Endocytosis and Recycling in C. elegans and Mammals

线虫和哺乳动物的内吞作用和回收

基本信息

批准号：
7932636
负责人：
Barth Demian Grant
金额：
$ 26.83万
依托单位：
RUTGERS, THE STATE UNIV OF N.J.
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-09-30 至 2011-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7932636
关键词：
Acquired Immunodeficiency Syndrome Actins Adipocytes Adipose tissue Animals Architecture Area Behavior Binding Binding Proteins Biochemical Biological Biological Assay Caenorhabditis elegans Cell Polarity Cell membrane Cell surface Cells Cellular biology Complex Cytokinesis Defect Disease Early Endosome Endocytosis Endosomes Engineering Environment Epidemic Epithelial Epithelium Event Failure Family GTP Binding GTPase-Activating Proteins Genetic Glucose Transporter Glutamate Receptor Grant Growth Factor Receptors Guanosine Triphosphate Phosphohydrolases HIV Budding Hand Human Insulin Intestines Knock-out Laboratories Link MDCK cell Maintenance Malignant Neoplasms Mammals Mediating Membrane Proteins Membrane Transport Proteins Microbe Modeling Molecular Molecular Conformation Molecular Genetics Monomeric GTP-Binding Proteins Movement Muscle Muscle Cells Neoplasm Metastasis Nervous system structure Non-Insulin-Dependent Diabetes Mellitus Organism Orthologous Gene Pathway interactions Phenotype Phylogenetic Analysis Physiological Point Mutation Postsynaptic Membrane Process Proline-Rich Domain Protein Family Protein Region Proteins Receptor Signaling Recruitment Activity Recycling Regulation Research Research Support Role Running SH3 Domains Signal Transduction Source System Tertiary Protein Structure Testing Therapeutic Time Tissues Transmembrane Transport United States Wasps Work cell motility combat in vivo insight knockout animal macromolecule mutant novel protein degradation protein function receptor recycling research study therapeutic target unpublished works

项目摘要

DESCRIPTION (provided by applicant): Cells and tissues establish and maintain their unique architectures in large part through the tight regulation of protein and membrane transport. One key aspect of this process is endocytic recycling, the selective return of internalized macromolecules to the cell surface from endosomes. Understanding endocytic recycling is of fundamental importance to cell biology and has broad relevance to many areas of biomedicine. Endocytic recycling is particularly critical to the maintenance of cell polarity, a defining and essential feature of epithelial tissues. Our general approach has been to exploit powerful features of C. elegans genetics to characterize proteins that are required for the recycling process in vivo. During the previous granting period we gained new understanding of how RME-1/Ehd1 family proteins, identified in our previous screens, function in recycling. We also identified new proteins (ALX-1/Alix, SDPN-1/Syndapin, and ARF-6/Arf6) that function with RME-1 in this process. We went on to establish the C. elegans intestine as a model for elucidating endocytic membrane transport pathways in polarized epithelia, and showed for the first time that the small GTPase RAB-10/Rab10 is required for basolateral recycling in the worm intestine and polarized mammalian MDCK cells. To gain mechanistic insight into how RAB-10 drives recycling we identified five proteins that specifically interact with RAB-10 in the active, GTP-bound, conformation. These new proteins are likely RAB-10 effectors, directly mediating RAB-10 driven transport. We propose three new aims to further elucidate the molecular mechanisms underlying endocytic recycling. First we propose to test key predictions of the hypothesis that our newly identified RAB-10 binding proteins are RAB-10 effectors. This will be accomplished by better defining their physical interactions with RAB-10, determining the effects on recycling when each candidate effector is knocked out, and testing the ability of engineered interaction defective forms of these proteins to rescue recycling defects in knockout animals. Second we propose to test a model for C. elegans orthologs of Alix and Syndapin, that we developed during the prior granting period, suggesting that they control recycling through the recruitment and activation of actin regulators on endosomes. Finally we propose to leverage a newly isolated knockout for the only C. elegans Arf6 ortholog, to test the role of this key GTPase in endocytic transport in polarized epithelia, and to understand how it is regulated in this complex environment. The experiments proposed here should provide significant new insights into how endocytic recycling works. Given the high level of phylogenetic conservation of such pathways from worms to mammals, our work should provide extensive predictive insight into equivalent pathways in human cells. Our research focuses on the molecular mechanisms controlling endocytic recycling - the return of internalized macromolecules to the cell surface from endosomes. Understanding endocytic recycling is of fundamental importance to many areas of biomedicine. For instance, endocytic recycling is a key control point in the insulin- stimulated movement of glucose transporters (Glut4) from endosomes to the plasma membrane of adipose and muscle cells. Failure in this recycling event is thought to be a major cause of type II diabetes, a disease that has recently reached epidemic proportions in the United States. A better understanding of how endocytic recycling functions will be profoundly important in identifying therapeutic targets to combat this and other diseases.

描述（由申请人提供）：细胞和组织通过严格的蛋白质和膜转运来建立和维护其独特的建筑。该过程的一个关键方面是内吞回收，这是内部大分子从内体中从内体表面的选择性返回。了解内吞回收对细胞生物学至关重要，并且与许多生物医学领域具有广泛的相关性。内吞回收对维持细胞极性特别重要，这是上皮组织的定义和基本特征。我们的一般方法是利用秀丽隐杆线虫遗传学的强大特征来表征体内回收过程所需的蛋白质。在上一个授予期间，我们对RME-1/EHD1家族蛋白的回收功能有了新的了解。在此过程中，我们还确定了新的蛋白质（ALX-1/ALIX，SDPN-1/SYNDAPIN和ARF-6/ARF6），在此过程中与RME-1一起起作用。我们继续建立了秀丽隐杆线虫肠道作为阐明偏振上皮中的内吞膜传输途径的模型，并首次表明，在蠕虫和极化的乳腺Mammalian MDCK细胞中，小型GTPase RAB-10/RAB10需要小的GTPase RAB-10/RAB10。为了洞悉RAB-10如何驱动回收利用，我们鉴定了五种在活性，GTP结合的构象中特异性相互作用的五种蛋白质。这些新蛋白可能是RAB-10效应子，直接介导RAB-10驱动的运输。我们提出了三个新的目标，以进一步阐明内吞回收基础的分子机制。首先，我们建议测试我们新鉴定的RAB-10结合蛋白是RAB-10效应子的假设的关键预测。这将通过更好地定义其与RAB-10的物理相互作用来实现，从而确定当每个候选效应器被淘汰时对回收的影响，并测试这些蛋白质工程相互作用缺陷形式的能力，以挽救基因敲除动物中的回收缺陷。其次，我们建议测试我们在上一批授予期间开发的Alix和Syndapin的秀丽隐杆线虫直系同源物的模型，这表明它们通过肌动蛋白调节剂在内体上的募集和激活来控制回收利用。最后，我们建议为唯一的秀丽隐杆线虫ARF6直系同源物利用新隔离的敲除，以测试该关键GTPase在偏振上皮中内吞传输中的作用，并了解在这种复杂环境中如何调节它的作用。这里提出的实验应提供有关内吞回收如何工作的重要新见解。鉴于从蠕虫到哺乳动物的这种途径的系统发育量很高，我们的工作应提供对人类细胞中等效途径的广泛预测性见解。我们的研究重点是控制内吞回收的分子机制 - 内部大分子从内体中返回细胞表面。了解内吞回收对许多生物医学领域至关重要。例如，内吞回收是葡萄糖转运蛋白（GLUT4）从内体到脂肪和肌肉细胞的质膜的胰岛素刺激运动中的关键控制点。在这一回收事件中的失败被认为是II型糖尿病的主要原因，该疾病最近在美国已经达到了流行病的疾病。更好地理解内吞回收功能如何在识别与这种疾病和其他疾病作斗争的治疗靶标方面非常重要。