title: Association between myristoylated alanin-rich C kinase substrate (MARCKS) translocation and cortical granule exocytosis in rat eggs.
abstract: Cortical granule exocytosis (CGE), following egg activation, is a secretory process that blocks polyspermy and enables successful embryonic development. CGE can be triggered independently by either a rise in intracellular calcium concentration ([Ca2+]i) or activation of protein kinase C (PKC). The present study investigates the signal transduction pathways leading to CGE through activation of PKC or stimulation of a rise in [Ca2+]i. Using Western blot analysis, co-immunoprecipitation and immunohistochemistry, combined with various inhibitors or activators, we investigated the link between myristoylated alanin-rich C kinase substrate (MARCKS) translocation and CGE. We were able to demonstrate translocation of MARCKS from the plasma membrane to the cortex, in fertilized as well as in parthenogenetically activated eggs. MARCKS phosphorylation was demonstrated upon PKC activation, whereas a PKC inhibitor (myrPKCpsi) prevented both MARCKS translocation and CGE in 12-O-tetradecanoyl phorbol-13-acetate (TPA)-activated eggs. We have further shown that upon egg activation the amount of phosphorylated MARCKS (p-MARCKS) and the amount of calmodulin bound to MARCKS were increased. MARCKS translocation in ionomycin activated eggs was also inhibited by the calmodulin inhibitor N-(6-aminohexyl)-5-chloro-1-napthalenesulfonamide hydrochloride (W7). These results complement other studies showing MARCKS requirement for exocytosis and imply that upon fertilization, MARCKS translocation is followed by CGE. These findings present a significant contribution to our understanding of CGE in mammalian eggs in particular, as well as cellular exocytosis in general.
authors.affiliation: Department of Cell and Developmental Biology, Sackler Faculty of Medicine, Tel Aviv University, Ramat Aviv, Tel Aviv 69978, Israel.
authors.forename: Efrat
authors.initials: E
authors.lastname: Eliyahu
authors.forename: Nataly
authors.initials: N
authors.lastname: Shtraizent
authors.forename: Alina
authors.initials: A
authors.lastname: Tsaadon
authors.forename: Ruth
authors.initials: R
authors.lastname: Shalgi
mesh_terms: D000818:Animals; D001692:Biological Transport; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000037:antagonists & inhibitors; D002462:Cell Membrane / Q000378:metabolism; D003594:Cytoplasmic Granules / Q000502:physiology*; D004075:Diglycerides / Q000494:pharmacology; D004789:Enzyme Activation; D005089:Exocytosis; D005260:Female; D015151:Immunoblotting; D047468:Immunoprecipitation; D047908:Intracellular Signaling Peptides and Proteins / Q000032:analysis / Q000378:metabolism*; D015759:Ionomycin / Q000494:pharmacology; D008565:Membrane Proteins / Q000032:analysis / Q000378:metabolism*; D018613:Microscopy, Confocal; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D010063:Ovum / Q000201:enzymology / Q000502:physiology*; D010312:Parthenogenesis; D011247:Pregnancy; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D015398:Signal Transduction / Q000502:physiology*; D013084:Sperm-Ovum Interactions / Q000502:physiology; D013449:Sulfonamides / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
chemical_list: D002147:Calmodulin; D004075:Diglycerides; D047908:Intracellular Signaling Peptides and Proteins; C000619712:Marcks protein, rat; D008565:Membrane Proteins; D013449:Sulfonamides; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D015759:Ionomycin; C017967:W 7; C038237:1-oleoyl-2-acetylglycerol; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium