Endosome to Golgi Retrograde Recycling in C.Elegans

线虫中的内体到高尔基体的逆行回收

• 批准号: 8264581
• 负责人: Anne Norris
• 金额: $ 5.39万
• 依托单位: RUTGERS, THE STATE UNIV OF N.J.
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-01 至 2013-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8264581
• 关键词: ATP phosphohydrolase; Alleles; Amino Acids; Apoptotic; Architecture; Binding; Biological Assay; Caenorhabditis elegans; Cell Adhesion Molecules; Cell Polarity; Cell Surface Proteins; Cell physiology; Cells; Clathrin; Complex; DNA; Data; Defect; Degradation Pathway; Development; Disease; E-Cadherin; Early Endosome; Endocytosis; Endosomes; Energy Transfer; Epithelial Cells; Equilibrium; Event; Generations; Genes; Genetic; Genetic Epistasis; Genetic Screening; Goals; Golgi Apparatus; Homeostasis; Immunoprecipitation; Intercellular Junctions; Intestines; Knowledge; Laboratories; Late Onset Alzheimer Disease; Left; Link; Lipids; Location; Lysosomes; Maintenance; Malignant Neoplasms; Mammalian Cell; Mammals; Mediating; Membrane; Membrane Protein Traffic; Modeling; Molecular; Molecular Chaperones; Multivesicular Body; Nutrient; Organism; Pathway interactions; Process; Protein Binding; Protein Region; Proteins; RNA Interference; Recycling; Regulation; Research Design; Role; Route; Saccharomyces cerevisiae; Screening procedure; Shiga Toxin; Signal Transduction; Signaling Molecule; Sorting - Cell Movement; Technology; Tertiary Protein Structure; Testing; Tissues; Transmembrane Transport; Work; Yeasts; base; genetic analysis; in vivo; insight; mutant; novel; protein transport; public health relevance; research study; retrograde transport; trafficking; uptake

DESCRIPTION (provided by applicant): Title of Project: Endosome to Golgi Retrograde Recycling in C. elegans Background: Cells and tissues establish and maintain their unique architectures in large part through the tight regulation of protein and membrane transport. Key aspects of this process include the decision to recycle or degrade a given molecule, particularly signaling and adhesion molecules, a process that goes awry in many cancers. After endocytosis lipids and trans membrane cargo enter the early endosome for sorting. Cargo may be directed to degrade via targeting to the lysosome, recycled to the PM directly or through the recycling endosome, or sorted for delivery to the Golgi via the retrograde route. A number of observations hint that these routes compete with each other for various cargo, and that the decision to recycle or degrade is a key point of regulation. Our focus is on the much underappreciated branch point between the retrograde route and the degradative route to the lysosome. It has been suggested that competition between these two routes is mediated by microdomains that form on the early endosome and multivesicular body. The clathrin/HGRS-1(Hrs)/ESCRT complexes form one microdomain that promotes degradation via the lysosome, and the retromer/RME-8 complex forms a competing microdomain and promotes the retrograde pathway, recycling cargo from the endosome to the Golgi. The goal of our proposal is to better understand the molecular mechanisms controlling the decision of cargo to traffic via the retrograde or degradative pathways, as well as to find new retrograde components. Objective/Hypothesis: Our lab has recently made advances in our understanding of the molecular mechanism of this competition between the clathrin/HGRS-1(Hrs)/ESCRT and RME- 8/retromer microdomains. Our data suggest that SNX-1, a retromer component, potentiates the binding of RME-8 to the chaperone HSP-1(Hsc70), an interaction that antagonizes the buildup of endosomal clathrin and the associated degradative subdomain via clathrin uncoating activity. Furthermore preliminary evidence suggests that HGRS-1(Hrs) directly competes with this clathrin uncoating process potentially by titrating SNX-1 away from RME-8, leaving clathrin free to build up and promote lysosomal degradation. We aim to test this microdomain maintenance hypothesis, as well as dramatically extend our knowledge of the retrograde pathway in a multicellular organism by large-scale RNAi-based screening. Specific Aims: (1) To determine the functional consequence of SNX-1/RME-8 interaction (2) Distinguish between two models to explain the HGRS-1(Hrs)/SNX-1 interaction, especially as it relates to RME-8/HSP-1(Hsc70) mediated endosomal sorting and (3) To identify novel components of the retrograde sorting pathway. Study design: We propose to analyze our competition model using a combination of in vivo rescue experiments with interaction impaired mutants of RME-8, epistasis analysis, and Fvrster resonance energy transfer (FRET) analysis. Additionally we propose a simple yet powerful large scale RNAi screen to identify novel retrograde pathway components. Significance: All of the components of retrograde and lysosomal sorting we are studying are conserved in mammals. Defects in retrograde transport are linked to late-onset Alzheimer's disease, E-cadherin mis-localization, defects in the generation of Wnt signaling gradients, and defects in the clearance of dead apoptotic cells. Additionally the retrograde sorting route is the mode of entry for Shiga Toxin. We believe that a better understanding the molecular mechanism that target MIG-14 to its proper location within the cell, as well as identifying new genes that have a role in this process, will help us to understand the underlying molecular events leading a vast array of disease states. PUBLIC HEALTH RELEVANCE: Endocytosis and endosomal sorting involves the uptake of lipids and proteins from the cell surface followed by their proper distribution in the cell. This "trafficking" is critical for diverse cellular functions such as signaling, cell-cell junction maintenance, nutrient uptake, and development. Defects in transport are linked to late- onset Alzheimer's disease, defects in cellular polarity (knowing its top from its bottom) and defects in the clearance of dead cells.

描述（由申请人提供）： 项目名称：线虫内体到高尔基体的逆行回收 背景：细胞和组织在很大程度上通过严格调节蛋白质和膜运输来建立和维持其独特的结构。该过程的关键方面包括决定回收或降解给定分子，特别是信号分子和粘附分子，这一过程在许多癌症中都会出错。内吞作用后，脂质和跨膜货物进入早期内体进行分类。货物可以通过靶向溶酶体直接降解，直接或通过回收内体回收到 PM，或通过逆行路线分类输送到高尔基体。大量观察结果表明，这些航线相互竞争各种货物，回收或降解的决定是监管的关键点。我们的重点是逆行路线和溶酶体降解路线之间被低估的分支点。有人认为，这两种途径之间的竞争是由早期内体和多泡体上形成的微结构域介导的。网格蛋白/HGRS-1(Hrs)/ESCRT复合物形成一个微域，促进通过溶酶体的降解，而逆转录体/RME-8复合物形成竞争性微域并促进逆行途径，将货物从内体循环到高尔基体。我们建议的目标是更好地理解控制货物通过逆行或降解途径运输的决定的分子机制，以及寻找新的逆行成分。目的/假设：我们的实验室最近在了解网格蛋白/HGRS-1(Hrs)/ESCRT 和 RME-8/retromer 微域之间竞争的分子机制方面取得了进展。我们的数据表明，SNX-1（一种逆转录体成分）增强了 RME-8 与伴侣 HSP-1（Hsc70）的结合，这种相互作用通过网格蛋白脱壳活性拮抗内体网格蛋白的积累和相关的降解亚结构域。此外，初步证据表明，HGRS-1(Hrs) 可能通过滴定 SNX-1 远离 RME-8 来直接与网格蛋白脱衣过程竞争，使网格蛋白自由积累并促进溶酶体降解。我们的目标是测试这种微域维持假说，并通过大规模基于 RNAi 的筛选，极大地扩展我们对多细胞生物体逆行途径的了解。具体目标： (1) 确定 SNX-1/RME-8 相互作用的功能结果 (2) 区分两个模型以解释 HGRS-1(Hrs)/SNX-1 相互作用，特别是与 RME-8 相关的相互作用/HSP-1(Hsc70) 介导的内体分选；(3) 鉴定逆行分选途径的新成分。研究设计：我们建议结合体内救援实验与 RME-8 相互作用受损突变体、上位分析和 Fvrster 共振能量转移 (FRET) 分析来分析我们的竞争模型。此外，我们提出了一种简单而强大的大规模 RNAi 筛选来识别新的逆行途径成分。意义：我们正在研究的逆行和溶酶体分选的所有组成部分在哺乳动物中都是保守的。逆行转运缺陷与迟发性阿尔茨海默病、E-钙粘蛋白错误定位、Wnt 信号梯度生成缺陷以及死亡凋亡细胞清除缺陷有关。此外，逆行分选路线是志贺毒素的进入方式。我们相信，更好地了解将 MIG-14 定位到细胞内正确位置的分子机制，以及识别在此过程中发挥作用的新基因，将有助于我们了解导致大量疾病的潜在分子事件。疾病状态。 公共卫生相关性：内吞作用和内体分选涉及从细胞表面摄取脂质和蛋白质，然后在细胞中适当分布。这种“运输”对于多种细胞功能至关重要，例如信号传导、细胞间连接维持、营养吸收和发育。运输缺陷与迟发性阿尔茨海默病、细胞极性缺陷（区分顶部和底部）以及死细胞清除缺陷有关。