Endosome to Golgi Retrograde Recycling in C.Elegans

项目名称：秀丽隐杆线虫内核体到高尔基逆行循环背景：细胞和组织建立和维持其独特的结构在很大程度上是通过对蛋白质和膜运输的严格调控。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都出错了。内吞作用后，脂质和跨膜货物进入早期核内体进行分选。货物可以通过靶向溶酶体来直接降解，直接回收到PM或通过回收内体，或通过逆行路线分类交付到高尔基体。许多观察表明，这些航线在各种货物上相互竞争，而回收或降解的决定是监管的一个关键点。我们的重点是被低估的分支点之间的逆行路线和降解路线的溶酶体。这两种途径的竞争是由早期核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复合物形成一个微结构域，通过溶酶体促进降解，而反转录物/RME-8复合物形成一个竞争的微结构域，促进逆行途径，将货物从核内体再循环到高尔基体。我们的建议的目标是更好地理解控制货物通过逆行或降解途径的交通决策的分子机制，以及寻找新的逆行组件。目的/假设：我们的实验室最近在网格蛋白/HGRS-1(Hrs)/ESCRT和RME- 8/逆转录微域之间竞争的分子机制的理解方面取得了进展。我们的数据表明，SNX-1，一个逆转录成分，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膜活性对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使其远离RME-8，从而直接与网格蛋白剥离过程竞争，使网格蛋白自由积累并促进溶酶体降解。我们的目标是验证这种微结构域维持假说，并通过大规模基于rna的筛选大大扩展我们对多细胞生物逆行途径的了解。具体目的：(1)确定SNX-1/RME-8相互作用的功能后果(2)区分两种模型来解释HGRS-1(Hrs)/SNX-1相互作用，特别是当它与RME-8/HSP-1（Hsc70）介导的内体分选有关时；(3)鉴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，我们提出了一个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：我们所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。逆行转运缺陷与晚发性阿尔茨海默病、e-钙粘蛋白错误定位、Wnt信号梯度产生缺陷以及死亡凋亡细胞清除缺陷有关。此外，逆行分类路线是志贺毒素的进入模式。我们相信，更好地了解靶向米格-14在细胞内正确位置的分子机制，以及识别在这一过程中起作用的新基因，将有助于我们了解导致大量疾病状态的潜在分子事件。

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.