Terasaki M, Jaffe L A, Hunnicutt G R, Hammer J A
Marine Biological Laboratory, Woods Hole, Massachusetts 02543, USA.
Dev Biol. 1996 Nov 1;179(2):320-8. doi: 10.1006/dbio.1996.0263.
Green fluorescent protein (GFP) was targeted to the lumen of the endoplasmic reticulum (ER) of starfish eggs by injecting mRNA coding for a chimeric protein containing a signal sequence and the KDEL ER retention sequence. By confocal microscopy, the GFP chimeric protein was localized in intracellular cisternae (membrane sheets) and the nuclear envelope, showing that it had been successfully targeted to the ER. The labeling pattern closely resembled that produced by the fluorescent dicarbocyanine DiI, which has been used previously to label the ER (Jaffe and Terasaki, Dev. Biol. 164, 579-587, 1994). Eggs expressing the GFP chimera were used to examine whether there is a loss of ER continuity at fertilization. The time required for recovery of fluorescence after photobleaching for both the GFP chimera and DiI was much longer in eggs at 1 min postfertilization than in unfertilized eggs or in 20-min-postfertilized eggs. This result provides strong evidence for a transient loss of continuity of the ER associated with Ca release at fertilization.
通过注射编码包含信号序列和KDEL内质网滞留序列的嵌合蛋白的mRNA，绿色荧光蛋白（GFP）被靶向到海星卵的内质网（ER）腔中。通过共聚焦显微镜观察，GFP嵌合蛋白定位于细胞内池（膜片层）和核膜，表明它已成功靶向内质网。标记模式与荧光双碳菁染料DiI产生的模式非常相似，DiI先前已用于标记内质网（Jaffe和Terasaki，《发育生物学》164，579 - 587，1994）。表达GFP嵌合体的卵用于研究受精时内质网的连续性是否丧失。受精后1分钟的卵中，GFP嵌合体和DiI光漂白后荧光恢复所需的时间比未受精卵或受精后20分钟的卵长得多。这一结果为受精时与钙释放相关的内质网连续性短暂丧失提供了有力证据。
