The Role of CP110 in Ciliogenesis

描述（由申请人提供）：我们的工作旨在了解调控有丝分裂纺锤体和纤毛组装的组装和组织的控制。这两个过程都是由中心体控制的，中心体是一个很大程度上仍然是谜的细胞器。我们特别感兴趣的是阐明控制中心粒和基底体功能之间切换的分子机制，因为来自基底体的纤毛的组装对于大量的发育决策至关重要。许多人类出生缺陷、发育异常和纤毛病是由纤毛发生缺陷或纤毛蛋白突变引起的。最近，我们已经确定和特点的中心体蛋白，CP 110，和干部的相关蛋白质，在中心体复制，有丝分裂纺锤体的组装，纤毛发生中发挥重要作用。最终，我们希望了解这种蛋白质网络如何调节初级纤毛组装和功能，以及这一过程如何在人类发育疾病中受到干扰。最近，我们发现了几种与CP 110和Cep 97相互作用的新蛋白，包括Talpid 3和WDR 35，并且我们已经开始表征每种蛋白在纤毛组装中的作用。Talpid 3和WDR 35的突变在不同的动物模型中引起发育缺陷，WDR 35的突变在人类中引起出生缺陷。然而，这些蛋白质的功能机制尚不清楚。我们的研究表明，这两种蛋白质都参与纤毛组装和功能。我们的建议是基于三个目标，使用生物化学，蛋白质组学，细胞生物学和RNAi的组合。首先，我们将研究Talpid 3和WDR 35的功能，并询问这些蛋白质如何与CP 110复合物相互作用以及它们如何指导纤毛组装。接下来，我们将探讨这些蛋白质的缺陷如何导致纤毛缺陷。我们将研究Talpid 3和WDR 35是否在囊泡和基底体/纤毛之间的蛋白质转运中发挥作用，以及它们是否调节细胞生长和静止。第三，我们将进行生化筛选，以鉴定与Talpid 3和WDR 35相互作用的蛋白质。除了解释有关CP 110相关蛋白的作用、蛋白质进出纤毛的运输和纤毛发生过程的基本生化细节外，我们还将开始了解人类纤毛病和先天缺陷的机制，这些机制是由纤毛疾病基因突变和与这些疾病相关的表型引起的。

DESCRIPTION (provided by applicant): Our work seeks to understand the controls that regulate assembly and organization of the mitotic spindle and cilia assembly. Both of these processes are governed by the centrosome, an organelle that remains largely enigmatic. We are particularly interested in elucidating the molecular mechanisms that govern the switch between centriolar and basal body function, as the assembly of cilia from basal bodies is vital for a large number of developmental decisions. Many human birth defects, developmental abnormalities, and ciliopathies result from defective ciliogenesis or mutations in ciliary proteins. Recently, we have identified and characterized a centrosomal protein, CP110, and a cadre of associated proteins that play important roles in centrosome duplication, assembly of the mitotic spindle, and ciliogenesis. Ultimately, we hope to understand how this network of proteins regulates primary cilia assembly and function and how this process is disturbed in human developmental diseases. Recently, we identified several novel proteins that interact with CP110 and Cep97, including Talpid3 and WDR35, and we have begun to characterize the role of each protein in the context of cilia assembly. Mutations in Talpid3 and WDR35 give rise to developmental defects in diverse animal models, and mutations in WDR35 give rise to birth defects in humans. However, the mechanisms underlying the function of these proteins are not known. Our studies indicate that both proteins are involved in cilia assembly and function. Our proposal is based on three aims that use a combination of biochemistry, proteomics, cell biology, and RNAi. First, we will investigate the function of Talpid3 and WDR35 and ask how these proteins interact with the CP110 complex and how they direct cilia assembly. Next, we will ask how defects in these proteins lead to ciliary defects. We will investigate whether Talpid3 and WDR35 play roles in protein transport between vesicles and basal bodies/cilia and whether they regulate cell growth and quiescence. Third, we will perform biochemical screens to identify proteins that interact with Talpid3 and WDR35. In addition to explaining basic biochemical details regarding the role of CP110-associated proteins, trafficking of proteins into and out of the cilium, and ciliogenesis-processes that remain poorly understood--we will begin to understand the mechanisms underlying human ciliopathies and birth defects resulting from mutations in ciliary disease genes and the phenotypes associated with these diseases.