Jeremic Aleksandar, Kelly Marie, Cho Sang-Joon, Stromer Marvin H, Jena Bhanu P
Department of Physiology, Wayne State University School of Medicine, Detroit, Michigan 48201, USA.
Biophys J. 2003 Sep;85(3):2035-43. doi: 10.1016/S0006-3495(03)74631-1.
Fusion pores or porosomes are basket-like structures at the cell plasma membrane, at the base of which, membrane-bound secretory vesicles dock and fuse to release vesicular contents. Earlier studies using atomic force microscopy (AFM) demonstrated the presence of fusion pores at the cell plasma membrane in a number of live secretory cells, revealing their morphology and dynamics at nm resolution and in real time. ImmunoAFM studies demonstrated the release of vesicular contents through the pores. Transmission electron microscopy (TEM) further confirmed the presence of fusion pores, and immunoAFM, and immunochemical studies demonstrated t-SNAREs to localize at the base of the fusion pore. In the present study, the morphology, function, and composition of the immunoisolated fusion pore was investigated. TEM studies reveal in further detail the structure of the fusion pore. Immunoblot analysis of the immunoisolated fusion pore reveals the presence of several isoforms of the proteins, identified earlier in addition to the association of chloride channels. TEM and AFM micrographs of the immunoisolated fusion pore complex were superimposable, revealing its detail structure. Fusion pore reconstituted into liposomes and examined by TEM, revealed a cup-shaped basket-like morphology, and were functional, as demonstrated by their ability to fuse with isolated secretory vesicles.
融合孔或孔体是位于细胞质膜上的篮状结构，在其基部，膜结合的分泌囊泡对接并融合以释放囊泡内容物。早期使用原子力显微镜（AFM）的研究表明，在许多活的分泌细胞的细胞质膜上存在融合孔，揭示了它们在纳米分辨率下的形态和动力学，并能实时观察。免疫AFM研究表明囊泡内容物通过这些孔释放。透射电子显微镜（TEM）进一步证实了融合孔的存在，免疫AFM和免疫化学研究表明t-SNARE定位于融合孔的基部。在本研究中，对免疫分离的融合孔的形态、功能和组成进行了研究。TEM研究更详细地揭示了融合孔的结构。对免疫分离的融合孔进行免疫印迹分析，除了氯离子通道的关联外，还揭示了先前鉴定的几种蛋白质同工型的存在。免疫分离的融合孔复合体的TEM和AFM显微照片可叠加，揭示了其详细结构。重构到脂质体中并通过TEM检查的融合孔呈现杯状篮状形态，并且具有功能，这通过它们与分离的分泌囊泡融合的能力得以证明。
