Disassembly of Subplasmalemmal Actin Filaments Induces Cytosolic Ca2+ Increases in Astropecten aranciacus Eggs
Cellular Physiology and Biochemistry
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 Ca 2+ 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
... s of phospholipase C (PLC), and the changes of the intracellular Ca 2+ 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-ɣ, 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. Owing to the large cell size and other unique characters, oocytes and eggs have been utilized for a variety of biological researches on a single cell basis  . Like nerve and muscle cells, oocytes and eggs are electrically excitable, as their plasma membrane is studded with various voltage-sensitive ion channels  . In addition, their cytoplasm comprises an excitable matrix that can propagate intracellular Ca 2+ waves by mobilizing the internal stores such as endoplasmic reticulum (ER). This is due to the concerted actions of intracellular ion channels on the Ca 2+ store that respond to Ca 2+ -releasing second messengers such as inositol 1,4,5-trisphosphate (InsP 3 ), cyclic ADP-ribose (cADPr), and nicotinic acid adenine dinucleotide (NAADP)     . Thus, occurrence of active ion fluxes across the intracellular and plasma membranes during physiological processes is one of the characteristic features of oocytes and eggs. Indeed, the first visual demonstration of intracellular Ca 2+ waves was made with the fertilized eggs of fish and echinoderms by use of Ca 2+ -sensitive fluorescent probes [7, 8] . Fertilized eggs of all animal species manifest a massive increase of intracellular Ca 2+ mainly in two modes, i.e. influx from the extracellular media and the ligand-gated release from the intracellular stores, and the Ca 2+ signals play important roles in the resumption of cell cycle and the subsequent embryonic development [5, 6,      . When fertilized, echinoderm eggs loaded with calcium dye readily display a rapid, but short-lived, synchronized increase of Ca 2+ underneath the entire plasma membrane. This Ca 2+ signal is called 'cortical flash' and is known to be linked to swift depolarization of the membrane potential and the opening of L-type Ca 2+ channels    . Cortical flash alone, however, is not sufficient to activate eggs at fertilization, and requires a prolonged and more intense Ca 2+ response that starts at the sperm interaction site and propagates to the antipode. This Ca 2+ wave is accompanied by a contraction wave and exocytosis of cortical granules. The extruded contents of cortical granules are thus deposited outside the plasma membrane, which elevates the vitelline layer and form fertilization envelope to protect the embryo. Furthermore, the Ca 2+ wave in the fertilized eggs triggers a series of biochemical and cytological changes that are collectively termed 'egg activation' [13,    . While the crucial roles played by Ca 2+ in egg activation were demonstrated by use of calcium ionophores or chelators [20, 21] , the precise mechanisms by which the successful sperm triggers the Ca 2+ wave in fertilized eggs have not been fully understood and may substantially differ from species to species. Among several hypotheses set forth, the 'receptor model' suggests a biochemical pathway involving a signal-transducing receptor on the egg surface, whereas the 'soluble sperm factor model' proposes sperm-borne signaling substances diffusing into the egg to evoke a Ca 2+ increase. Nonetheless, the common denominator of the two models is the de novo synthesis or increment of the aforementioned Ca 2+ -releasing second messengers, e.g. InsP 3 , cADPr, and NAADP, at the end of the signaling cascade, which bind to their cognate ligand-gated ion channels on the Ca 2+ stores [5, 22] . However, the contribution made by each second messenger may be quite different depending on the animal species. For example, whereas cADPr may provide an alternative path to generate the Ca 2+ wave in fertilized eggs of sea urchin, its contribution in the fertilized eggs of certain species of starfish (e.g. Asterina pectinifera) appears to be negligible. Furthermore, while NAADP evokes a strong Ca 2+ response in A. pectinifera, its effect inside the eggs of another species of starfish (e.g. Astropecten aranciacus) is remarkably weaker      . On the other hand, the seemingly universal pathway involving InsP 3 as the second messenger may have diverse modes of generating Ca 2+ waves in the fertilized eggs of different animal species  . This is because the enzyme that synthesizes InsP 3 and diacylglycerol from phosphatidylinositol 4,5-bisphosphate (PIP2) exists in multiple isoforms [22, 29, 30] . In mammalian eggs, for example, a sperm-specific isoform PLCζ is accountable for generating Ca 2+ waves at fertilization [31, 32] . PLCζ, however, does not seem to exist in the genome or transcriptome of sea urchin and starfish    , and the InsP 3 -dependent Ca 2+ increase in the fertilized eggs of echinoderms is mostly attributable to PLCɣ    , which is shown to be activated by Src-family kinase that interacts with the SH domain of PLCɣ within 15 sec after Preparation of oocytes Astropecten aranciacus were captured in the Gulf of Naples or at the sea near Gaeta during the breeding season (January to April) and transported to the Stazione Zoologica in Naples, Italy. Germinal vesicle (GV)stage oocytes were obtained by a small slit on the central dorsal area of female adult animals, and transferred to filter-sterilized seawater. Individual oocytes released from the gonad were sieved in gauze and rinsed several times. To induce meiotic maturation, oocytes were stimulated with 10 µM 1-methyladenine (1-MA) in seawater for 70 min. The oocytes at this stage were referred to as 'eggs' in this study. Actin drugs, chemicals, and reagents LAT-A and Jasplakinolide were purchased from Molecular Probes, and phalloidin from Invitrogen. Cytochalasin B, U-73122, U-73343, neomycin, and all other chemicals and agents were purchased from Sigma-Aldrich, unless specified otherwise, e.g., mycalolide B (Santa Cruz Biotechnology). All chemicals and reagents were utilized as described in manufacturers' instruction. Microinjection, photo-activation of caged InsP 3 , and Ca 2+ imaging Oocytes were microinjected with various agents and dyes, as previously described [44, 47] . The pipette concentrations of fluorescent calcium dye (Calcium Green conjugated with 10 kDa dextran, Molecular Probes) and caged InsP 3 (Molecular Probe) prepared in the injection buffer (10 mM Hepes, 0.1M potassium aspartate, pH 7.0) were 5 mg/ml and 2 µM, respectively. The caged InsP 3 was photo-liberated by irradiating the eggs with 330 nm UV light by use of the computer-controlled shutter system Lambda 10-2 (Sutter Instruments, Co., Novato, CA). Cytosolic Ca 2+ changes were recorded with a cooled CCD camera (MicroMax, Princeton Instruments, Inc., Trenton, NJ) mounted on a Zeiss Axiovert 200 microscope with a Plan-Neofluar 20x/0.50 objective. The quantified Ca 2+ signal was normalized with the baseline fluorescence (F 0 ) following the formula F rel = [F-F 0 ]/F 0 , where F represents the average fluorescence level over the entire oocyte. The incremental changes of the Ca 2+ rise was analyzed by applying the formula F inst = [(F t -F t-1 )/F t-1 ] to visualize the site of instantaneous Ca 2+ release. Fluorescent Ca 2+ images were analyzed with MetaMorph software package 7.7 (Universal Imaging Corporation, West Chester, PA, USA). Fluorescent fusion proteins ligated to the Pleckstrin Homology (PH) domain of rat PLC-δ1 The cDNA fragment encoding the PH domain (140 amino acid residues) that specifically interacts with PIP2 was prepared and fused to the expression vector (pET28, Novagen) containing GFP or RFP as described previously  . The fusion proteins expressed from the two plasmid were referred to as PH-GFP and RFP-PH, respectively, in respect of the configuration. F-actin staining and confocal microscopy F-actin was visualized in living oocytes by microinjecting AlexaFluor568-conjugated phalloidin (50 µM, pipette concentration) and examined with Zeiss LSM 510 META laser-scanning confocal microscope (Jena, Germany) by use of a Planar-Neofluar 25x/0.80 objective water lens through a BP 560/610 emission filter. On the other hand, PH-GFP was visualized by use of the same confocal microscope with excitation at 488 nm and emission at 500/555 nm, wheras LifeAct-GFP and RFP-SH2 were visualized by Leica TCS SP8 X with the WLL laser. Transmitted light and fluorescence confocal images were acquired from the equatorial planes. Cloning of the Src Homology 2 (SH2) domains from A. aranciacus PLC-ɣ The cDNA fragment encoding the tandem SH2 domains were cloned through RT-PCR by use of degenerate primers elected from the most conserved regions. To this end, the protein sequences of PLC-ɣ from diverse animal species were aligned (zebrafish, GenBank: AY163168; Asterina miniata, AY486068; sponge, BAA76275; human, ABB84466; Paracentrotus lividus, CAB38087; Drosophila melanogaster, BAA06189), and a pair of degenerate primers were prepared: the forward primer (5'-GCGCGGGAATTCGAyTGyTGGGAyGG -3') from the target amino acid residues DCWDG, and the reverse primer (5'-GCGCGCAAGCTTyTTnCCrTGrAACCA-3') from the peptide sequence WFHGK Statistical analysis The average and variation of the data were reported as 'mean ± standard deviation (SD)' in all cases in this manuscript. Oneway ANOVA and t-tests were performed by use of Prism 3.0 (GraphPad Software), and P<0.05 was considered as statistically significant. For ANOVA results showing P<0.05, statistical significance Fig. 10 . Massive depolymerization of subplasmalemmal actin filaments in the starfish eggs exposed to LAT-A. A. aranciacus eggs microinjected with LifeAct-GFP fusion protein (6 µg/µl, pipette concentration) were exposed to 6 µM LAT-A or to the solvent of the drug (0.1% DMSO, final concentration), and the changes of the actin filaments were monitored by confocal microscopy. Whereas LAT-A induced extensive depolymerization of subplasmalemmal actin filaments by 10 min (arrow), the control eggs exposed to 0.1% DMSO lacked such changes even after extended incubation. Thus, the drastic changes of the actin filaments and egg activation are due to the legitimate effect of the actin drug, and not to the spontaneous changes of the actin cytoskeleton or to the side effect of DMSO.