STRUCTURAL-CHANGE OF THE ENDOPLASMIC-RETICULUM DURING FERTILIZATION -EVIDENCE FOR LOSS OF MEMBRANE CONTINUITY USING THE GREEN FLUORESCENT PROTEIN

Green fluorescent protein (GFP) was targeted to the lumen of the endop lasmic reticulum (ER) of starfish eggs by injecting mRNA coding for a chimeric protein containing a signal sequence and the KDEL ER retentio n sequence. By confocal microscopy, the GFP chimeric protein was local ized in intracellular cisternae (membrane sheets) and the nuclear enve lope, showing that it had been successfully targeted to the ER. The la beling pattern closely resembled that produced by the fluorescent dica rbocyanine 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 eg gs at 1 min postfertilization than in unfertilized eggs or in 20 min-p ostfertilized eggs. This result provides strong evidence for a transie nt loss of continuity of the ER associated with Ca release at fertiliz ation. (C) 1996 Academic Press, Inc.