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: 10379375
• 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/10379375
• 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; 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; Respiratory Tract Infections; 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 纤毛是进化上保守的微管结构，从顶端细胞表面突出， 具有多种生物学功能。初级纤毛存在于人体的大多数细胞类型上， 在胚胎和出生后的各种器官系统的发育中起着重要作用， 机械感觉中枢初级纤毛功能障碍与许多遗传疾病有关，例如 器官偏侧缺陷、多指（趾）畸形和多囊性肾病（PKD），统称为纤毛病。 因此，更好地了解纤毛发生对于发展全面的策略至关重要， 纤毛病的预防和治疗。 蛋白质和脂质的极性囊泡从高尔基体网络（TGN）的运输和再循环 内体在纤毛发生中起重要作用。当睫状体基部或周围的囊泡融合时， 在睫状体周围区域，脂质和蛋白质被并入睫状体隔室中。然而，分子 纤毛结合囊泡的形成、货物分选和运输的机制非常复杂， 仍然知之甚少。 Chibby 1（Cby 1）是一种保守的小卷曲螺旋蛋白，定位于纤毛基部，在纤毛的生长过程中起重要作用。 在纤毛发生中的重要作用。cby 1基因敲除小鼠出现了几种纤毛病变特征，如慢性气道炎症， 感染、多指畸形和PKD。通过亲和纯化/质谱，我们鉴定了卷曲螺旋 结构域186（CCDC 186）是一个新的与Cby 1相互作用的蛋白。CCDC 186含有分子 golgins与小GTP酶在出芽、运输、拴系和对接中起作用的特性， 高尔基体衍生的囊泡我们发现CCDC 186在中心体和TGN上与Cby 1结合并共定位。 培养细胞中CCDC 186的消耗导致Cby 1向睫状体基底的募集减少， 损害纤毛发生。与其在纤毛发生中的关键作用一致，CCDC 186基因敲除小鼠出现囊性变， 肾脏，纤毛缺陷的标志。因此，我们的初步数据表明，CCDC 186促进靶向 的Cby 1囊泡的睫状体的基础上，从而提高纤毛发生。 本申请的总体目标是阐明Cby 1的分子和功能机制。 纤毛发生过程中CCDC 186在囊泡运输中的相互作用。为了实现这一目标，我们建议 具体目标：具体目标1。研究CCDC 186的物理和功能相互作用 与Cby 1和小GTP酶在纤毛发生;特异性目的2。肾囊肿的治疗 CCDC 186基因敲除小鼠肾纤毛的表型和可能的缺陷;特异性目的3.隔离 使用邻近标记和亲和纯化的新型CCDC 186相互作用物，并表征其 在纤毛发生过程中起作用。我们预计，这些实验将有助于一个基本的 了解纤毛发生过程中囊泡和货物运输的分子和细胞机制。