Department of Cell Biology and Anatomy, New York Medical College, Valhalla, New York 10595.
East Hospital, Tongji University School of Medicine, Shanghai 200120, China.
J Biol Chem. 2013 Jun 7;288(23):16872-16881. doi: 10.1074/jbc.M113.468231. Epub 2013 Apr 25.
Formation of the fusion pore is a central question for regulated exocytosis by which secretory cells release neurotransmitters or hormones. Here, by dynamically monitoring exocytosis of large vesicles (2-7 μM) in astrocytes with two-photon microscopy imaging, we found that the exocytotic fusion pore was generated from the SNARE-dependent fusion at a ring shape of the docked plasma-vesicular membrane and the movement of a fusion-produced membrane fragment. We observed two modes of fragment movements, 1) a shift fragment that shifted to expand the fusion pore and 2) a fall-in fragment that fell into the collapsed vesicle to expand the fusion pore. Shift and fall-in modes are associated with full and partial collapses of large vesicles, respectively. The astrocytic marker, sulforhodamine 101, stained the fusion-produced membrane fragment more brightly than FM 1-43. Sulforhodamine 101 imaging showed that double fusion pores could simultaneously occur in a single vesicle (16% of large vesicles) to accelerate discharge of vesicular contents. Electron microscopy of large astrocytic vesicles showed shift and fall-in membrane fragments. Two modes of fusion pore formation demonstrate a novel mechanism underlying fusion pore expansion and provide a new explanation for full and partial collapses of large secretory vesicles.
融合孔的形成是调控型胞吐作用的核心问题，通过这种作用，分泌细胞释放神经递质或激素。在这里，我们通过双光子显微镜成像动态监测星形胶质细胞中大囊泡（2-7 μM）的胞吐作用，发现胞吐融合孔是由 SNARE 依赖性融合在对接的质膜和融合产生的膜片段的运动下在环形状下产生的。我们观察到两种片段运动模式，1）移位片段，它移动以扩大融合孔，2）落入片段，它落入塌陷的囊泡以扩大融合孔。移位和落入模式分别与大囊泡的完全和部分塌陷相关。星形胶质细胞标记物，磺基罗丹明 101，比 FM 1-43 更亮地染色融合产生的膜片段。磺基罗丹明 101 成像显示，两个融合孔可以同时在单个囊泡（大囊泡的 16%）中发生，以加速囊泡内容物的释放。大星形胶质细胞囊泡的电子显微镜显示移位和落入的膜片段。融合孔形成的两种模式展示了融合孔扩展的新机制，并为大分泌囊泡的完全和部分塌陷提供了新的解释。
