Mechanism of membrane fission at the recycling endosome

回收内体的膜裂变机制

基本信息

批准号：
9331711
负责人：
HAYS S RYE
金额：
$ 29.17万
依托单位：
TEXAS A&M AGRILIFE RESEARCH
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-09-01 至 2019-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9331711
关键词：
ATP Hydrolysis Actins Affect Alpha Cell Back Binding Binding Proteins Biochemical Genetics Biological Biological Assay Biological Models Blindness Caenorhabditis elegans Capsid Proteins Cardiac Cell surface Cell-Free System Cells Chemistry Clathrin Collaborations Coupled Cryoelectron Microscopy DNA Sequence Alteration Defect Deformity Detection Development Diabetes Mellitus Dictyostelium discoideum dynamin A Dimensions Disease Elements Endocytosis Endosomes Exocytosis Fluorescence Fluorescence Resonance Energy Transfer Glucose Transporter Grant Growth and Development function Human Resources Hydrolysis Immunologics In Vitro Individual Insulin Lipids Liposomes Malignant Neoplasms Measures Membrane Mental Retardation Metabolic Methods Microfluidics Microtubules Modality Molecular Molecular Conformation Mutation Nature Nucleotides Organelles Organism Particle Size Phenotype Physics Physiology Polymers Process Production Proteins Reaction Recycling Role Sampling Signal Pathway Signal Transduction Signaling Molecule Sorting - Cell Movement Spectrum Analysis Structure Techniques Tertiary Protein Structure Testing Time Transforming Growth Factor beta Tubular formation Vesicle Work X-Ray Crystallography Yeasts amphiphysin cancer type cell growth deafness design electron crystallography electron tomography epsin experimental study in vivo membrane biogenesis mutant nervous system disorder novel nucleotide analog particle protein protein interaction public health relevance receptor mediated endocytosis reconstitution skeletal therapeutic development tool vesicular release

项目摘要

DESCRIPTION (provided by applicant): Membrane-enclosed transport carriers, such as vesicles, sort biological molecules between stations in the cell in a dynamic process that is fundamental to the physiology of organisms. While much is known about the protein coats that sculpt membranes into vesicles, what remains to be resolved is the mechanism for vesicle release, a membrane fission reaction. In this proposal, we show how a membrane fission mechanism can be dissected by combining powerful biochemical and genetic tools with a versatile and highly sensitive new tool that we have developed: Burst Analysis Spectroscopy (BAS). BAS is a single-particle fluorescence technique that measures changes in particle size and concentration in free solution, allowing detection of intermediates and products during a membrane fission reaction. In the worm model system, C. elegans, release of transport carriers from the signaling organelle known as the recycling endosome, requires a dynamin-like, Eps15-homology domain (EHD) protein, RME-1, functioning with AMPH-1, the worm Bin/ Amphiphysin/Rvs (N-BAR) domain protein. While it is known that cytoskeletal elements, in particular microtubules and actin, participate with vesicle fission machinery in the cell, they are not necessary for membrane fission, in cell-free systems. In vitro, liposome membranes are deformed into rigid, tubular structures, wrapped by the RME-1/AMPH-1 polymer, when locked in an ATP-bound configuration. Although the role of these tubules remains to be discovered, our preliminary experiments with liposomes support a role for RME-1 and AMPH-1 in membrane fission. Here, we seek to reconstitute the minimal fission-active protein machinery in a cell-free assay, to understand how ATP hydrolysis is coupled to membrane binding, rearrangement, and fission. We will further inform our hypotheses for the protein and membrane dynamics required for fission using three-dimensional information provided by additional structural studies performed in collaboration with electron cryo-microscopy and crystallography collaborators. Our hypotheses will be tested in live cells, using genetic mutations in a well-established recycling assay, developed in the C. elegans model system.

描述（由申请人提供）：膜封闭的传输载体，例如囊泡，在细胞中的站点之间在动态过程中对生物分子进行排序，这是生物体生理学基础的动态过程。虽然对将膜塑造成囊泡的蛋白质涂层有很多了解，但仍有要解决的是囊泡释放的机制，即一种膜裂变反应。在此提案中，我们展示了如何通过将强大的生化和遗传工具与我们开发的多功能且高度敏感的新工具相结合：爆发分析光谱（BAS）来阐明膜裂变机制。 BAS是一种单粒子荧光技术，可测量自由溶液中粒度和浓度的变化，从而可以在膜裂变反应期间检测中间体和产物。在蠕虫模型系统中，秀丽隐杆线虫，从称为回收内体的信号细胞器中释放传输载体，需要类似元素的EPS15------------------------ bin/ amphysin/ rvs（n-bar）蛋白质蛋白。虽然众所周知，细胞骨架元素，特别是微管和肌动蛋白，参与细胞中的囊泡裂变机械，但在无细胞系统中，它们对于膜裂变不是必需的。在体外，当锁定在ATP结合的构型中时，脂质体膜变形为由RME-1/AMPH-1聚合物包裹的刚性，管状结构。尽管这些小管的作用仍然有待发现，但我们与脂质体的初步实验支持RME-1和AMPH-1在膜裂变中的作用。在这里，我们试图在无细胞测定中重建最小的裂变活性蛋白质机械，以了解如何将ATP水解与膜结合，重排和裂变耦合。我们将进一步向我们的假设提供有关裂变所需的蛋白质和膜动力学的假设，该蛋白质和膜动力学使用与电子冷冻微镜和晶体学合作者合作进行的其他结构研究提供的三维信息。我们的假设将在活细胞中测试，使用在秀丽隐杆线虫模型系统中开发的良好回收测定中的基因突变。