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A cell-free system for regulated exocytosis in PC12 cells.

作者信息

Avery J, Ellis D J, Lang T, Holroyd P, Riedel D, Henderson R M, Edwardson J M, Jahn R

机构信息

Department of Neurobiology, Max-Planck-Institute for Biophysical Chemistry, Am Fassberg, D-37077 Göttingen, Germany.

出版信息

J Cell Biol. 2000 Jan 24;148(2):317-24. doi: 10.1083/jcb.148.2.317.

DOI:10.1083/jcb.148.2.317
PMID:10648564
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC2174285/
Abstract

We have developed a cell-free system for regulated exocytosis in the PC12 neuroendocrine cell line. Secretory vesicles were preloaded with acridine orange in intact cells, and the cells were sonicated to produce flat, carrier-supported plasma membrane patches with attached vesicles. Exocytosis resulted in the release of acridine orange which was visible as a disappearance of labeled vesicles and, under optimal conditions, produced light flashes by fluorescence dequenching. Exocytosis in vitro requires cytosol and Ca(2+) at concentrations in the micromolar range, and is sensitive to Tetanus toxin. Imaging of membrane patches at diffraction- limited resolution revealed that 42% of docked granules were released in a Ca(2+)-dependent manner during 1 min of stimulation. Electron microscopy of membrane patches confirmed the presence of dense-core vesicles. Imaging of membrane patches by atomic force microscopy revealed the presence of numerous particles attached to the membrane patches which decreased in number upon stimulation. Thus, exocytotic membrane fusion of single vesicles can be monitored with high temporal and spatial resolution, while providing access to the site of exocytosis for biochemical and molecular tools.

摘要
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b85b/2174285/88faa5944615/JCB9907073.f9.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b85b/2174285/b7d592b1fa78/JCB9907073.f1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b85b/2174285/b3c3a2618a16/JCB9907073.f2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b85b/2174285/0a3738d0f3a7/JCB9907073.f3.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b85b/2174285/deb5a41d644f/JCB9907073.f4.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b85b/2174285/dac66eee342f/JCB9907073.f5.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b85b/2174285/5f512763bf7c/JCB9907073.f7.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b85b/2174285/389916cd1741/JCB9907073.f6.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b85b/2174285/5ce950dcd8e1/JCB9907073.f8.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b85b/2174285/88faa5944615/JCB9907073.f9.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b85b/2174285/b7d592b1fa78/JCB9907073.f1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b85b/2174285/b3c3a2618a16/JCB9907073.f2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b85b/2174285/0a3738d0f3a7/JCB9907073.f3.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b85b/2174285/deb5a41d644f/JCB9907073.f4.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b85b/2174285/dac66eee342f/JCB9907073.f5.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b85b/2174285/5f512763bf7c/JCB9907073.f7.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b85b/2174285/389916cd1741/JCB9907073.f6.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b85b/2174285/5ce950dcd8e1/JCB9907073.f8.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b85b/2174285/88faa5944615/JCB9907073.f9.jpg

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本文引用的文献

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Stages of regulated exocytosis.受调控的胞吐作用的阶段。
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Membrane fusion and exocytosis.膜融合与胞吐作用。
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