Endosome to Golgi Retrograde Recycling in C.Elegans

描述(由申请人提供)：项目标题：线虫内体到高尔基体的逆行循环背景：细胞和组织在很大程度上通过对蛋白质和膜运输的严格调控来建立和保持其独特的结构。这一过程的关键方面包括决定回收或降解给定的分子，特别是信号和黏附分子，这一过程在许多癌症中都会出错。内吞作用后，脂类和跨膜货物进入早期的内吞体内进行分选。货物可以通过定向到溶酶体来降解，直接或通过回收内体回收到PM，或者分拣后通过逆行路线运送到高尔基体。一些观察暗示，这些航线在各种货物上相互竞争，决定回收或降级是监管的关键。我们的重点是在逆行途径和到溶酶体的降解途径之间的分支点上。有人认为，这两条途径之间的竞争是由早期内体和多囊泡体上形成的微区介导的。Clathrin/HGRS-1(HRs)/ESCRT复合体形成一个微域，通过溶酶体促进降解，而逆转体/RME-8复合体形成竞争微域，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了解控制货物通过逆行或退化路径运输的分子机制，以及寻找新的逆行成分。目的/假设：本实验室最近对clathrin/HGRS-1(HRs)/ESCRT和RME-8/Retrmer微区之间竞争的分子机制的理解取得了进展。我们的数据表明，SNX-1，一个逆转聚体成分，加强了RME-8与伴侣HSP-1(Hsc70)的结合，这种相互作用通过分子筛去涂层的活性来对抗内体蛋白及其相关降解亚域的积聚。此外，初步证据表明，HGRS-1(HRS)可能通过滴定SNX-1从RME-8中分离出来，直接竞争这种笼状蛋白的去包被过程，使笼状蛋白自由积聚并促进溶酶体的降解。我们的目标是验证这一微域维持假说，并通过大规模的基于RNAi的筛选来极大地扩展我们对多细胞生物体中逆行途径的知识。具体目的：(1)确定SNX-1/RME-8相互作用的功能结果；(2)区分解释HGRS-1(HRS)/SNX-1相互作用的两种模型，特别是与RME-8/HSP-1(Hsc70)介导的内体分选有关的模型；(3)确定逆行分选途径的新成分。研究设计：我们建议结合RME-8相互作用受损突变体的体内救援实验、上位性分析和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但功能强大的大规模RNAi筛选来识别新的逆行途径组件。意义：我们正在研究的逆行和溶酶体分选的所有成分在哺乳动物中都是保守的。逆行转运的缺陷与晚发性阿尔茨海默病、E-钙粘蛋白定位错误、Wnt信号梯度的产生缺陷以及死亡的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靶向细胞内适当位置的分子机制，以及识别在这一过程中发挥作用的新基因，将有助于我们理解导致大量疾病状态的潜在分子事件。 与公共卫生相关：内吞作用和内体分选涉及从细胞表面吸收脂类和蛋白质，然后将它们适当地分布在细胞中。这种“贩运”对多种细胞功能至关重要，如信号传递、细胞-细胞连接维持、营养吸收和发育。运输缺陷与晚发性阿尔茨海默病、细胞极性缺陷(自上而下了解细胞)和清除死亡细胞的缺陷有关。

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.