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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）：膜封闭的运输载体，如囊泡，在生物体生理学基础的动态过程中在细胞中的站之间对生物分子进行分类。虽然我们对将膜塑造成囊泡的蛋白质外壳了解很多，但仍有待解决的是囊泡释放的机制，即膜分裂反应。在这个建议中，我们展示了如何通过将强大的生物化学和遗传工具与我们开发的多功能和高灵敏度的新工具相结合来解剖膜裂变机制：突发分析光谱（BAS）。BAS是一种单颗粒荧光技术，可测量游离溶液中颗粒尺寸和浓度的变化，从而检测膜裂变反应期间的中间体和产物。在蠕虫模型系统中，C.在线虫中，从称为再循环内体的信号细胞器释放运输载体需要动力蛋白样Eps 15同源结构域（EHD）蛋白RME-1，其与AMPH-1（蠕虫Bin/ Amphiphysin/Rvs（N-BAR）结构域蛋白）一起发挥功能。虽然已知细胞骨架元件，特别是微管和肌动蛋白，参与细胞中的囊泡分裂机制，但在无细胞系统中，它们不是膜分裂所必需的。在体外，脂质体膜变形为刚性的管状结构，被RME-1/AMPH-1聚合物包裹，当锁定在ATP结合的配置。虽然这些小管的作用仍有待发现，我们的初步实验脂质体支持RME-1和AMPH-1在膜分裂中的作用。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制，以了解ATP水解如何与膜结合，重排和裂变相结合。我们将进一步告知我们的假设裂变所需的蛋白质和膜动力学使用的三维信息，通过与电子冷冻显微镜和晶体学合作者合作进行的额外的结构研究提供。我们的假设将在活细胞中进行测试，使用在C. elegans模型系统。