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

• 批准号: 8723843
• 负责人: Mara C Duncan
• 金额: $ 28.97万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-30 至 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8723843
• 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

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）：细胞行为的差异对于正常人类发育以及损伤、病原体和突变如何导致功能障碍至关重要。细胞行为差异的许多方面可归因于蛋白质定位的差异，例如粘附分子减少或酶分泌增加。跨高尔基体网络 (TGN) 和内体之间的网格蛋白依赖性交通在对细胞迁移、细胞信号传导和生存至关重要的蛋白质的定位中发挥着重要作用。该提案中的实验符合一个长期目标，即了解发育程序、突变和细胞信号传导如何作用于交通机械，从而导致不同的细胞行为。这是了解导致 TGN 和内涵体高效、准确运输的分子机制的第一步。目前尚不清楚网格蛋白及其许多接头蛋白和辅助蛋白如何结合以提供交通中涉及的复杂步骤所需的所有活性。这些步骤包括选择和集中货物、生成并瞄准体内的运输载体。先前对作用于 TGN 和内体的相互作用的网格蛋白接头网络的鉴定为理解网格蛋白依赖性交通的分子机制开辟了新的途径。在该提案中，将检查导致膜运输保真度的分子机制和调节酵母膜运输的信号通路。在目标1中，将测试有关网格蛋白和接头如何确保涂层在正确的膜上组装、事件以正确的顺序发生以及正确的蛋白质被运输的具体假设。要测试的假设是 1) 协同结合和 2) 紧密堆积的网格蛋白涂层中的空间竞争决定了接头何时发挥作用。测试这些假设的综合方法将使用体外生物化学、细胞分级分离、膜运输分析和接头蛋白特定突变的活细胞显微镜检查。在 Aim2 中，将研究低营养条件下新发现的网格蛋白接头的调节。要测试的假设是：1) 低营养物质抑制膜运输，2) 抑制作用在接头修饰水平上起作用，3) 保守的信号通路协调运输与其他细胞对低营养物质的反应。