A fluorescence dequenching method for monitoring exocytotic membrane fusion in fertilization of single sea urchin eggs

Abstract

A method for monitoring exocytotic membrane fusion by using a fluorescent membrane probe is presented. The method is based on the relief from concentration‐dependent self‐quenching (dequenching) of fluorescence of 5‐N‐(octadecanoyl)aminofluorescein (AF18), an amphiphilic derivative of fluorescein. The validity and usefulness of this method were shown by the following results: 1) self‐quenching of AF18 fluorescence occurred in the plasma membrane of unfertilized eggs of a sea urchin, Pseudocentrotus depressus, which were heavily stained with the fluorescent dye; 2) dequenching of AF18 fluorescence occurred upon fertilization in normal eggs but not in EGTA‐injected eggs; 3) Ca2+ induced both AF18 fluorescence dequenching and cortical granule disappearance in the isolated sea urchin egg cortex; and 4) simultaneous measurements of the intracellular Ca2+ concentration ([Ca2+]i) and dequenching of AF18 fluorescence by using a simple one‐excitation and two‐emission wavelength system.