Visualization of exocytosis during sea urchin egg fertilization using confocal microscopy.
Visualization of exocytosis during sea urchin egg fertilization using confocal microscopy.
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发表时间：
1995-06
影响因子：
4
通讯作者：
Mark Terasaki
中科院分区：
文献类型：
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作者：
Mark Terasaki
受精时的钙波触发海胆卵的皮质颗粒胞吐。利用荧光探针和共聚焦显微镜开发了显示单个皮质颗粒胞吐作用的新方法。电子显微镜以前提供的证据表明，皮质颗粒胞吐导致细胞表面形成长期凹陷。海水中的荧光葡聚糖或卵清蛋白似乎标记了这些凹陷，并在共聚焦显微镜下显示为圆盘。FM1-43是一种水溶性荧光染料，用来标记与海水接触的膜，似乎标记了这些凹陷的膜，看起来像是环。在双标记实验中，两种荧光染料的圆盘标记和环标记在0.5秒内重合。荧光标记与透射光学显微镜下皮质颗粒的消失一致，表明该标记对应于皮质颗粒的胞吐作用。荧光标记与皮质颗粒所占空间的扩大是同步的，而荧光葡聚糖标记所需时间为0.1-0.2秒。这些结果与先前的电子显微镜证据一致，并强化了由胞吐作用形成的长寿凹陷；此外，新方法为研究活鸡蛋中的皮质颗粒胞吐提供了新的方法。用荧光标记法结合钙指示剂Ca Green-葡聚糖检测钙离子与皮质颗粒胞吐在时间和空间上是否密切相关。在任何给定的皮质区域，钙离子的增加都相对较慢。
A Ca2+ wave at fertilization triggers cortical granule exocytosis in sea urchin eggs. New methods for visualizing exocytosis of individual cortical granules were developed using fluorescent probes and confocal microscopy. Electron microscopy previously provided evidence that cortical granule exocytosis results in the formation of long-lived depressions in the cell surface. Fluorescent dextran or ovalbumin in the sea water seemed to label these depressions and appeared by confocal microscopy as disks. FM 1-43, a water-soluble fluorescent dye which labels membranes in contact with the sea water, seemed to label the membrane of these depressions and appeared as rings. In double-labeling experiments, the disk and ring labeling by the two types of fluorescent dyes were coincident to within 0.5 second. The fluorescent labeling is coincident with the disappearance of cortical granules by transmitted light microscopy, demonstrating that the labeling corresponds to cortical granule exocytosis. Fluorescent labeling was simultaneous with an expansion of the space occupied by the cortical granule, and labeling by the fluorescent dextran was found to take 0.1-0.2 second. These results are consistent with, and reinforce the previous electron microscopic evidence for, long-lived depressions formed by exocytosis; in addition, the new methods provide new ways to investigate cortical granule exocytosis in living eggs. The fluorescence labeling methods were used with the Ca2+ indicator Ca Green-dextran to test if Ca2+ and cortical granule exocytosis are closely related spatially and temporally. In any given region of the cortex, Ca2+ increased relatively slowly.(ABSTRACT TRUNCATED AT 250 WORDS)