Xu Weiming, Nathwani Bhavik, Lin Chenxiang, Wang Jing, Karatekin Erdem, Pincet Frederic, Shih William, Rothman James E
Wyss Institute for Biologically Inspired Engineering and Biological Chemistry and Molecular Pharmacology, Harvard Medical School, and Department of Cancer Biology, Dana Farber Cancer Institute , Boston, Massachusetts 02115, United States.
Laboratoire de Neurophotonique, Université Paris Descartes, Faculté des Sciences Fondamentales et Biomédicales, Centre National de la Recherche Scientifique (CNRS) UMR8250, 45, rue des Saints Pères, 75270 Cedex 06 Paris, France.
J Am Chem Soc. 2016 Apr 6;138(13):4439-47. doi: 10.1021/jacs.5b13107. Epub 2016 Mar 23.
Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes are the core molecular machinery of membrane fusion, a fundamental process that drives inter- and intracellular communication and trafficking. One of the questions that remains controversial has been whether and how SNAREs cooperate. Here we show the use of self-assembled DNA-nanostructure rings to template uniform-sized small unilamellar vesicles containing predetermined maximal number of externally facing SNAREs to study the membrane-fusion process. We also incorporated lipid-conjugated complementary ssDNA as tethers into vesicle and target membranes, which enabled bypass of the rate-limiting docking step of fusion reactions and allowed direct observation of individual membrane-fusion events at SNARE densities as low as one pair per vesicle. With this platform, we confirmed at the single event level that, after docking of the templated-SUVs to supported lipid bilayers (SBL), one to two pairs of SNAREs are sufficient to drive fast lipid mixing. Modularity and programmability of this platform makes it readily amenable to studying more complicated systems where auxiliary proteins are involved.
可溶性N - 乙基马来酰亚胺敏感因子附着蛋白受体（SNARE）复合物是膜融合的核心分子机制，膜融合是驱动细胞间和细胞内通讯及运输的一个基本过程。一个仍存在争议的问题是SNAREs是否以及如何协同作用。在这里，我们展示了利用自组装的DNA纳米结构环来构建均匀大小的小单层囊泡，这些囊泡含有预定最大数量的面向外部的SNAREs，以研究膜融合过程。我们还将脂质共轭的互补单链DNA作为连接物掺入囊泡膜和靶膜中，这使得融合反应的限速对接步骤得以绕过，并允许在低至每个囊泡一对SNARE密度下直接观察单个膜融合事件。利用这个平台，我们在单个事件水平上证实，在模板化的小单层囊泡与支持脂质双层（SBL）对接后，一到两对SNAREs足以驱动快速脂质混合。该平台的模块化和可编程性使其易于用于研究涉及辅助蛋白的更复杂系统。
