IMAGING PROTEIN-KINASE-C ACTIVATION IN LIVING SEA-URCHIN EGGS AFTER FERTILIZATION

The fluorescent dye NBD-phorbol acetate was used to visualize the acti vation of protein kinase C (PKC) in living Lptechinus pictus eggs duri ng fertilization. This dye interacts directly with PKC as determined u sing a competitive binding assay. Quantitative image analysis of seque ntial images from laser-scanning confocal microscopy showed a signific ant reorganization of the signal in the vicinity of the cortical granu les and the plasma membrane that began immediately following fertiliza tion and persisted up to 1 hr (P < 0.0001). At the concentrations empl oyed, the NBD-phorbol dye was not capable of inducing a significant tr anslocation of the fluorescent signal to the membrane, nor did it appe ar to interfere with the cell cycle. It therefore seems likely that th e present in vivo results reflect the previously reported in vitro act ivation of protein kinase C immediately subsequent to fertilization. S uch an interpretation is parsimonious with the results of parallel sub cellular fractionation experiments using an N-terminal polyclonal anti body to sea urchin PRC which showed a significant (P < 0.037) transloc ation of the enzyme from the cytosolic fraction to the membrane fracti on 40 min subsequent to fertilization. This study supports and extends previous in vitro data suggesting that PKC activation subsequent to f ertilization occurs at or near the egg plasma membrane, perhaps in ass ociation with arachadonic acid-rich cortical granules. (C) 1995 Academ ic Press, Inc.