MOLECULAR STUDIES OF PRIMARY CILIUM BIOGENESIS

描述(申请人提供)：初级纤毛，一个突出在细胞表面的“信号天线”，是传递Hedgehog和平面细胞极性信号所必需的，并在其表面集中许多信号受体。此外，在遗传性疾病Bardet-Biedl综合征(BBS)中，睫状体功能障碍会导致肥胖、视网膜变性和肾囊肿。然而，信号受体转运到睫状膜的机制尚不清楚。我们最近发现了一个稳定的由七种BBS蛋白组成的复合体，我们将其命名为BBSome，并基于其与Rab8的功能相互作用参与了囊泡向纤毛的运输。Rab8是一种具有成熟的运输功能的GTP酶，其操作直接影响纤毛的生长。在这里，我们提出一个假设，即BBSome将特定的跨膜蛋白分类到初级纤毛。在这个研究方案中，我们将通过以下目的剖析BBSome在囊泡转运到纤毛的背景下的功能：1-鉴定BBSome向纤毛运输的跨膜蛋白。已知的纤毛跨膜蛋白将被测试BBSome对纤毛运输的要求，我们将建立一个时间分辨的运输分析来确定BBSome从中选择其货物的供体间隔。2.剖析BBSome靶向纤毛的机制。我们将检测BBSome结合蛋白在介导BBSome向纤毛募集中的作用。3-描述BBSome和Rab8之间的功能相互作用。在初步研究中，我们发现BBSome与Rab的GDP/GTP交换因子Rabin8相互作用。在这里，我们将试图了解BBSome如何调节Rabin8的活性和定位，以使Rab8进入纤毛。总之，我们的BBSome功能模型对Bardet-Biedl综合征的病因学具有重要意义：在BBSome将特定的信号受体定位于纤毛的模型中，BBS的每个单独症状都是由于特定的纤毛信号通路中断造成的。

DESCRIPTION (provided by applicant): The primary cilium, a "signaling antenna" projecting at the surface of the cell, is required for the transduction of Hedgehog and planar cell polarity signals and concentrates many signaling receptors on its surface. Furthermore, ciliary dysfunction leads to obesity, retinal degeneration and kidney cysts in the inherited disorder Bardet-Biedl Syndrome (BBS). Yet, the mechanisms of signaling receptor trafficking to the ciliary membrane are not understood. We recently discovered a stable complex of seven BBS proteins, that we named the BBSome and have implicated in vesicular trafficking to the cilium based on its functional interaction with Rab8, a GTPase with a well-established trafficking function whose manipulations directly impact cilium growth. Here, we advance the hypothesis that the BBSome sorts specific transmembrane proteins to the primary cilium. In this research proposal, we will dissect BBSome function within the context of vesicular transport to the cilium through the following aims: 1- Identify the transmembrane proteins transported by the BBSome towards the cilium. Known ciliary transmembrane proteins will be tested for a BBSome requirement in their trafficking to cilia and we will establish a time-resolved trafficking assay to identify the donor compartment from which the BBSome selects its cargoes. 2- Dissect the mechanisms of BBSome targeting to cilia. We will assay the function of BBSome-binding proteins in mediating the recruitment of the BBSome to cilia. 3- Characterize the functional interplay between the BBSome and Rab8. In preliminary studies, we have found that the BBSome interacts with Rabin8, the GDP/GTP exchange factor for Rab. Here, we will seek to understand how the BBSome modulates the activity and localization of Rabin8 to enable Rab8 entry into the cilium. In conclusion, our model for BBSome function has significant implications for the etiology of Bardet-Biedl syndrome: in a model where the BBSome targets specific signaling receptors to the cilium, each individual symptom of BBS results from the disruption of a specific ciliary signaling pathway.