Novel Cilia Trafficking Mechanisms

描述（由申请人提供）：最近的证据表明纤毛异常在多囊肾病的病因学中很重要。我们的实验室已经确定了极性蛋白在纤毛发生中的重要作用，如顶端跨膜蛋白crumb3。在我们对crumb3的研究过程中，我们发现了一种新的纤毛和中心体运输机制，涉及Ran/输入蛋白系统。Ran是一种小的GTPase，它集中在细胞核中，并与进口蛋白结合调节蛋白质进入细胞核的进口。进口蛋白带着它们的货物进入细胞核，当它们与RanGTP结合时，货物被释放，导致整个核进口。然而，Ran也集中在细胞的其他点，包括中心体。我们发现，Importin β 1可以结合到Crumbs3的剪接形式，称为Crumbs3- clpi，并将这种碎屑同工异构体定位到中心体。我们还获得了另一种纤毛蛋白RP2的证据，该蛋白也通过进口蛋白系统被贩运到纤毛上。我们的假设指导了这一建议，即Ran/进口蛋白系统在纤毛和中心体贩运的调节中起主要作用。为了验证这一假设，我们提出了三个具体目标。第一个具体目标将是确定Crumb3-CLPI与Importins相互作用的分子基础，并确定改变这种相互作用的突变如何影响crumb3异构体的运输。第二个具体目标将检验其他进口蛋白，包括进口蛋白α在这一贩运机制和一般纤毛功能中的作用。在最后一个具体目标中，我们将操纵中心体的RanGTP水平，并确定这种操纵对中心体和纤毛贩运的影响。我们还将在中心体上鉴定Ran和Importin α结合蛋白。在这些研究的结论中，我们将对Ran/Importin系统在纤毛贩运网络中的作用有更深入的了解。这将对我们了解多囊肾病和其他纤毛病具有重要意义。公共卫生相关性：本提案的目的是研究控制蛋白质进入称为纤毛的细胞器的运动的机制。最近的研究表明，纤毛功能缺陷导致肾脏囊性疾病。肾脏囊性疾病，即正常肾脏被无功能的充满液体的囊肿所取代，是肾衰竭的主要原因。

DESCRIPTION (provided by applicant): Recent evidence implicates cilia abnormalities as important in the etiology of Polycystic Kidney Disease. Our laboratory has defined an important role for polarity proteins, such as the apical transmembrane protein Crumbs3, in ciliogenesis. In the course of our study on Crumbs3, we have identified a novel cilia and centrosomal trafficking mechanism that involves the Ran/Importin system. Ran is a small GTPase that is concentrated in the nucleus and regulates the import of proteins into the nucleus in combination with Importins. Importins travel into the nucleus carrying their cargo and when they bind to RanGTP, the cargo is released leading to overall nuclear import. However Ran is also concentrated at other points in the cell including the centrosome. We have found that Importin beta1 can bind to a splice form of Crumbs3, called Crumbs3-CLPI, and target this isoform of Crumbs to the centrosome. We also have obtained evidence that another cilia protein, called RP2, also uses the Importin system for trafficking to the cilia. Our hypothesis that guides this proposal is that the Ran/Importin system plays a major role in regulation of cilia and centrosomal trafficking. To test this hypothesis, we propose three specific aims. The first specific aim will be to identify the molecular basis for the Crumb3-CLPI interaction with Importins and determine how mutations that alter this interaction affect the trafficking of this isoform of Crumbs3. The second specific aim will test the role of other Importins, including Importin alphas, in this trafficking mechanism and in cilia function in general. In the last specific aim we will manipulate the levels of RanGTP at the centrosome and determine the effect of this manipulation on centrosome and cilia trafficking. We will also work to identify Ran and Importin alpha binding proteins at the centrosome. At the conclusion of these studies we will have a greater understanding of the role of the Ran/Importin system in cilia trafficking networks. This will have important implications for our understanding of Polycystic Kidney Disease and other ciliopathies. PUBLIC HEALTH RELEVANCE: The aim of this proposal is to study mechanisms that control the movements of proteins into a cell organelle called the cilia. Recent studies have suggested that defective function of the cilia leads to cystic diseases of the kidney. Cystic diseases of the kidney, where the normal kidney is replaced by non-functioning fluid filled cysts, are a major cause of kidney failure.