Genetic Analysis of Centrioles and Cilia

中心粒和纤毛的遗传分析

• 批准号: 10414933
• 负责人: SUSAN K DUTCHER
• 金额: $ 39.38万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-15 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10414933
• 关键词: Address; Algae; Aneuploidy; Biochemistry; Cell Cycle; Cells; Centrioles; Chlamydomonas; Cilia; Cryo-electron tomography; Cystic kidney; Cytoplasm; Defect; Diabetes Mellitus; Diploidy; Docking; Dwarfism; Dynein ATPase; Exhibits; Genes; Genetic; Genetic Screening; Genomics; Goals; Haploidy; Infertility; Knowledge; Licensing; Malignant Neoplasms; Membrane; Membrane Proteins; Microcephaly; Microscopy; Microtubule Triplet; Mothers; Mutation; Obesity; Organelles; Organism; Partner in relationship; Pathway interactions; Patients; Persons; Phenotype; Play; Polydactyly; Primary Ciliary Dyskinesias; Production; Protein Isoforms; Proteins; Proteomics; RNA Splicing; Regulation; Retinal Degeneration; Role; Scheme; Signal Transduction; Site; Structure; Translating; Vesicle; arm; bone; ciliopathy; congenital heart disorder; extracellular vesicles; genetic analysis; human disease; mutant; novel; protein complex; protein function; protein transport; respiratory

Project Summary Cilia and centrioles are evolutionarily conserved organelles that require over 2000 proteins for their assembly and function. We use the unicellular alga, Chlamydomonas, to study these organelles. Chlamydomonas allows us to use both haploid and diploid mutant strains obtained from unbiased forward genetic screens together with outstanding genomics, biochemistry and microscopy to gain knowledge about these organelles. The conservation of these organelles allows us to translate our findings to other organisms where it is harder to do biochemistry and or genetic screens. The goal of my lab is to understand how these organelles are built and function. As we and others have shown, ciliopathies can exhibit a wide range of phenotypes in people that include retinal degeneration, polydactyly, cystic kidneys, diabetes, obesity, respiratory clearance defects, shortened bones, congenital heart disease and infertility. Defects in centrioles result in microcephaly and dwarfism. Duplication of centrioles once per cell cycle is key; many cancers have more than two centrioles and form transient multipolar spindle structures with more than two poles that can generate aneuploidy. I propose to address three questions using genetics, genomics, biochemistry, and microscopy. We want to know how inner dynein arms are correctly placed. How do they find the right address in the cilia? How are inner dynein arms assembled in the cytoplasm? Using genes that we identified in patients with primary ciliary dyskinesia, we are examining proteins that are needed to first assemble and then dock the large megadalton inner dynein arms at the right address so that a functional waveform is produced. We will also perform a large-scale mating scheme to ask if digenic inheritance can identify genes involved in dynein arm assembly and function. We will use proteomics and cryo-EM tomography to characterize these mutations. We want to explore new and novel pathways for regulating the precise duplication of centrioles; what redundancies are present to guarantee only two centrioles per cell? We think that cells have layers of control of centriole duplication. We will determine if splice site isoforms of centriolar proteins sequester other centriolar proteins to regulate duplication. We will use proximity mapping to ask if the mother centriole provides another level of regulation via a unique licensing site on specific triplet microtubules. We want to examine the role of the ciliary necklace, which is a unique membrane compartment at the transition zone; does it may play a role in membrane protein trafficking and/or in production of extracellular vesicles? Using mutants that have a greatly reduced ciliary necklace, we will ask if extracellular vesicle production is altered and what proteins make up the cargo of these vesicles.

项目摘要 纤毛和中心粒是进化上保守的细胞器，需要2000多种蛋白质才能 装配和功能。我们使用单细胞藻衣藻来研究这些细胞器。 衣藻允许我们使用单倍体和二倍体突变株，这些突变株是从无偏见的Forward获得的 基因筛查与杰出的基因组学、生物化学和显微技术一起获得关于 这些细胞器。这些细胞器的保护使我们能够将我们的发现转化为其他 较难进行生物化学和/或基因筛选的生物。我实验室的目标是 了解这些细胞器是如何构建和运作的。正如我们和其他人所表明的，纤毛病可以 在人群中表现出广泛的表型，包括视网膜变性，多指，囊性肾脏， 糖尿病、肥胖症、呼吸清除缺陷、骨骼缩短、先天性心脏病和不孕症。 中心粒缺陷会导致小头畸形和侏儒症。每个细胞周期复制一次中心粒是 许多癌症有两个以上的中心粒，并形成瞬时的多极纺锤体结构，其中 而不是两个能产生非整倍体的极点。我建议用遗传学来回答三个问题， 基因组学、生物化学和显微镜。我们想知道内部动力学臂是如何正确的 放置好了。他们如何在纤毛中找到正确的地址？内侧动力蛋白手臂是如何组装的 细胞质？利用我们在原发性睫状肌运动障碍患者中识别的基因，我们正在 检查首先组装然后对接大的百万吨的内部动力蛋白手臂所需的蛋白质 在正确的地址，从而产生功能波形。我们还将进行大规模的交配 询问双基因遗传是否可以识别与动力蛋白手臂组装和功能有关的基因的方案。我们 将使用蛋白质组学和冷冻EM断层扫描来表征这些突变。我们想要探索新的 和调控中心粒精确复制的新途径；什么是冗余 保证每个细胞只有两个中心粒？我们认为细胞有中心粒的控制层 复制。我们将确定中心粒蛋白的剪接位置异构体是否隔离其他中心粒 调节复制的蛋白质。我们将使用邻近映射来询问母中心粒是否提供 通过对特定的三联体微管的独特许可站点进行另一级别的监管。我们想要检查 睫状链的作用，它是过渡区的一个独特的膜室； 它是否可能在膜蛋白运输和/或胞外生产中发挥作用？ 水泡？使用纤毛项链大大减少的突变体，我们会问细胞外小泡 产物和构成这些小泡货物的蛋白质都发生了变化。