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：武丸、健一 7.项目总结 纤毛是进化上保守的基于微管的结构，从顶端细胞表面突出到 执行多种生物功能。初级纤毛存在于人体内的大多数细胞类型上， 作为一种动态的化学和化学物质，在各种器官系统的胚胎和出生后发育中起着重要的作用 机械传感中心。功能失调的初级纤毛与许多遗传性疾病有关，例如 器官偏侧缺陷、多指和多囊肾病(PKD)，统称为纤毛疾病。 因此，更好地了解纤毛发生对于制定全面的治疗策略是至关重要的。 纤毛病的防治。 跨高尔基网络(TGN)中蛋白质和脂类的极化囊泡运输和再循环 内体在纤毛发生中起着重要作用。在睫状体底部或周围的囊泡融合时 睫状区、脂类和蛋白质被结合到睫状室。然而，分子 纤毛结合的囊泡的形成、货物分类和运输的机制非常复杂和 人们对此仍然知之甚少。 CHIBBY 1(Cby1)是一种保守的卷曲状小卷曲蛋白，定位于纤毛的底部，发挥着 在纤毛发生中起着至关重要的作用。Cby1基因敲除小鼠出现几种纤毛病变特征，如慢性呼吸道疾病 感染、多指和PKD。通过亲和纯化/质谱分析，我们鉴定了盘绕线圈 含结构域186(CCDC186)是一种新的Cby1相互作用蛋白。CCDC186含有分子 与小GTP酶共同作用的高尔基体在发芽、运输、系留和对接中的特性 高尔基体衍生的囊泡。我们发现CCDC186在中心体和TGN与Cby1结合并与Cby1共定位。 CCDC186在培养细胞中的耗尽导致Cby1在睫状体基底部的募集减少 会损害纤毛的形成。与其在纤毛发生中的关键作用一致，CCDC186基因敲除小鼠发展为囊性 肾脏，纤毛缺陷的标志。因此，我们的初步数据表明，CCDC186有助于靶向 Cby1囊泡到纤毛底部，从而促进纤毛发生。 本应用的总体目标是阐明Cby1-1的分子和功能机制。 纤毛发生过程中CCDC186与囊泡运输的相互作用为了实现这一目标，我们建议 具体目标：具体目标1.研究CCDC186的物理和功能相互作用 纤毛发生过程中的Cby1和小GTP酶；特定目的2.研究囊性肾 CCDC186基因敲除小鼠肾纤毛的表型和可能的缺陷；特定目的3.分离 利用邻近标记和亲和纯化的新型CCDC186相互作用元件及其特性 纤毛发生过程中的功能。我们预计，这些实验将有助于 理解纤毛发生过程中囊泡和货物运输的分子和细胞机制。