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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：
9135458
负责人：
HAYS S RYE
金额：
$ 28.41万
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/9135458
关键词：
ATP Hydrolysis Actins Affect 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 Crystallography DNA Sequence Alteration Defect Deformity Detection Development Diabetes Mellitus Disease Dynamin Endocytosis Endosomes Exocytosis Fluorescence Fluorescence Resonance Energy Transfer Glucose Transporter Grant Growth and Development function Health Human Resources Hydrolysis In Vitro Indium Individual Insulin Life Lipids Liposomes Malignant Neoplasms Measures Membrane Membrane Proteins Mental Retardation Metabolic Methods Microfluidics Microtubules Molecular Mutation Nature Nucleotides Organelles Organism Particle Size 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 Tubular formation Vesicle Work X-Ray Crystallography Yeasts amphiphysin cancer type cell growth deafness design electron tomography epsin in vivo membrane biogenesis mutant nervous system disorder novel nucleotide analog particle protein protein interaction receptor mediated endocytosis reconstitution research study 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(申请人提供)：膜封闭的运输载体，如囊泡，在细胞内各站之间的动态过程中对生物分子进行分类，这是生物体生理学的基础。虽然人们对将膜雕刻成囊泡的蛋白质涂层了解很多，但仍需解决的是囊泡释放的机制，这是一种膜裂变反应。在这个提案中，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和高灵敏度的新工具：爆发分析光谱(BAS)相结合来解剖膜裂变机制。BAS是一种单颗粒荧光技术，可以测量自由溶液中颗粒大小和浓度的变化，从而可以在膜裂变反应中检测中间体和产物。在蠕虫模型系统中，线虫从信号细胞器释放运输载体称为循环内小体，需要一个类似动力蛋白的Eps15同源结构域(EHD)蛋白RME-1，与Amph-1，蠕虫Bin/AmphiPhyin/RVS(N-bar)结构域蛋白一起发挥作用。虽然已知细胞骨架元素，特别是微管和肌动蛋白，参与细胞内的囊泡分裂机制，但在无细胞系统中，它们不是膜分裂所必需的。在体外，当脂质体膜被锁定在ATP结合的构型中时，被RME-1/AMPH-1聚合物包裹，变形为刚性的管状结构。尽管这些小管的作用仍有待发现，但我们用脂质体进行的初步实验支持RME-1和AMPH-1在膜分裂中的作用。在这里，我们试图在无细胞实验中重建最小的裂变活性蛋白质机制，以了解ATP水解是如何与膜结合、重排和分裂相耦合的。我们将使用与电子冷冻显微镜和结晶学合作者合作进行的额外结构研究提供的三维信息，进一步说明我们对分裂所需的蛋白质和膜动力学的假设。我们的假设将在活细胞中进行测试，使用在线虫模型系统中开发的成熟的循环试验中的基因突变。