We targeted Green Fluorescent Protein to the ER lumen of starfish eggs. By confocal microscopy, the GFP chimera labeled the ER cisternae in the interior and the germinal vesicle membrane. At fertilization, the ER structure became altered by about 2 minutes, then returned by about 20 minutes. FRAP experiments provide strong evidence that the ER became transiently discontinuous as it released Ca.
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This work was done in collaboration with John Hammer, Laurinda Jaffe and Gary Hunnicutt.
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Email correspondence: John Hammer, Laurinda Jaffe, and Gary Hunnicutt.
Abstract from the paper
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 ("GFP-KDEL") containing a signal sequence and the KDEL ER retention sequence. By confocal microscopy, GFP-KDEL was localized in intracellular 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 (Jaffe and Terasaki, Dev. Biol. 164: 579-587, 1994), confirming the previous use of DiI to label the ER. GFP-KDEL expressing eggs 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 GFP-KDEL and DiI was much longer in eggs 1 min post-fertilization than in unfertilized eggs or 20 min post-fertilized eggs. This result provides strong evidence for a transient loss of continuity of the ER associated with Ca release at fertilization. We also demonstrate that GFP chimeras can be quantitated by methods that are potentially useful for protein synthesis studies. GFP-KDEL is likely to be a highly specific and generally useful fluorescent marker for the ER in many cells.