Mechanism of membrane fission at the recycling endosome

描述（由申请人提供）：膜封闭运输载体，如囊泡，在动态过程中对细胞内各站之间的生物分子进行分选，这是生物体生理学的基础。虽然我们对将膜雕刻成囊泡的蛋白质外壳了解甚多，但仍有待解决的是囊泡释放的机制，即膜裂变反应。在这个提议中，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相结合来解剖膜裂变机制：突发分析光谱（BAS）。BAS是一种单粒子荧光技术，可测量自由溶液中颗粒大小和浓度的变化，允许检测膜裂变反应中的中间体和产物。在蠕虫模型系统中，秀丽隐杆线虫从被称为循环内体的信号细胞器释放运输载体，需要一个动力蛋白样的eps15同源结构域（EHD）蛋白RME-1，该蛋白与蠕虫Bin/ Amphiphysin/Rvs （N-BAR）结构域蛋白AMPH-1协同工作。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，但在无细胞系统中，它们不是膜裂变所必需的。在体外，脂质体膜被锁定在atp结合结构中时，被RME-1/AMPH-1聚合物包裹成刚性管状结构。虽然这些小管的作用仍有待发现，但我们对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，我们试图在无细胞实验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，重排和裂变相结合。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，进一步告知我们关于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，在秀丽隐杆线虫模型系统中开发的一种成熟的循环试验中使用基因突变。

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.