Regulatory mechanisms that control vesicle secretion at the endoplasmic reticulum

控制内质网囊泡分泌的调节机制

• 批准号: 9205237
• 负责人: Anjon Audhya
• 金额: $ 27.64万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-03-01 至 2019-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9205237
• 关键词: Address; Affect; Animal Model; Architecture; Axonal Transport; Binding; Biochemical; Biochemistry; Biogenesis; Biological Assay; COPII-Coated Vesicles; Caenorhabditis elegans; Caliber; Carrier Proteins; Cells; Chemicals; Coated vesicle; Complex; Cryoelectron Microscopy; Defect; Degenerative Disorder; Development; Disease; Electrons; Endoplasmic Reticulum; Ensure; Eukaryotic Cell; Fluorescence; Genes; Germ Cells; Goals; Golgi Apparatus; Growth and Development function; Guanosine Triphosphate Phosphohydrolases; Hereditary Motor and Sensory Neuropathies; Hereditary Spastic Paraplegia; Homeostasis; Human; Human Development; Image; Immune System Diseases; Immune System and Related Disorders; Immunoelectron Microscopy; In Vitro; Lipids; Location; Maintenance; Malignant Neoplasms; Mediating; Membrane; Methodology; Microscopy; Mitosis; Molecular; Monomeric GTP-Binding Proteins; Motor Neurons; Mutation; Nerve Degeneration; Neurodegenerative Disorders; Neurons; Organelles; Pathway interactions; Phosphorylation; Point Mutation; Polymers; Population; Positioning Attribute; Prevention; Process; Proteins; Recruitment Activity; Regulation; Resolution; Roentgen Rays; Role; Route; Scaffolding Protein; Set protein; Site; Spatial Distribution; Speed; Stem cells; Structural Models; Structure; Testing; Time; To specify; Transmembrane Transport; Transport Vesicles; Vesicle; Vesicle Transport Pathway; X-Ray Crystallography; anterograde transport; base; cell growth; combat; defined contribution; depolymerization; developmental disease; electron tomography; endoplasmic reticulum stress; experimental study; human tissue; improved; in vitro Assay; in vivo; induced pluripotent stem cell; insight; live cell imaging; novel therapeutic intervention; polymerization; protein complex; protein transport; public health relevance; reconstitution; role model; tissue/cell culture; trafficking

DESCRIPTION (provided by applicant): The long-term goals of this proposal are to define the molecular mechanisms that regulate the spatial distribution of organelles in the early secretory pathway and to determine the importance of this architecture to normal membrane trafficking during cell growth and development. Most biosynthetic cargo molecules destined for secretion initiate their journey within specific subdomains of the endoplasmic reticulum (ER), known as ER exit sites. At these locations, COPII-coated vesicles are first generated, packaging cargoes for transport to ER- Golgi intermediate compartments (ERGIC), stable organelles that are juxtaposed to ER exit sites. The COPII coat is composed of two multimeric protein complexes, Sec23/24 and Sec13/31, and the small GTPase Sar1. Although these factors are sufficient to reconstitute vesicle budding from chemically defined membranes in vitro, additional proteins are required to promote COPII vesicle biogenesis and anterograde transport in cells. This proposal focuses on the role of TFG, a metazoan-specific protein required for the robust recruitment of COPII coat subunits to ER exit sites. Based on our preliminary results, we hypothesize that TFG forms a highly regulated meshwork at the ER/ERGIC interface that facilitates COPII coat stability and vesicle egress. Importantly, mutations in TFG have been implicated in progressive neurodegenerative disease, suggesting a role for COPII-mediated transport in maintaining neuron function. We propose a combination of in vivo and in vitro approaches to achieve our aims, taking advantage of assays developed in human cells and biochemical methodologies established to study the structure and function of early secretory pathway components. Using electron tomography, we recently defined the architecture of the early secretory pathway in germ cells derived from the model organism C. elegans. Our findings revealed the presence of an electron-dense meshwork, filled with molecules of TFG, which encompasses the region between ER exit sites and ERGIC membranes. We hypothesize that this meshwork functions in the regulation of COPII dynamics and helps to maintain the organization of the early secretory pathway. In a similar fashion, our preliminary studies in human cells have demonstrated a conserved role for TFG in controlling early secretory pathway architecture and function. Additionally, we have recently defined a structural model for TFG using cryo-electron microscopy and small angle X-ray scattering, which has led us to propose a testable model for the role of TFG at the interface between ER and ERGIC membranes. The specific aims of this proposal are to: 1) determine the structural basis for TFG assembly and disassembly at the ER/ERGIC interface, 2) define the contributions of TFG to COPII-mediated vesicle transport, and 3) define mechanisms by which TFG contributes to neuronal maintenance. Together, the experiments outlined in this proposal will provide fundamental new insights into how the organization of the early secretory pathway promotes the rapid anterograde transport of newly synthesized cargoes in COPII-coated vesicles, which is necessary for normal human development and neuronal homeostasis.

描述（由申请人提供）：该提案的长期目标是定义调节器细胞器在早期分泌途径中的空间分布的分子机制，并确定该体系结构对细胞生长和发育过程中正常膜运输的重要性。大多数要进行分泌物的生物合成货物分子都在内质网（ER）的特定子域中启动其旅程，称为ER出口位点。在这些位置，首先生成了Copii涂层的囊泡，包装货物，用于运输到Ergolgi中间室（ERGIC），稳定的细胞器与ER出口位点并列。 COPII涂层由两个多聚体蛋白复合物，Sec23/24和Sec13/31，以及小的GTPase SAR1。尽管这些因素足以在体外从化学定义的膜从化学定义的膜中萌芽，但需要额外的蛋白质来促进细胞中的复合囊泡生物发生和顺行转运。该提案的重点是TFG的作用，TFG是一种可重大募集Copii外套亚基到ER出口部位所需的后生动物特异性蛋白质。基于我们的初步结果，我们假设TFG在ER/ERGIC界面上形成了高度调节的网状功能，可促进Copii外套的稳定性和囊泡出口。重要的是，TFG中的突变已与进行性神经退行性疾病有关，这表明COPII介导的转运在维持神经元功能中的作用。 我们提出了体内和体外方法的结合，以利用在人类细胞中开发的测定以及为研究早期分泌途径成分的结构和功能而建立的生化方法论的优势。我们最近使用电子层析成像来定义了来自模型有机体秀丽隐杆线虫的生殖细胞中早期分泌途径的结构。我们的发现揭示了充满了TFG分子的电子致密网状功能，其中涵盖了ER出口位点和ERGIC膜之间的区域。我们假设该网格工作在调节COPII动力学中起作用，并有助于维持早期分泌途径的组织。以类似的方式，我们在人类细胞中的初步研究表明，TFG在控制早期分泌途径结构和功能方面具有保守的作用。此外，我们最近使用冷冻电子显微镜和小角度X射线散射定义了TFG的结构模型，这使我们提出了一个可测试模型，以实现TFG在ER和ERGIC膜之间的界面中的作用。该提案的具体目的是：1）确定在ER/ERGIC接口处TFG组装和拆卸的结构基础，2）定义TFG对CopII介导的囊泡运输的贡献，3）定义了TFG对神经元维持的机制。总之，本提案中概述的实验将为早期分泌途径的组织如何促进新近合成的囊泡的快速顺序运输提供基本的新见解，这对于正常的人类发展和神经元稳态是必不可少的。