累。KINA是一种与传统动力蛋白同源的A. nidulans，它是动力蛋白和动力蛋白微管正端定位所必需的，但不需要NUDF。有趣的是，在没有NUDF的情况下，动力蛋白和动力蛋白的正端积累增加。基于这些和其他结果，我们假设细胞质动力蛋白由KINA运输到微管正端，在那里它接收货物并被NUDF/LIS 1激活，从正端出发到负端。本应用程序提出了实验来测试这样的假设，并表征细胞质动力蛋白功能的更多调节因子。我们的第一个具体目标是通过使用活细胞成像和光漂白技术，以及通过开发一个体外系统来测试哪些蛋白质足以用于动力蛋白或动力蛋白的微管末端定位，研究kina依赖的细胞质动力蛋白微管末端定位的机制。我们的第二个目的是检查缺乏NUDF的突变体和动力蛋白正端定位缺陷的突变体的动力蛋白运动活性，以确定细胞质动力蛋白运动活性是否依赖于其正端定位和NUDF的存在。我们的第三个目标是创建动力蛋白复合物中几种蛋白质的零突变体，以研究它们在动力蛋白调控中的功能，并通过克隆额外的nud基因来鉴定新的动力蛋白调节因子。

DESCRIPTION (provided by applicant): Cytoplasmic dynein, a multi-subunit complex, is a minus-end-directed microtubule motor. With its accessory complex, dynactin, cytoplasmic dynein performs multiple cellular functions including retrograde vesicle transport and organelle distribution. The molecular mechanisms involved in regulating the activity of cytoplasmic dynein are not well understood. Our long-term goal is to understand how the intracellular targeting and motor activity of cytoplasmic dynein is regulated in vivo by using the filamentous fungus Aspergillus nidulans as a genetic model system. We have identified multiple genes that function in the cytoplasmic dynein pathway through the genetic analyses of A. nidulans mutants defective in nuclear distribution (nud). While some nud genes encode components of the cytoplasmic dynein and dynactin complexes, novel regulators have also been discovered. For example, the nudF gene is homologous to Lis1, a human lissencephaly (smooth brain) disease gene involved in neuronal migration. We have recently found that GFP labeled cytoplasmic dynein, dynactin, and NUDF, all accumulate at the plus ends of microtubules in vivo. KINA, an A. nidulans homolog of the conventional kinesin, is required for the microtubule plus-end localization of dynein and dynactin, but not NUDF. Interestingly, the plus-end accumulation of dynein and dynactin increases in the absence of NUDF. Based on these and other results, we hypothesize that cytoplasmic dynein is transported by KINA to the microtubule plus end where it receives its cargo and is activated by NUDF/LIS 1 to depart from the plus end for the minus end. This application proposes experiments to test such a hypothesis and to characterize more regulators of cytoplasmic dynein function. Our first specific aim is to study the mechanism of KINA-dependent microtubule plus-end localization of cytoplasmic dynein, by using living cell imaging and photobleaching techniques, as well as by developing an in vitro system to test which proteins are sufficient for dynein or dynactin's microtubule plus-end localization. Our second aim is to examine dynein motor activity in mutants that lack NUDF and mutants that are defective in plus-end dynein localization, to determine whether cytoplasmic dynein motor activity is dependent upon its plus-end localization and the presence of NUDF. Our third aim is to create null-mutants of several proteins in the dynein complex to study their functions in dynein regulation, and identify novel dynein regulators by cloning additional nud genes.