Supplementary Material for: Disassembly of Subplasmalemmal Actin Filaments Induces Cytosolic Ca<sup>2+</sup> Increases in <b><i>Astropecten aranciacus</i></b> Eggs

<b><i>Background/Aims:</i></b> Eggs of all animal species display intense cytoplasmic Ca<sup>2+</sup> increases at fertilization. Previously, we reported that unfertilized eggs of <i>Astropecten aranciacus</i> exposed to an actin drug latrunculin A (LAT-A) exhibit similar Ca<sup>2+</sup> waves and cortical flashes after 5-10 min time lag. Here, we have explored the molecular mechanisms underlying this unique phenomenon. <b><i>Methods:</i></b> Starfish eggs were pretreated with various agents such as other actin drugs or inhibitors of phospholipase C (PLC), and the changes of the intracellular Ca<sup>2+</sup> 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. <b><i>Results:</i></b> We have shown that the LAT-A-induced Ca<sup>2+</sup> increases are related to the disassembly of actin flaments: <i>i)</i> not only LAT-A but also other agents depolymerizing F-actin (i.e. cytochalasin B and mycalolide B) induced similar Ca<sup>2+</sup> increases, albeit with slightly lower efficiency; <i>ii)</i> drugs stabilizing F-actin (i.e. phalloidin and jasplakinolide) either blocked or significantly delayed the LAT-A-induced Ca<sup>2+</sup> increases. Further studies utilizing pharmacological inhibitors of PLC (U-73122 and neomycin), dominant negative mutant of PLC-ɣ, specific sequestration of PIP2 (RFP-PH), InsP<sub>3</sub> uncaging, and quantitation of endogenous InsP<sub>3</sub> all indicated that LAT-A induces Ca<sup>2+</sup> increases by stimulating PLC rather than sensitizing InsP<sub>3</sub> receptors. In support of the idea, it bears emphasis that LAT-A timely increased intracellular contents of InsP<sub>3</sub> with concomitant decrease of PIP2 levels in the plasma membrane. <b><i>Conclusion:</i></b> 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<sup>2+</sup> signaling.