Mother Centriole Appendages Regulate the AMIS Compartment and Cilogenesis.

描述（由申请人提供）：人体中几乎所有细胞类型都表现出细胞极性，并从细胞表面延伸出一种基于微管（MT）的毛发样细胞器，称为纤毛。极化细胞使用初级纤毛作为在发育、形态发生和体内平衡过程中组织特异性功能的通用工具，解释了为什么纤毛/极性相关疾病（纤毛病）可以影响许多器官系统。许多涉及囊性疾病的蛋白质定位于初级纤毛和/或其相关的基体（a.k.a.中心体），包括鞭毛内转运蛋白（IFTs）、与小GTP酶Rab 11和Rab 8相互作用的Bardet-Biedl综合征复合物、以及囊泡栓系复合物、外囊。虽然特定纤毛病变突变的后果是在生物体水平上描述的，但细胞过程尚不清楚。由于许多已知的纤毛病突变位于中心体的蛋白质，我将研究中心体在极性建立和纤毛发生中的共同作用。母亲中心粒附属蛋白，中心蛋白，与Rab 11效应器的外囊相互作用。外囊可以定位于Rab 11修饰的再循环内体（RE），并有助于在极化的肾上皮细胞中形成瞬时顶膜起始位点（AMIS）。此外，Rab 11、中心蛋白和外囊都被报道参与了初级纤毛的形成，已知初级纤毛的形成与管腔形成和极性建立同时发生。引人注目的是，我最近发现Rab 11，外囊，Rab 11 GT3激活蛋白（GAP）Evi 5与RE一起定位于母亲中心粒附件沿着（Hehloun，Chen，Powers，Liu，& Doxsey，2012）。基于此，我推测，母亲中心粒附属蛋白所需的极性形成和纤毛在管腔形成通过调节Rab 11活性。我们将使用结构化照明显微镜和活细胞成像检查1）纤毛发生和管腔发生是否同时发生以及管腔发生是否依赖于纤毛发生。此外，我们将使用EM断层扫描检查AMIS是否实际上是睫状囊泡。2)我们将使用超分辨率显微镜技术（如STORM）来检查是否需要母亲中心粒附属蛋白cenexin锚中心粒，Rab 11，和外囊在中心体调节管腔形成和纤毛发生。3)我们将使用我在实验室中开发的新型体外结合测定来检查母中心粒调节Rab 11活性的能力是否影响MT极性和动力学（Hehlynn等人，2012）和超分辨率显微镜技术。总之，我们的初步结果为中心体的内体组织和功能的分子机制提供了意想不到的见解，未来的发现将对中心体，膜运输，纤毛组织/功能等感兴趣的广泛受众具有重要意义。这一建议可能提供一些见解，描述所提出的分子相互作用的破坏如何导致纤毛病的疾病表型。患者

DESCRIPTION (provided by applicant): Almost all cell types in the human body demonstrate cellular polarity and extend a microtubule (MT)-based hair-like organelle from the cell surface called the cilium. Polarized cells use the primary cilium as a versatile tool for tissue-specific functions during development, morphogenesis and homeostasis, explaining why cilia/polarity-related disorders (ciliopathies) can affect many organ systems. A number of proteins involved in cystic diseases localize to both the primary cilium and/or its associated basal body (a.k.a. the centrosome) including the intraflagellar transport proteins (IFTs), the Bardet-Biedl syndrome complex that interacts with the small GTPases Rab11 and Rab8, and the vesicle tethering complex, the exocyst. While the consequences of specific ciliopathy mutations are described on an organismal level, the cellular processes are not understood. Since many known ciliopathy mutations lie in proteins that localize to the centrosome, I will examine the concurrent role of th centrosome in polarity establishment and ciliogenesis. The mother centriole appendage protein, centriolin, interacts with the Rab11 effector the exocyst. The exocyst can localize to the Rab11 decorated recycling endosome (RE) and contributes to the formation of the transient apical membrane initiation site (AMIS) in polarized kidney epithelial cells. Furthermore, Rab11, centriolin, and the exocyst are all reported to be involved in primary cilia formation, which is known to occur simultaneously with lumen formation and polarity establishment. Strikingly, I recently found Rab11, the exocyst, and the Rab11 GTPase Activating Protein (GAP) Evi5 to localize to the mother centriole appendages along with REs (Hehnly, Chen, Powers, Liu, & Doxsey, 2012). Based on this, I hypothesize that the mother centriole appendage proteins are required for polarity formation and ciliogenesis during lumenogenesis by regulating Rab11 activity. We will examine 1) whether ciliogenesis and lumenogenesis occur simultaneously and whether lumenogenesis is dependent on ciliogenesis using structured illumination microscopy and live cell imaging. In addition, we will examine whether the AMIS is actually the ciliary vesicl by using EM tomography. 2) We will use super resolution microscopy techniques (e.g. STORM) to examine whether the mother centriole appendage protein cenexin is required to anchor centriolin, Rab11, and the exocyst at the centrosome to regulate lumen formation and ciliogenesis. 3) We will examine whether the mother centrioles ability to regulate Rab11 activity effects MT polarity and dynamics using a novel in vitro binding assay that I have developed in the lab(Hehnly et al., 2012) and super resolution microscopy techniques. In conclusion, our preliminary results provide unexpected insights into the molecular mechanism of endosome organization and function at the centrosome, and future findings will be of importance to a wide audience with interests in centrosomes, membrane trafficking, cilia organization/function, etc. Most importantly, this proposal may provide insights describing how disruption of the proposed molecular interactions may lead to disease phenotypes in ciliopathy patients.