Schwenen Lando L G, Hubrich Raphael, Milovanovic Dragomir, Geil Burkhard, Yang Jian, Kros Alexander, Jahn Reinhard, Steinem Claudia
Institute for Organic and Biomolecular Chemistry, University of Göttingen, Tammannstr. 2, 37077 Göttingen, Germany.
Max-Planck-Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany.
Sci Rep. 2015 Jul 13;5:12006. doi: 10.1038/srep12006.
Even though a number of different in vitro fusion assays have been developed to analyze protein mediated fusion, they still only partially capture the essential features of the in vivo situation. Here we established an in vitro fusion assay that mimics the fluidity and planar geometry of the cellular plasma membrane to be able to monitor fusion of single protein-containing vesicles. As a proof of concept, planar pore-spanning membranes harboring SNARE-proteins were generated on highly ordered functionalized 1.2 μm-sized pore arrays in Si3N4. Full mobility of the membrane components was demonstrated by fluorescence correlation spectroscopy. Fusion was analyzed by two color confocal laser scanning fluorescence microscopy in a time resolved manner allowing to readily distinguish between vesicle docking, intermediate states such as hemifusion and full fusion. The importance of the membrane geometry on the fusion process was highlighted by comparing SNARE-mediated fusion with that of a minimal SNARE fusion mimetic.
尽管已经开发了许多不同的体外融合测定法来分析蛋白质介导的融合,但它们仍然只能部分捕捉体内情况的基本特征。在这里,我们建立了一种体外融合测定法,该方法模拟细胞质膜的流动性和平面几何形状,以便能够监测单个含蛋白质囊泡的融合。作为概念验证,在Si3N4中高度有序的功能化1.2μm大小的孔阵列上生成了含有SNARE蛋白的平面跨孔膜。通过荧光相关光谱法证明了膜成分的完全流动性。通过双色共聚焦激光扫描荧光显微镜以时间分辨的方式分析融合,从而能够轻松区分囊泡对接、半融合和完全融合等中间状态。通过将SNARE介导的融合与最小SNARE融合模拟物的融合进行比较,突出了膜几何形状对融合过程的重要性。