Endosome to Golgi Retrograde Recycling in C.Elegans

描述（由申请人提供）：项目名称：C.内体到高尔基体的逆行再循环。elegans背景：细胞和组织在很大程度上通过蛋白质和膜转运的严格调节来建立和维持其独特的结构。这一过程的关键方面包括决定回收或降解给定的分子，特别是信号和粘附分子，这一过程在许多癌症中出错。内吞作用后，脂质和跨膜货物进入早期内体进行分选。货物可以通过靶向溶酶体而被引导降解，直接或通过再循环内体再循环至PM，或通过逆行途径被分选以递送至高尔基体。一些观察结果表明，这些路线相互竞争各种货物，回收或降解的决定是监管的一个关键点。我们的重点是在逆行路线和溶酶体降解路线之间的分支点。已经表明，这两种途径之间的竞争是由在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复合物形成一个促进通过溶酶体降解的微结构域，而逆转录酶/RME-8复合物形成竞争性微结构域并促进逆行途径，将货物从内体再循环到高尔基体。我们建议的目标是更好地了解控制货物通过逆行或降解途径运输的分子机制，以及寻找新的逆行成分。目的/假设：我们的实验室最近取得了进展，在我们的理解之间的网格蛋白/HGRS-1（Hrs）/ESCRT和RME- 8/retromer微结构域的这种竞争的分子机制。我们的数据表明，SNX-1，一个retromer组件，加强RME-8的伴侣HSP-1（Hsc 70），拮抗内体网格蛋白和相关的降解亚结构域通过网格蛋白脱壳活性的积累的相互作用的结合。此外，初步证据表明，HGRS-1（Hrs）可能通过滴定SNX-1使其远离RME-8，从而使网格蛋白游离以积聚并促进溶酶体降解，从而直接与该网格蛋白脱壳过程竞争。我们的目标是测试这个微域维护假说，以及通过大规模的基于RNAi的筛选，极大地扩展我们对多细胞生物体中逆行途径的了解。具体目标：（1）确定SNX-1/RME-8相互作用的功能后果（2）区分两种模型以解释HGRS-1（Hrs）/SNX-1相互作用，特别是当它与RME-8/HSP-1（Hsc 70）介导的内体分选相关时，以及（3）鉴定逆行分选途径的新组分。研究设计：我们建议分析我们的竞争模型，结合使用在体内救援实验与相互作用受损的突变体的RME-8，上位性分析，和Fvrster共振能量转移（FRET）分析。此外，我们提出了一个简单而强大的大规模RNAi筛选，以确定新的逆行通路组件。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信号梯度生成缺陷和死亡凋亡细胞清除缺陷有关。此外，逆行分选途径是滋贺毒素的进入模式。我们相信，更好地了解靶向β-14到其在细胞内的适当位置的分子机制，以及确定在这一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状态的潜在分子事件。 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，然后将其适当分布在细胞中。这种“运输”对于多种细胞功能如信号传导、细胞-细胞连接维持、营养吸收和发育至关重要。运输缺陷与迟发性阿尔茨海默病、细胞极性缺陷（从底部知道顶部）和死亡细胞清除缺陷有关。

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.