A fluorescence assay for monitoring and analyzing fusion biological membrane vesicles in vitro.
A new technique has been developed to study fusion of biological membrane vesicles. Bovine chromaffin granule ghosts (CGG) were loaded with fluorescein isothiocyanate-dextran (FITC-dextran) at self-quenching concentrations. Loaded ghosts were then made to fuse with empty CGG. Fusion was induced by synexin, a protein previously proposed to be involved in exocytosis. The fusion process was monitored by measuring the dequenching of the fluorescence. Dequenching occurred as FITC-dextran was diluted into the increased volume due to fusion with empty ghosts. Spurious signals from leakage or breakage of vesicles were removed by including a specific anti-fluorescein antibody in the reaction medium. This new technique may prove to be of more general use for studying membrane fusion processes in other systems.
Stutzin
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D000313:Adrenal Medulla; D000818:Animals; D017310:Annexin A7; D002417:Cattle; D002837:Chromaffin Granules / Q000648:ultrastructure; D003911:Dextrans; D016650:Fluorescein-5-isothiocyanate / Q000031:analogs & derivatives*; D005452:Fluoresceins; D005456:Fluorescent Dyes; D007158:Immunologic Techniques; D007425:Intracellular Membranes / Q000502:physiology; D008561:Membrane Fusion / Q000187:drug effects; D011506:Proteins / Q000494:pharmacology; D012235:Rhodamines / Q000378:metabolism; D013050:Spectrometry, Fluorescence
D017310:Annexin A7; D003911:Dextrans; D005452:Fluoresceins; D005456:Fluorescent Dyes; D011506:Proteins; D012235:Rhodamines; C015219:fluorescein isothiocyanate dextran; D016650:Fluorescein-5-isothiocyanate; C044542:octadecyl Rhodamine B chloride