Regulatory mechanisms of clathrin dependant traffic at the TGN and endosomes

TGN 和内体上网格蛋白依赖性交通的调节机制

基本信息

批准号：
8917971
负责人：
Mara C Duncan
金额：
$ 28.9万
依托单位：
UNIVERSITY OF MICHIGAN AT ANN ARBOR
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-09-30 至 2017-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8917971
关键词：
Adaptor Signaling Protein Address Affinity Alleles Animals Behavior Binding Biochemical Biochemical Genetics Biochemistry Biological Assay Biological Models Capsid Proteins Cell Adhesion Molecules Cell Fractionation Cell Survival Cell physiology Cells Cellular Assay Clathrin Clathrin Adaptors Cyclic AMP-Dependent Protein Kinases Data Development Disease Endosomes Ensure Environment Enzymes Eukaryota Event Exclusion Feedback Fluorescence Microscopy Functional disorder Genetic Transcription Goals Health Homeostasis Human Human Development In Vitro Inherited Injury Lead Life Link Malignant Neoplasms Mediating Membrane Membrane Protein Traffic Mental Retardation Microscopy Mitosis Modeling Modification Molecular Molecular Biology Mutation Nutrient Oranges Organelles Peptide Hydrolases Phosphorylation Play Process Property Protein Isoforms Proteins Regulation Risk Factors Role Saccharomyces cerevisiae Schizophrenia Signal Pathway Signal Transduction Staging Structure System Testing Therapeutic Intervention Time Work Yeasts adhesion receptor cell behavior cell motility genome wide association study human disease in vitro Assay in vivo insight intercellular communication pathogen programs protein transport research study response trafficking trans-Golgi Network yeast genetics

项目摘要

DESCRIPTION (provided by applicant): Differences in cellular behavior are central to normal human development and how injury, pathogens and mutations cause dysfunction. Many aspects of differential cellular behavior can be attributed to differences in protein localization such as reduced adhesion molecules or increased secretion of enzymes. Clathrin dependent traffic between the trans-Golgi network (TGN) and endosomes plays an important role in localization of proteins important for cell migration, cell signaling and survival. The experiments in this proposal fit into a long term goal to understand how developmental programs, mutations and cell signaling act on the traffic machinery to cause differential cellular behavior. This is the first step will be understand the molecular mechanisms leading to efficient and accurate traffic at the TGN and endosomes. It is still unclear how clathrin and its many adaptor and accessory proteins combine to provide all activities required for the complicated steps involved in traffic. These steps include selecting and concentrating cargo, generating and targeting a transport carrier in vivo. Previous identification of a network of interacting clathrin adaptors that act at TGN and endosomes has opened new avenues for understanding the molecular mechanism of clathrin dependent traffic. In this proposal, molecular mechanisms leading to fidelity in membrane traffic and a signaling pathway regulating membrane traffic in yeast will be examined. In aim1, specific hypotheses will be tested about how clathrin and adaptors ensure coats assemble at the correct membrane, that events occur in the correct order and the correct proteins are transported. Hypotheses to be tested are that 1) cooperative binding and 2) competition for space in the tightly packed clathrin coat determine when an adaptor functions. A combined approach to test these hypotheses will use in vitro biochemistry, cell fractionation, assays of membrane traffic and live-cell microscopy of specific mutations in adaptor proteins. In Aim2, a newly identified regulation of clathrin adaptors in low nutrient conditions will be investigated. Hypotheses to be tested are that 1) low nutrients inhibits membrane traffic, 2) inhibition acts at the level of adaptor modification and 3) conserved signaling pathways coordinate traffic with other cellular responses to low nutrients.

描述（由申请人提供）：细胞行为的差异是正常人类发育以及损伤，病原体和突变如何引起功能障碍的核心。差异细胞行为的许多方面可以归因于蛋白质定位的差异，例如粘附分子减少或酶的分泌增加。跨加利基网络（TGN）和内体之间的网格蛋白依赖性流量在对细胞迁移，细胞信号和生存重要的蛋白质定位中起重要作用。该提案中的实验符合长期目标，以了解发展程序，突变和细胞信号如何在交通机械上作用以引起差异性细胞行为。这是第一步是了解导致TGN和内体的有效和准确流量的分子机制。目前尚不清楚网格蛋白及其许多适配器和附件蛋白如何合并以提供涉及流量中复杂步骤所需的所有活动。这些步骤包括选择和集中货物，在体内产生和靶向运输载体。以前对在TGN和内体作用的相互作用网格蛋白适配器网络的识别已开辟了新的途径，以理解网格蛋白依赖性流量的分子机制。在此提案中，将检查导致膜流量忠诚度的分子机制和调节酵母膜流量的信号通路。在AIM1中，将测试特定的假设，以确保在正确的膜上组装外套和适配器，以确保事件以正确的顺序发生，并且正确的蛋白质被运输。要测试的假设是1）合作结合和2）紧密包装的网格蛋白外套中空间的竞争确定适配器何时功能。测试这些假设的合并方法将使用体外生物化学，细胞分馏，膜流量的测定和适配蛋白特异性突变的活细胞显微镜。在AIM2中，将研究在养分较低的情况下对网格蛋白适配器的新确定的调节。要测试的假设是1）低营养素抑制膜流量，2）在适配器修饰水平上抑制作用和3）保守信号通路协调流量与其他细胞对低营养物质的反应。