Imaging protein kinase C activation in living sea urchin eggs after fertilization

The fluorescent dye NBD-phorbol acetate was used to visualize the activation of protein kinase C (PKC) in living Lytechinus pictus eggs during fertilization. The dye interacts directly with PKC as determined using a competitive binding assay. Quantitative image analysis of sequential images from laser-scanning confocal microscopy showed a significant reorganization of the signal in the vicinity of the cortical granules and the plasma membrane that began immediately following fertilization and persisted up to 1 hr (P<0.0001). At the concentrations employed, the NBD-phorbol dye was not capable of inducing a significant translocation of the fluorescent signal to the membrane, nor did it appear to interfere with the cell cycle. It therefore seems likely that the present in vivo results reflect the previously reported in vitro activation of protein kinase C immediately subsequent to fertilization. Such an interpretation is parsimonious with the results of parallel subcellular fractionation experiments using an N-terminal polyclonal antibody to sea urchin PKC which showed a significant (P<0.037) translocation of the enzyme from the cytosolic fraction to the membrane fraction 40 min subsequent to fertilization. This study supports and extends previous in vitro data suggesting that PKC activation subsequent to fertilization occurs at or near the egg plasma membrane, perhaps in association with arachadonic acid-rich cortical granules.