Disassembly of subplasmalemmal actin filaments induces cytosolic Ca 2+ increases in astropecten aranciacus eggs

Abstract

Background/Aims: Eggs of all animal species display intense cytoplasmic Ca 2+ increases at fertilization. Previously, we reported that unfertilized eggs of Astropecten aranciacus exposed to an actin drug latrunculin A (LAT-A) exhibit similar Ca2+ waves and cortical flashes after 5-10 min time lag. Here, we have explored the molecular mechanisms underlying this unique phenomenon. Methods: Starfish eggs were pretreated with various agents such as other actin drugs or inhibitors of phospholipase C (PLC), and the changes of the intracellular Ca2+ levels were monitored by use of Calcium Green in the presence or absence of LAT-A. The concomitant changes of the actin cytoskeleton were visualized with fluorescent F-Actin probes in confocal microscopy. Results: We have shown that the LAT-A-induced Ca 2+ increases are related to the disassembly of actin filaments: i) not only LAT-A but also other agents depolymerizing F-Actin (i.e. cytochalasin B and mycalolide B) induced similar Ca 2+ increases, albeit with slightly lower efficiency; ii) drugs stabilizing F-Actin (i.e. phalloidin and jasplakinolide) either blocked or significantly delayed the LAT-A-induced Ca 2+ increases. Further studies utilizing pharmacological inhibitors of PLC (U-73122 and neomycin), dominant negative mutant of PLC-8, specific sequestration of PIP2 (RFP-PH), InsP 3 uncaging, and quantitation of endogenous InsP 3 all indicated that LAT-A induces Ca 2+ increases by stimulating PLC rather than sensitizing InsP 3 receptors. In support of the idea, it bears emphasis that LAT-A timely increased intracellular contents of InsP 3 with concomitant decrease of PIP2 levels in the plasma membrane. Conclusion: Taken together, our results suggest that suboolemmal actin filaments may serve as a scaffold for cell signaling and modulate the activity of the key enzyme involved in intracellular Ca 2+ signaling.