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: 10486834
• 负责人: Christopher Westlake
• 金额: $ 123.46万
• 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/10486834
• 关键词: 3-Dimensional; AKT Signaling Pathway; Ablation; Address; Affect; Apical; Astrocytoma; Authorship; Binding; Bioinformatics; Brain; CCR; Cancer Patient; Cancer cell line; Cell Line; Cells; Cellular Membrane; Cellular Structures; Centrioles; Cilia; Clustered Regularly Interspaced Short Palindromic Repeats; Collaborations; Communicable Diseases; Communication; Complex; Defect; Development; Docking; Electron Microscopy; Embryo; Endocrine System Diseases; Epidermis; Erinaceidae; Event; Faculty; Fluorescence; Fluorescence Microscopy; Functional disorder; Genes; Genetic Diseases; Glioblastoma; Goals; Growth; Homeostasis; Human; Human Genetics; Human body; Hyperactivity; Investigation; Knock-in; Laboratories; Lead; Lighting; Link; Lung; Male Infertility; Malignant Neoplasms; Manuscripts; Membrane; Membrane Fusion; Methods; Microscope; Microscopy; Molecular; Molecular Biology; Mothers; Movement; National Heart, Lung, and Blood Institute; Nature; Normal Cell; Null Lymphocytes; PTEN gene; Paper; Pathway interactions; Platelet-Derived Growth Factor; Play; Process; Proteins; Protocols documentation; Publications; Publishing; RNA interference screen; Rana; Regulation; Reporting; Reproductive system; Research; Resolution; Role; SNAP receptor; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Small Interfering RNA; Structure; Testing; Tissues; Transforming Growth Factor beta; Up-Regulation; Vesicle; Work; Zebrafish; apical membrane; base; cancer cell; cell motility; cellular imaging; ciliopathy; cilium biogenesis; cilium motility; confocal imaging; fluorescence imaging; human disease; imaging approach; imaging study; in vivo; inhibitor/antagonist; interdisciplinary approach; kinetosome; knock-down; link protein; malignant endocrine gland neoplasm; membrane assembly; microscopic imaging; migration; nervous system disorder; rab GTP-Binding Proteins; recruit; restoration; sperm cell; trafficking; tumor; tumorigenesis

In FY21, my laboratory continued to investigate the molecular pathways important for primary cilium assembly and connections to human disease (Project 1 and 2). We also initiated work investigating the molecular mechanism of motile cilia assembly (Project 3). Project 1) Membrane trafficking regulation of primary ciliogenesis: Our previously published work (Westlake et al., 2011, PNAS; Lu et al., Nat Cell Biol. 2015; Insinna et al., Nature Communication. 2019; Walia et a., Dev Cell, 2019; Cuenca et al., JBC, 2019) has helped establish the importance of membrane trafficking in the initiation and progression of ciliogenesis. This work has lead us to conclude that the docking of small preciliary vesicles to the mother centriole is required to initiate ciliogenesis processes at the mother centriole and. to assemble a larger ciliary vesicle (CV), upstream of ciliary axoneme growth. Moreover, we have shown that the CV stage is associated with ciliopathy (Shimada et al., Cell Rep. 2017). A primary objective of the laboratory in FY21 was to understand the mechanism of CV membrane assembly (Project 1a) and requirements for SNARE membrane fusion regulators (Project 1b) a) Dr. Lu and Dr. Huijie Zhao have investigated CV formation using super resolution fluorescence microscopy and 3D electron microscopy (FIB-SEM). We pioneered the use of CLEM confocal imaging and FIB-SEM in studying ciliogenesis structures as described in our 2019 paper by Insinna et al. (Nature Commun). In our current work, we have extended this approach to include super resolution structured illumination microscopy (SIM) to investigate the relationship between membrane structure and ciliogenesis initiation and progression. We have tested the function of EHD1 and Rab8a/b in regulating CV formation and have discovered new intermediate membrane structures between the preciliary vesicles and the CV stage requiring these membrane trafficking regulators. We have also demonstrated that EHD1 and membrane structure is directly involved in MC uncapping. Finally, we have determined that the transition zone, a region at the base of the cilium critical for regulating primary cilium signaling trafficking, is associated with assembly of early ciliary membrane structures. We also initiated live cell SIM imaging studies for this project to investigate ciliogenesis structure/function studies using a Zeiss Elyra7 microscope at NHLBI. A manuscript is being prepared for submission in 2021. Our FIB-SEM work is being done in collaboration with Dr. Kedar Narayan at the Center for Molecular Microscopy, Frederick National Laboratory. Based in part on the positive results achieved using the NHLBI microscope an RRS request was submitted and approved by CCR to purchase an Elyra7 to be housed at NCI-Frederick. b) Dr. Lu is also continuing to investigate the role of membrane fusion regulators in ciliogenesis. Based on our previous and current work we hypothesized that SNARES play important roles in the assembly of the ciliary membrane. Membrane fusion requires four SNAREs forming a trans-complex between two membrane compartments. We previously established that the Qbc SNARE SNAP29, functions in ciliogenesis (Lu et al., 2015 Nat Cell Biol) and therefore, we expect that at least two additional SNAREs will be required for ciliary fusion events. From an RNAi screen of the 38 human SNAREs in RPE-1 cells using 4 siRNA per gene we have identified 6 SNAREs with ciliogenesis defects that we are characterizing using advanced microscopy imaging described above and in vivo using zebrafish embryos. Dr. Lu has been joined in this study by Dr. Ipsita Saha who will be investigating membrane trafficking networks associated with ciliogenesis. The Elyra7 microscope will be an important approach used in this work utilizing CRISPR knock-in cells lines generated with fluorescence tags inserted into different membrane trafficking regulators to enable examination of endogenous protein ciliogenic function. Project 2) Ciliogenesis initiation dysfunction in cancer Many ciliary signaling pathways are important drivers of cancer. Notably, there have been many reports describing ciliogenesis dysfunction in cancer cells. However, it is known why these tumors lack cilia and what the consequences of this dysfunction are on tumorigenesis. Interestingly, it has been reported that cilium assembly dysfunction is associated with CV-like stages in glioblastoma/astrocytoma lines. To investigate the molecular mechanism of ciliogenesis dysregulation in cancer cells we have carried out studies that extend from our recently published work describing PI3K/Akt signaling as a negative regulator of ciliogenesis initiation in normal cells (Walia et al., Dev Cell. 2019). Importantly, The PI3K-Akt signaling pathway is frequently upregulated in cancer. We reported that Akt directly negatively regulates Rab11-dependent preciliary trafficking by stabilizing binding between Rab11 and its effector, and Akt substrate, WDR44. Based on this work we hypothesized that PI3K/Akt signaling upregulation could cause ciliogenesis dysfunction in cancer cells. From our analysis of ciliation in 60 cancer cell lines, most from the NCI-60 panel, we determined that 50% of the cell lines had ciliogenesis dysfunction. Importantly, PI3K and Akt inhibitors could enhance ciliation in some of these cell lines. We next examined 9 cancer cells null for PTEN, a highly studied negative regulator of PI3K, and discovered that 7 of these cells lines failed to undergo uncapping of the mother centriole needed to initiate ciliogenesis. Remarkably, ciliogenesis initiation could be observed in all of the PTEN null cells following treatment with Akt inhibitors. Moreover, PTEN re-expression in the two lines we tested completely restored ciliogenesis. Currently, we are examining whether ablation of WDR44 by siRNA and CRISPR affects ciliogenesis in cancer cells with hyperactive PI3K/Akt signaling to determine if this protein could be a target for ciliation restoration in cancer cells. Project 3) Investigation of initiating processes in motile multiciliogenesis To investigate motile multiciliogenesis mechanisms we are also employing 3D FIB-SEM studies and our preliminary studies indicate that motile multiciliated cells (MCC) use a similar process as the primary cilium. Thus, we are investigating roles of membrane trafficking regulators in MCC ciliogenesis that have been linked to primary cilium assembly. In addition, Dr. Zhao has used bioinformatics approaches to identify potential uncharacterized multiciliogenesis factors. Using localization and knockdown studies he discovered that Ccdc108, a protein linked to sperm dysfunction and male infertility, has an evolutionarily conserved requirement in motile ciliation. Using frog embryos, Ccdc108 was found to be required for the migration and docking of basal bodies to the apical membrane in epidermis MCCs. His studies indicate that Ccdc108 and IFT-B complex components cooperate in centriole apical migration during multiciliogenesis. In FY21 a manuscript was submitted to EMBO Reports entitled "Ccdc108 Regulates Multiciliogenesis via Interaction with the Intraflagellar Transport Machinery" and is currently under revision. This work is a collaboration and co-corresponding authorship with Dr. Ira Daar (CCR, CDBL). FY21 Publications: A review article was published in Faculty Review (10:16, 2021) authored by Dr. Saurabh Shakya, and Dr. Christopher Westlake. Dr. Westlake and Dr. Quanlong Lu were co-authors on a publication in Developmental Cell (56:325-340, 2021). Dr. Lu and Dr. Westlake also have an article accepted for publication in Methods in Molecular Biology. Rab GTPases: Methods and Protocols entitled "CLEM characterization of Rab8 and associated membrane trafficking regulators at primary cilium". Current Research:

在21财年，我的实验室继续研究对原发性纤毛组装和与人类疾病的联系很重要的分子途径（项目1和2）。我们还开始研究纤毛组装的分子机制（项目3）。项目1）主要纤毛生成的膜贩运调节：我们先前发表的工作（Westlake等，2011，PNAS； PNAS; Lu等，Nat CellBiol。2015; Insinna等，自然传播，2019; Walia等人，Walia等人，Dev Cell，2019; Cuenca et al。纤毛发生。这项工作使我们得出结论，需要将小的预先固定囊泡对接到母亲中心，以便在母亲中心启动纤毛生成过程。要组装一个较大的睫状囊泡（CV），睫状轴增长的上游。此外，我们已经证明了CV阶段与纤毛病有关（Shimada等，CellRep。2017）。实验室在FY21中的主要目的是了解CV膜组件（项目1A）的机理（项目1A）以及对SNARE膜融合调节剂的要求（项目1B）A）a）Lu博士和Huijie Zhao博士研究了使用超级分辨率荧光显微镜和3D电子显微镜（Fib-Sem）的CV形成。我们开创了Clem共聚焦成像和FIB-SEM在研究纤毛发生结构中的使用，如我们的2019年论文所述。 （自然社区）。在我们目前的工作中，我们扩展了这种方法，以包括超级分辨率结构化照明显微镜（SIM），以研究膜结构与纤毛发生的起始和进展之间的关系。我们已经测试了EHD1和RAB8A/B在调节CV形成中的功能，并发现了前提囊泡和需要这些膜运输调节剂的CV阶段之间的新的中间膜结构。我们还证明了EHD1和膜结构直接参与MC解膜。最后，我们确定过渡区是纤毛基础的一个区域，对于调节原发性纤毛信号运输至关重要，与早期纤毛膜结构的组装有关。我们还为该项目启动了活细胞成像研究，以使用NHLBI的Zeiss Elyra7显微镜研究纤毛生成结构/功能研究。一项手稿正在为2021年的提交准备。我们的FIB-SEM工作正在与弗雷德里克国家实验室分子显微镜中心的Kedar Narayan博士合作完成。部分基于使用NHLBI显微镜取得的积极结果，CCR提交并批准了RRS请求，以购买在NCI-Frederick中容纳的Elyra7。 b）Lu博士还继续研究膜融合调节剂在纤毛发生中的作用。基于我们以前的工作和当前的工作，我们假设网罗在纤毛膜的组装中起着重要作用。膜融合需要四个小圈，在两个膜隔室之间形成跨复合物。我们先前确定QBC SNARE SNAP29在纤毛生成中起作用（Lu等，2015 Nat Cell Biol），因此，我们期望纤毛融合事件至少需要另外两个弹药。从每个基因的4个siRNA的RPE-1细胞中38个人类鼻的RNAi筛选中，我们已经鉴定出6个具有纤毛生成缺陷的网罗，我们使用上述高级显微镜成像和使用Zebrafish Embryos在体内进行了表征。 Ipsita Saha博士加入了Lu博士，他将研究与纤毛发生相关的膜贩运网络。 Elyra7显微镜将是利用CRISPR敲击细胞系列的重要方法，该方法是用插入不同膜运输调节剂插入荧光标签生成的，以便检查内源性蛋白质纤毛作用。项目2）癌症的纤毛发生功能障碍许多睫状信号通路是癌症的重要驱动因素。值得注意的是，有许多报道描述了癌细胞中纤毛发生功能障碍。但是，知道为什么这些肿瘤缺乏纤毛以及这种功能障碍对肿瘤发生的影响。有趣的是，据报道，纤毛组装功能障碍与胶质母细胞瘤/星形细胞瘤线中的CV样阶段有关。为了研究癌细胞中纤毛生成失调的分子机制，我们进行了研究，这些研究从最近发表的工作延伸，该研究描述了PI3K/AKT信号传导是正常细胞中纤毛生成起始的负调节剂（Walia等人，DevCell。2019）。重要的是，在癌症中，PI3K-AKT信号通路经常上调。我们报告说，AKT通过稳定RAB11及其效应子之间的结合以及Akt底物WDR44来直接对RAB11依赖性的预算运输进行负面调节。基于这项工作，我们假设PI3K/AKT信号传导上调可能导致癌细胞中的纤毛发生功能障碍。根据我们对60个癌细胞系的纤毛分析，大部分来自NCI-60面板，我们确定50％的细胞系具有纤毛生成功能障碍。重要的是，PI3K和AKT抑制剂可以增强其中一些细胞系的纤维化。接下来，我们检查了9个癌细胞对PTEN（PTEN）无效，PTEN是PIN的高度调节剂，并发现这些细胞系中的7条未能经历启动纤毛生成所需的母中心菌的固定。值得注意的是，在用Akt抑制剂处理后，在所有PTEN无效细胞中都可以观察到纤毛发生的起始。此外，在两条线中，PTEN重新表达了完全恢复的纤毛生成。目前，我们正在研究siRNA和CRISPR对WDR44的消融是否会影响具有多动PI3K/AKT信号传导的癌细胞中的纤毛生成，以确定该蛋白是否可以成为癌细胞中纤毛恢复的靶标。项目3）调查动态多硅生成​​中的启动过程以研究运动多硅生成机制，我们还采用了3D FIB-SEM研究，我们的初步研究表明，机动多硅细胞（MCC）使用类似的过程与原代粉状。因此，我们正在研究与原发性纤毛组装有关的膜运输调节剂在MCC纤毛发生中的作用。此外，Zhao博士使用生物信息学方法来识别潜在的未表征的多重生成因子。他使用定位和敲低研究发现，与精子功能障碍和男性不育症相关的蛋白质CCDC108在通气纤维化方面具有进化保守的需求。使用青蛙胚胎，发现CCDC108是将基体迁移和对接到表皮MCC中的顶膜所必需的。他的研究表明，在多层生成过程中，CCDC108和IFT-B复合物成分在中心顶端迁移中合作。在21财年，一份手稿被提交给题为“ CCDC108通过与flagellar运输机械的相互作用”的EMBO报告，目前正在修订中。这项工作是与Ira Daar博士（CCR，CDBL）的合作和相应的作者身份。 FY21出版物：由Saurabh Shakya博士和Christopher Westlake博士撰写的教师评论（10:16，2021）发表了一篇评论文章。 Westlake博士和Quanlong Lu博士是发育细胞出版物的合着者（56：325-340，2021）。 Lu博士和Westlake博士还接受了一篇文章，以发表分子生物学方法。 RAB GTPASES：标题为“ Rab8和相关膜运输调节剂的CLEM表征”的方法和方案。当前的研究：