MOLECULAR STUDIES OF PRIMARY CILIUM BIOGENESIS

描述（由申请人提供）：初级纤毛是突出在细胞表面的“信号天线”，是Hedgehog和平面细胞极性信号转导所必需的，并且在其表面集中了许多信号受体。此外，纤毛功能障碍可导致遗传性疾病Bardet-Biedl综合征（BBS）的肥胖、视网膜变性和肾囊肿。然而，信号受体转运到纤毛膜的机制尚不清楚。我们最近发现了一个由7种BBS蛋白组成的稳定复合物，我们将其命名为BBSome，并基于其与Rab8的功能相互作用，涉及到纤毛的囊泡运输。Rab8是一种具有完善的运输功能的GTPase，其操作直接影响纤毛的生长。在这里，我们提出了一个假设，即BBSome将特定的跨膜蛋白分类到初级纤毛。在本研究计划中，我们将通过以下目的来剖析BBSome在囊泡运输到纤毛的背景下的功能：1-鉴定BBSome向纤毛运输的跨膜蛋白。我们将测试已知的纤毛跨膜蛋白在向纤毛运输过程中是否需要BBSome，我们将建立一个时间分辨的运输试验，以确定BBSome选择其货物的供体隔室。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向纤毛募集中的功能。表征BBSome和Rab8之间的功能相互作用。在初步研究中，我们发现BBSome与Rabin8相互作用，Rabin8是rabb的GDP/GTP交换因子。在这里，我们将试图了解BBSome如何调节Rabin8的活性和定位，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靶向特定纤毛信号受体的模型中，BBS的每个个体症状都是由于特定纤毛信号通路的破坏。

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.