Regulation of Ciliogenesis and Ciliary-related signaling

纤毛发生和纤毛相关信号传导的调节

• 批准号: 9153893
• 负责人: Christopher Westlake
• 金额: $ 121.67万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9153893
• 关键词: Animal Husbandry; Aquaculture; Architecture; Basal cell carcinoma; Belief; Biological Models; Biology; CCR; Cancer Model; Cells; Cellular Membrane; Cellular biology; Centrioles; Cilia; Clustered Regularly Interspaced Short Palindromic Repeats; Collaborations; Color; Complex; Defect; Development; Disease; Disease Pathway; Distal; Electron Microscope; Electron Microscopy; Embryo; Equipment; Erinaceidae; Extramural Activities; Family; Fishes; Fluorescence Microscopy; Funding; Generations; Genes; Genetic; Housing; Human; Image; Investigation; Journals; Kinetics; Knock-in Mouse; Laboratories; Lasers; Lead; Life; Lighting; Link; Malignant Neoplasms; Mediating; Membrane; Membrane Protein Traffic; Methods; Microscope; Microscopy; Molecular; Mothers; Nature; Neurologic; Oral; Pathway interactions; Patients; Phosphorylation; Phototransduction; Process; Proteins; Publications; Publishing; Rana; Recruitment Activity; Regulation; Research; Research Personnel; Resolution; SNAP receptor; Signal Transduction; Signal Transduction Pathway; Sonic Hedgehog Pathway; Structure; System; Technology; Time; Transgenic Animals; Transmembrane Transport; Tumor Suppressor Proteins; United States National Institutes of Health; Universities; Vesicle; Work; Zebrafish; abstracting; appendage; base; ciliopathy; cilium biogenesis; falls; genetic approach; image processing; imaging system; insight; kinetosome; light microscopy; medulloblastoma; meetings; morphogens; novel; posters; protein function; receptor; smoothened signaling pathway; symposium; tool; trafficking

In FY15, my laboratory continued our investigations into molecular pathways important for primary cilium assembly and signaling. These studies involved the characterization of proteins important in regulating membrane trafficking, specifically proteins associated with the Rab family. Significant findings on ciliogenesis processes: We have made important new discovers into the cell signaling control of cilia assembly initiation via a previously uncharacterized preciliary Rab-dependent membrane trafficking pathway. This year we published our key findings in the journal Nature Cell Biology (Lu et al. 2015). In this work we demonstrated that 1) Rab8 is not required for pre-ciliary membrane trafficking steps leading to CV formation, but rather functions to grow this structure into the ciliary membrane at a time coincident with the assembly of the axoneme. 2) EHD1 and EHD3 have functional overlap in ciliogenesis in human cells and zebrafish embryos 3) using advanced imaging approaches (Correlative light and electron microscopy, CLEM, and super-resolution structured illumination microscopy, SIM) to demonstrate that EHD proteins have a unique localization to the ciliary pocket 4) using EM and SIM to show that EHD1/3 are needed for formation of the early ciliary vesicle (CV) structure from not previously described small distal appendage vesicles (DAV) 5) demonstrated that EHD proteins are need to reshape DAV to allow for SNARE mediated fusion to form the CV. In the process we identified a novel SNARE, SNAP29, required for ciliogenesis 6) showed that DAV to CV assembly is critical for mother centriole reorganization to become the basal body and for the recruitment of proteins needed to build the cilia, including Intraflagellar proteins and transition protein. We have also continued our work describing how cell signaling control of cilia assembly initiation via a previously uncharacterized preciliary Rab-dependent membrane trafficking pathway. Our major finding in FY15 have focused a Rab11-WDR44 effector switch dependent on phosphorylation. We anticipate submitting this work for publication by the end of FY15. Studies on regulation of Hedgehog ciliary signaling: We continue to investigates how these membrane trafficking pathways are involved in ciliary Hedgehog signaling regulation. We are using advanced imaging approaches including total internal fluorescence microscopy (TIRFM) to directly study Hedgehog regulator ciliary trafficking. Key equipment and methods: Key equipment and methods for our project include advanced microscope imaging systems and cell and zebrafish based genetics tools for characterizing protein functions. In FY11-12 we established live light microscopy systems to study ciliogenesis and ciliary signaling. The Marianas spinning disk confocal microscope acquired is unique at the NCI for its capacity to simultaneous image in two-color widefield, confocal or TIRF mode. Using this system we are able to image the processes of cilia assembly and disassembly, as well as to examine the transport of ciliary receptors important in signaling. This equipment/technology was also used for multiple collaborations with intramural and extramural researchers and lead to a recently published work in the Journal of Cell Biology (2013). We are upgrading the Marianas microscope to include a far red 640nm laser which will permit 4 color live imagine. In addition, we are collaborating with Dr. Jiji Chen (OML, Frederick National Labs) to develop STORM super resolution imaging to achieve spatial resolution of cilia structures at an even lower 20-50nm. We are also continuing to use Zebrafish is model system to investigate primary cilium function during development. Zebrafish encode 85% of the genes linked to ciliopathy in humans and is an established model for cancer studies. We have successful used genetic approaches (morpholinos) to investigate protein function in ciliogenesis. In FY15 we began developing CRISPR tools to generate genetic knock-in and knock-outs. In FY14 we received funding to establish a proper zebrafish facility. For this a aquatic tank system has been purchased and plans were finalized to construct an aquaculture facility in building to house the zebrafish and enable expansion of our fish capabilities to include generation of transgenic animals. CCR has since approved the renovation of a room in building 560. We anticipate this room will be ready in late 2015. This will greatly enhance our capabilities since we continue to perform our animal husbandry in numerous small domestic fish tanks. This facility will also house frogs from Dr. Ira Daar's laboratory. I have also been responsible for the purchase of a new 120kV electron microscope to be housed in a newly renovated room in building 560. We anticipate the arrival of this microscope in the fall of 2015 from FEI. We are collaborating with NIH researchers to carry out 3D electron microscopy studies. Meetings and Presentations: Abstracts on EHD-dependent regulation of ciliogenesis were accepted for oral presentation at the ASCB 2014 meeting (short talk, Dr. Christine Insinna presenter) and the FASEB Cilia meeting (talk, Dr. Chris Westlake presenter; Dr. Quanlong Lu poster presentation). An abstract on the Rab11-WDR44 effector switch were also presented at the ASCB 2014 meeting (e-poster, Dr. Vijay Walia). I gave invited talks on this work at the NCI-CCR Tenure Track Investigator Symposium, Department of Biology at Catholic University, and the ARVO 2015 meeting.

在2015财年，我的实验室继续研究对原代纤毛组装和信号传导重要的分子途径。这些研究涉及对调节膜运输的重要蛋白质的表征，特别是与RAB家族相关的蛋白质。关于纤毛生成过程的重要发现：我们已经通过先前未经特色的预科依赖性RAB依赖性膜运输途径使纤毛组装起始的细胞信号传导控制进行了重要的新发现。今年，我们在《自然细胞生物学》杂志上发表了关键发现（Lu等，2015）。在这项工作中，我们证明了1）固定前膜运输步骤并不需要Rab8，导致CV形成，而是在与Axoneme组装一致的时候起作用该结构到纤毛膜中。 2）EHD1和EHD3在人类细胞和斑马鱼胚胎中的纤毛生成中具有功能重叠3）使用先进的成像方法（相关光和电子显微镜，Clem和超分辨率结构化照明显微镜，SIM），以证明EHD蛋白具有eHD蛋白的唯一本地化，以表明EM em em em em em em em em em em em em em em em和sim sim sim先前未描述的小远端附属囊泡（DAV）5的纤毛囊泡（CV）结构表明，需要EHD蛋白来重塑DAV，以允许SNARE介导的融合形成CV。在此过程中，我们确定了纤毛生成所需的一种新颖的SNARE，SNAP29 6）表明，DAV至CV组装对于母亲Centriole重组至关重要，成为基础体体，以及募集纤毛所需的蛋白质（包括氟法内蛋白质和过渡蛋白）所需的蛋白质。我们还继续进行工作，描述了纤毛组装启动的细胞信号传导如何通过先前未经特章的预科依赖性膜运输途径来控制。我们在2015财年中的主要发现集中于RAB11 WDR44效应子开关，取决于磷酸化。我们预计在2015财年结束时将这项工作提交出版。刺猬睫状信号的调节研究：我们继续研究这些膜运输途径如何参与睫状刺猬信号调节。我们正在使用包括总内荧光显微镜（TIRFM）在内的先进成像方法来直接研究刺猬调节纤毛贩运。关键设备和方法：我们项目的关键设备和方法包括高级显微镜成像系统以及细胞和基于斑马鱼的遗传学工具，用于表征蛋白质功能。在FY11-12中，我们建立了活光学显微镜系统来研究纤毛发生和纤毛信号。获得的Marianas旋转磁盘共聚焦显微镜在NCI上是独一无二的，因为它可以在两色广场，共聚焦或TIRF模式下同时图像。使用该系统，我们能够对纤毛组装和拆卸的过程进行成像，并检查在信号传导中重要的睫状受体的运输。该设备/技术还用于与壁内和校外研究人员的多次合作，并导致了《细胞生物学杂志》（2013年）最近发表的工作。我们正在升级Marianas显微镜，包括一个远红色的640nm激光器，该激光器将允许4彩色现场想象。此外，我们正在与Jiji Chen博士（OML，Frederick National Labs）合作开发Storm Super分辨率成像，以在20-50nm较低的情况下实现纤毛结构的空间分辨率。我们还继续使用斑马鱼是模型系统来研究开发过程中的主要纤毛功能。斑马鱼编码与人类中纤毛病有关的85％的基因，是癌症研究的既定模型。我们成功地使用了遗传方法（形态学）来研究纤毛生成中的蛋白质功能。在2015财年，我们开始开发CRISPR工具来产生遗传敲击和淘汰赛。在2014财年，我们获得了建立适当的斑马鱼设施的资金。为此，已经购买了水上储罐系统，并完成了计划，以建造水产养殖设施，以建造斑马鱼，并使我们的鱼类能力扩展，以包括产生转基因动物。此后，CCR批准了在560号建筑物中的房间进行翻新。我们预计该房间将在2015年底准备就绪。这将极大地提高我们的能力，因为我们继续在许多小型家庭鱼缸中进行饲养的动物饲养。该设施还将容纳Ira Daar博士实验室的青蛙。我还负责购买新的120kV电子显微镜，该显微镜被安置在560号建筑物的新房间中。我们预计该显微镜将于2015年秋天从FEI到达。我们正在与NIH研究人员合作，进行3D电子显微镜研究。会议和演讲：在ASCB 2014会议上接受口头介绍的EHD依赖性调节摘要（简短讲话，Christine Insinna Erchener博士）和Faseb Cilia会议（Talk，Chris Westlake Erchenser博士； Chris Westlake主持人； ASCB 2014会议（电子寄养者Vijay Walia博士）也在RAB11 WDR44效应子开关上进行了摘要。我在NCI-CCR任期调查员研讨会，天主教大学生物学系和Arvo 2015会议上就这项工作进行了邀请演讲。