Supplementary Material for: Disassembly of Subplasmalemmal Actin Filaments Induces Cytosolic Ca²⁺ Increases in Astropecten aranciacus Eggs

Background/Aims: Eggs of all animal species display intense cytoplasmic Ca²⁺ 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 Ca²⁺ increases are related to the disassembly of actin flaments: i) not only LAT-A but also other agents depolymerizing F-actin (i.e. cytochalasin B and mycalolide B) induced similar Ca²⁺ 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 Ca²⁺ increases. Further studies utilizing pharmacological inhibitors of PLC (U-73122 and neomycin), dominant negative mutant of PLC-ɣ, specific sequestration of PIP2 (RFP-PH), InsP₃ uncaging, and quantitation of endogenous InsP₃ all indicated that LAT-A induces Ca²⁺ increases by stimulating PLC rather than sensitizing InsP₃ receptors. In support of the idea, it bears emphasis that LAT-A timely increased intracellular contents of InsP₃ 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 Ca²⁺ signaling.