Mother Centriole Appendages Regulate the AMIS Compartment and Cilogenesis.

描述（由申请人提供）：人体中几乎所有类型的细胞都表现出细胞极性，并从细胞表面延伸出一种基于微管（MT）的毛发状细胞器，称为纤毛。极化细胞利用初级纤毛作为一种多功能工具，在发育、形态发生和体内平衡过程中发挥组织特异性功能，这解释了为什么纤毛/极性相关疾病（纤毛病）可以影响许多器官系统。许多与囊性疾病相关的蛋白质都局限于初级纤毛和/或其相关的基底体（又称中心体），包括鞭毛内转运蛋白（IFTs）、与小gtp酶Rab11和Rab8相互作用的Bardet-Biedl综合征复合体、囊泡系结复合体、囊泡。虽然在机体水平上描述了特定纤毛病突变的后果，但细胞过程尚不清楚。由于许多已知的纤毛病突变存在于定位于中心体的蛋白质中，我将研究中心体在极性建立和纤毛发生中的同时作用。母中心粒附体蛋白，中心蛋白，与胞囊中的Rab11效应物相互作用。囊泡可定位于Rab11修饰的再循环内体（RE），并参与极化肾上皮细胞瞬时顶膜起始位点（AMIS）的形成。此外，Rab11、中心蛋白和囊泡都被报道参与初级纤毛的形成，这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Rab11、胞囊和Rab11 GTPase激活蛋白（GAP） Evi5与REs一起定位于母中心粒附着物（Hehnly, Chen, Powers, Liu, & Doxsey, 2012）。基于此，我假设母中心粒附属蛋白通过调节Rab11活性，在管腔形成过程中需要极性形成和纤毛发生。我们将使用结构照明显微镜和活细胞成像检查1)纤毛发生和流光发生是否同时发生，以及流光发生是否依赖于纤毛发生。此外，我们将通过EM断层扫描检查ami是否实际上是纤毛囊。2)我们将使用超分辨率显微镜技术（如STORM）检测母体中心粒附件蛋白cenexin是否需要锚定中心粒蛋白、Rab11和胞囊在中心体上调节管腔形成和纤毛发生。3)我们将使用我在实验室开发的一种新型体外结合试验（Hehnly et al., 2012）和超分辨率显微镜技术，研究母中心粒调节Rab11活性的能力是否会影响MT极性和动力学。总之，我们的初步结果为中心体内核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/功能等领域的广泛关注具有重要意义。最重要的是，这一建议可能提供描述所提出的分子相互作用的破坏如何导致纤毛病患者的疾病表型的见解。

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.