Mother Centriole Appendages Regulate the AMIS Compartment and Cilogenesis.

描述(申请人提供)：人体内几乎所有类型的细胞都表现出细胞的极性，并从细胞表面延伸出一个基于微管(MT)的毛状细胞器，称为纤毛。极化细胞使用初级纤毛作为一种多功能工具，在发育、形态发生和动态平衡过程中发挥组织的特定功能，解释了为什么纤毛/极性相关疾病(纤毛疾病)会影响许多器官系统。囊性疾病中的许多蛋白质定位于初级纤毛和/或其相关的基底体(又名。中心体)包括鞭毛内运输蛋白(IFTS)，与小GTP酶Rab11和Rab8相互作用的Bardet-Biedl综合征复合体，以及囊泡拴系复合体，即外囊。虽然特定纤毛病变突变的后果是在生物水平上描述的，但细胞过程并不清楚。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。母体中心粒附属蛋白中心素与Rab11效应器相互作用于外囊。外囊可定位于Rab11修饰的循环内小体(RE)，并有助于极化肾上皮细胞形成瞬时顶膜起始点(AMI)。此外，Rab11、中心粘附素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的形成和极性的建立同时发生。引人注目的是，我最近发现Rab11，即外囊，以及Rab11 GTP酶激活蛋白(GAP)Evi5与RES一起定位于母中心粒附件(Hehnly，Chen，Power，Liu，&Doxsey，2012)。在此基础上，我推测母中心粒附属物蛋白通过调节Rab11的活性在管腔形成过程中的极性形成和纤毛发生中是必需的。我们将使用结构照明显微镜和活细胞成像技术检查1)纤毛生成和腔生成是否同时发生，以及腔生成是否依赖于纤毛生成。此外，我们将使用EM断层扫描来检查AMI是否实际上是睫状囊泡样病变。2)我们将使用超分辨率显微镜技术(例如STORM)来研究母中心粒附属蛋白cenexin是否需要锚定中心素Rab11和中心体上的外囊来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的体外结合分析(Hehnly等人，2012)和超分辨率显微镜技术来检查母中心粒调节Rab11活性的能力是否影响MT的极性和动力学。总之，我们的初步结果提供了对中心体内体组织和功能的分子机制的意想不到的见解，未来的发现将对对中心体、膜运输、纤毛组织/功能等感兴趣的广泛受众具有重要意义。最重要的是，这一提议可能提供关于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.