Trafficking and Sorting Mechanisms of Golgi Vesicles to Cilia

高尔基体囊泡向纤毛的运输和分选机制

• 批准号: 10161782
• 负责人: KEN-ICHI TAKEMARU
• 金额: $ 34.79万
• 依托单位: STATE UNIVERSITY NEW YORK STONY BROOK
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-01 至 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10161782
• 关键词: Adult; Affinity Chromatography; Apical; Binding; Binding Proteins; Biological Process; Biology; Blindness; Cell Surface Extensions; Cell physiology; Cell surface; Centrioles; Centrosome; Characteristics; Chronic; Cilia; Coiled-Coil Domain; Complex; Cultured Cells; Cystic Lesion; Cystic kidney; Data; Defect; Development; Disease; Docking; Embryonic Development; Endosomes; Event; Family; Functional disorder; Genetic Diseases; Goals; Golgi Apparatus; Guanine Nucleotide Exchange Factors; Hair; Handedness; Homeostasis; Human body; Impairment; Infection; Intracellular Transport; Kidney; Knockout Mice; Label; Link; Lipids; Mass Spectrum Analysis; Mediating; Membrane; Mental disorders; Microtubules; Molecular; Monomeric GTP-Binding Proteins; Mutant Strains Mice; Obesity; Organ; Organism; Phenotype; Play; Polycystic Kidney Diseases; Polydactyly; Prevention strategy; Protein Biosynthesis; Proteins; Recycling; Reporting; Research; Retinal Degeneration; Role; Sorting - Cell Movement; Stimulus; Structure; Vesicle; Work; base; body system; cell type; ciliopathy; cilium biogenesis; experimental study; extracellular; golgin; human disease; insight; kinetosome; knock-down; lipid transport; mechanotransduction; membrane assembly; novel; postnatal development; protein transport; recruit; sensor; trafficking; trans-Golgi Network

PD/PI: Takemaru, Ken-Ichi 7. PROJECT SUMMARY Cilia are evolutionarily conserved microtubule-based structures that protrude from the apical cell surface to perform diverse biological functions. Primary cilia are present on most cell types in the human body and play essential roles in embryonic and postnatal development of various organ systems as a dynamic chemo- and mechanosensing center. Dysfunctional primary cilia have been linked to numerous genetic disorders, such as organ laterality defects, polydactyly, and polycystic kidney disease (PKD), collectively known as ciliopathies. Therefore, a better understanding of ciliogenesis is crucial for the development of comprehensive strategies for the prevention and treatment of ciliopathies. The polarized vesicle trafficking of proteins and lipids from the trans-Golgi network (TGN) and recycling endosomes plays important roles in ciliogenesis. Upon fusion of vesicles at the ciliary base or the surrounding periciliary region, lipids and proteins are incorporated into the ciliary compartment. However, the molecular mechanisms for the formation, cargo sorting, and trafficking of cilium-bound vesicles are highly complex and remain poorly understood. Chibby 1 (Cby1) is a conserved small coiled-coil protein that localizes to the base of cilia and plays a crucial role in ciliogenesis. Cby1-knockout mice develop several ciliopathy features such as chronic airway infection, polydactyly, and PKD. Through affinity purification/mass spectrometry, we have identified coiled-coil domain-containing 186 (CCDC186) as a new Cby1-interacting protein. CCDC186 harbors molecular characteristics of golgins that function with small GTPases in budding, transport, tethering, and docking of Golgi-derived vesicles. We found that CCDC186 binds to and colocalizes with Cby1 at centrosomes and TGN. Depletion of CCDC186 in cultured cells results in a reduction in the recruitment of Cby1 to the ciliary base and impairs ciliogenesis. Consistent with its crucial role in ciliogenesis, CCDC186-knockout mice develop cystic kidneys, a hallmark of ciliary defects. Thus, our preliminary data suggest that CCDC186 facilitates the targeting of Cby1 vesicles to the ciliary base, thereby enhancing ciliogenesis. The overall goal of this application is to elucidate the molecular and functional mechanisms of Cby1- CCDC186 interactions in vesicle trafficking during ciliogenesis. In order to achieve this goal, we propose the following Specific Aims: Specific Aim 1. Investigate the physical and functional interactions of CCDC186 with Cby1 and small GTPases during ciliogenesis; Specific Aim 2. Investigate cystic kidney phenotypes and possible defects in renal cilia in CCDC186-knockout mice; Specific Aim 3. Isolate novel CCDC186 interactors using proximity labeling and affinity purification and characterize their functions during ciliogenesis. We anticipate that these experiments will contribute to a fundamental understanding of the molecular and cellular mechanisms of vesicle and cargo trafficking during ciliogenesis.

PD/PI：Takemaru，Ken-ichi 7。项目摘要 纤毛是基于进化的微管结构，从根尖细胞表面伸出 执行多种生物学功能。主要纤毛在人体中的大多数细胞类型上都存在 在各种器官系统的胚胎和产后发展中的重要作用，作为动态化学和 机械感应中心。功能失调的原发性纤毛与许多遗传疾病有关，例如 器官横向缺陷，多态和多囊性肾脏疾病（PKD），统称为纤毛病。 因此，对纤毛发生的更好理解对于制定综合策略至关重要 纤毛病的预防和治疗。 来自trans-Golgi网络（TGN）和回收利用的蛋白质和脂质的极化囊泡运输 内体在纤毛发生中起重要作用。囊泡在睫状底或周围融合后 周围区域，脂质和蛋白质被纳入睫状室。但是，分子 纤毛结合囊泡的形成，货物排序和运输的机制非常复杂，并且 保持不当理解。 Chibby 1（CBY1）是一种保守的小盘绕蛋白，该蛋白位于纤毛底部并播放A 在纤毛发生中的关键作用。 CBY1-敲除小鼠开发了几种纤毛病特征，例如慢性气道 感染，多态和PKD。通过亲和力纯化/质谱法，我们已经确定 含域的186（CCDC186）作为新的CBY1相互作用蛋白。 CCDC186拥有分子 在萌芽，运输，束缚和对接的golgins的特征 高尔基体囊泡。我们发现CCDC186与中心体和TGN的CBY1结合并与CBY1结合并共定位。 CCDC186在培养细胞中的耗竭导致CBY1募集到睫状基碱和 损害纤毛发生。与其在纤毛发生中的关键作用一致，CCDC186-敲除小鼠发展囊性 肾脏，睫状缺陷的标志。因此，我们的初步数据表明CCDC186促进了目标 cby1囊泡的纤毛碱，从而增强了纤毛发生。 该应用的总体目标是阐明CBY1-的分子和功能机制 纤毛发生过程中囊泡运输中的CCDC186相互作用。为了实现这一目标，我们建议 以下特定目的：特定目标1。研究CCDC186的物理和功能相互作用 在纤毛发生过程中，CBY1和小GTPase；特定目标2。调查囊性肾脏 CCDC186-敲除小鼠的肾纤毛中的表型和可能的缺陷；特定目标3。分离株 新颖的CCDC186相互作用者使用接近标记和亲和力纯化并表征其 纤毛发生过程中的功能。我们预计这些实验将有助于基本 了解纤毛发生过程中囊泡和货物贩运的分子和细胞机制。