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Reconstituted Fusion Pore

Aleksandar Jeremic ^*, Marie Kelly ^*, Sang-Joon Cho ^*, Marvin H. Stromer  and Bhanu P. Jena ^*

^* Department of Physiology, Wayne State University School of Medicine, Detroit, Michigan 48201; and Department of Animal Science, Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, Iowa 50011

Correspondence: Address reprint requests to Bhanu P. Jena, Ph.D., Dept. of Physiology, Wayne State University School of Medicine, 5239 Scott Hall, 540 E. Canfield Ave., Detroit, MI 48201-4177. Tel.: 313-577-1532; Fax: 313-993-4177; E-mail: bjena{at}med.wayne.edu.

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.

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