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：
8861439
负责人：
HAYS S RYE
金额：
$ 29.98万
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/8861439
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 Solutions 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（由申请人提供）：膜封闭的运输载体，例如囊泡，在动态过程中对细胞中的站之间的生物分子进行分类，这对于生物体的生理学来说是基础。虽然人们对将膜塑造成囊泡的蛋白质涂层了解很多，但仍有待解决的是囊泡释放的机制，即膜裂变反应。在本提案中，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灵敏的新工具相结合来剖析膜裂变机制：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，从而能够检测膜裂变反应期间的中间体和产物。在线虫模型系统中，线虫从信号细胞器（称为循环内体）释放运输载体，需要一种类似动力的 Eps15 同源结构域 (EHD) 蛋白 RME-1，与 AMPH-1 一起发挥作用，AMPH-1 是蠕虫 Bin/ Amphiphyn/Rvs (N-BAR) 结构域蛋白。虽然已知细胞骨架元件，特别是微管和肌动蛋白，参与细胞中的囊泡裂变机制，但它们对于无细胞系统中的膜裂变不是必需的。在体外，当锁定在 ATP 结合构型时，脂质体膜变形为刚性管状结构，由 RME-1/AMPH-1 聚合物包裹。尽管这些小管的作用仍有待发现，但我们的脂质体初步实验支持 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机制，以了解 ATP 水解如何与膜结合、重排和裂变耦合。我们将利用与电子冷冻显微镜和晶体学合作者合作进行的额外结构研究提供的三维信息，进一步告知我们对裂变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回收测定中的基因突变。