In vivo Regulation of Cytoplasmic Dynein

描述(申请人提供)：细胞质动力蛋白是一种多亚单位复合体，是一种负端定向的微管马达。胞浆动力蛋白通过其附属复合体dynactin发挥多种细胞功能，包括逆行运输囊泡和细胞器分布。调控细胞质动力蛋白活性的分子机制还不是很清楚。我们的长期目标是通过丝状真菌Nidulans作为遗传模型系统，了解胞浆动力蛋白的胞内靶向和运动活性是如何在体内调节的。通过对核分布缺陷突变株(NUD)的遗传分析，我们发现了多个参与胞质动力蛋白途径的基因。虽然一些nud基因编码细胞质dynein和dynactin复合体的成分，但也发现了新的调节因子。例如，nudF基因与Lis1同源，Lis1是一种与神经元迁移有关的人类无脑(平滑脑)疾病基因。我们最近发现，GFP标记的胞浆动力蛋白、动力蛋白和NUDF都聚集在体内微管的正端。KINE是一种与传统的动蛋白同系物，它是微管+末端定位动力蛋白和动力蛋白所必需的，但不需要NUDF。有趣的是，在没有NUDF的情况下，dynein和dynactin的正端积累增加。根据这些和其他结果，我们假设细胞质动力蛋白由KENA运输到微管正端，在那里它接收货物，并被NUDF/LIS 1激活，从负端的正端离开。这项申请提出了实验来检验这一假设，并描述了细胞质动力蛋白功能的更多调节因素。我们的第一个特定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够用于动力蛋白或动力蛋白的微管+末端定位，来研究细胞质动力蛋白微管+端定位的机制。我们的第二个目标是检测缺乏NUDF的突变体和加端动力蛋白定位缺陷的突变体中的动力蛋白马达活性，以确定细胞质动力蛋白马达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dynein复合体中几种蛋白质的零突变，以研究它们在dynein调节中的功能，并通过克隆额外的nud基因来鉴定新的dynein调节基因。

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.