Mechanism of centrosome regulation from the Golgi

描述(申请人提供)：本项目的目标是了解哺乳动物细胞中调节邻近高尔基体中心体的控制机制。虽然高尔基体和中心体的接近是哺乳动物细胞独有的，在酵母、植物或苍蝇细胞中没有观察到，但它的功能意义一直难以捉摸。我们实验室的工作已经确定了一条中心体调控途径，在该途径中，高尔基体蛋白GM130导致一种名为Tuba的鸟嘌呤核苷酸交换因子激活高尔基体上的CDc42。这个项目试图确定高尔基体上的这些事件如何控制中心体的组织和功能，以及它们是否依赖于高尔基体-中心体的接近。 目的1将确定GM130是通过增加Tuba与CDc42的结合亲和力还是通过招募Cdc42与高尔基体结合来激活高尔基体中的CDc42。这一目标还将通过分析GM130是否控制高尔基体中已知受CDc42调控的额外细胞过程，如高尔基体到内质网的运输和高尔基体局部肌动蛋白的组装，来检验GM130是否是高尔基体中CDc42的主要调节因子。还将研究高尔基体相关的CdC42的潜在负调节因子。 目的2将研究GM130-CDC42通路如何在中心体上发挥作用。我们将研究CDC42效应器Par6？，以确定它是否是这一调控途径的下游组成部分，以及它是否通过与dynactin亚单位p150Glued的相互作用由发动机蛋白dynein运输到中心体。PCM-1是一种将更多的蛋白质招募到中心体的蛋白质，将被研究以确定它是否作用于Par6？通过GM130-CDC42途径介导中心体组织的调控。 目的3研究高尔基体-中心体亲近性的功能意义，检测是否可以将CDC42从高尔基体动员到中心体，从而调节中心体组织。高尔基体中心体周围的位置将被打乱，作为另一种测试高尔基体中心体邻近是否对GM130-CDC42途径调节中心体组织是必要的手段。

DESCRIPTION (provided by applicant): The objective of this project is to understand a control mechanism that regulates the centrosome from the adjacent Golgi apparatus in mammalian cells. While the proximity of the Golgi and centrosome is unique to mammalian cells, and not observed in yeast, plant or fly cells, its functional significance has been elusive. Work in our laboratory has identified a centrosome regulatory pathway in which a Golgi protein, GM130, causes a guanine nucleotide exchange factor called Tuba to activate Cdc42 at the Golgi. This project seeks to determine how these events at the Golgi control centrosome organization and function and whether they depend on Golgi-centrosome proximity. Aim 1 will determine whether GM130 activates Cdc42 at the Golgi by increasing the binding affinity of Tuba for Cdc42 or by recruiting Cdc42 to the Golgi. This aim will also examine if GM130 is the major regulator of Cdc42 at the Golgi by assaying whether GM130 controls additional cellular processes at the Golgi that are known to be regulated by Cdc42, such as Golgi to ER transport and local actin assembly at the Golgi. A potential negative regulator of Golgi-associated Cdc42 will also be studied. Aim 2 will examine how the GM130-Cdc42 pathway exerts its effect on the centrosome. A Cdc42 effector, Par6?, will be studied to determine if it is a downstream component of this regulatory pathway and if it is transported to the centrosome by the motor protein, dynein, through interactions with the dynactin subunit, p150Glued. PCM-1, a protein that recruits additional proteins to the centrosome, will be studied to determine if it acts downstream of Par6? to mediate the control of centrosome organization by the GM130-Cdc42 pathway. Aim 3 will study the functional significance of Golgi-centrosome proximity by examining if Cdc42 can be mobilized from the Golgi to the centrosome so that it can regulate centrosome organization. The pericentriolar location of the Golgi will be disrupted as another means of testing whether Golgi centrosome proximity is necessary for regulation of centrosome organization by the GM130-Cdc42 pathway.