VISUALIZATION OF EXOCYTOSIS DURING SEA-URCHIN EGG FERTILIZATION USINGCONFOCAL MICROSCOPY

A Ca2+ wave at fertilization triggers cortical granule exocytosis in s ea urchin eggs. New methods for visualizing exocytosis of individual c ortical granules were developed using fluorescent probes and confocal microscopy. Electron microscopy previously provided evidence that cort ical granule exocytosis results in the formation of long-lived depress ions in the cell surface. Fluorescent dextran or ovalbumin in the sea water seemed to label these depressions and appeared by confocal micro scopy as disks. FM1-43, a water-soluble fluorescent dye which labels m embranes in contact with the sea water, seemed to label the membrane o f these depressions and appeared as rings. In double-labeling experime nts, the disk and ring labeling by the two types of fluorescent dyes w ere coincident to within 0.5 second. The fluorescent labeling is coinc ident with the disappearance of cortical granules by transmitted light microscopy, demonstrating that the labeling corresponds to cortical g ranule exocytosis. Fluorescent labeling was simultaneous with an expan sion of the space occupied by the cortical granule, and labeling by th e fluorescent dextran was found to take 0.1-0.2 second. These results are consistent with, and reinforce the previous electron microscopic e vidence for, long-lived depressions formed by exocytosis; in addition, the new methods provide new ways to investigate cortical granule exoc ytosis in living eggs. The fluorescence labeling methods were used wit h the Ca2+ indicator Ca Green-dextran to test if Ca2+ and cortical gra nule exocytosis are closely related spatially and temporally. In any g iven region of the cortex, Ca2+ increased relatively slowly. Furthermo re, Ca2+ began to rise about 8 seconds before the first fluorescent la beling of exocytosis, suggesting that it takes a relatively long time for the Ca2+ concentration to reach a threshold value that triggers fu sion. No evidence was found for highly localized Ca2+ increases that c ould account for the pattern of individual exocytic events. These resu lts indicate that there is a long lag period between Ca2+ rise and cor tical granule exocytosis, and that Ca2+ and cortical granule exocytosi s are not tightly coupled spatially and temporally.