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PLoS One
2010 Nov 23;511:e14100. doi: 10.1371/journal.pone.0014100.
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The biphasic increase of PIP2 in the fertilized eggs of starfish: new roles in actin polymerization and Ca2+ signaling.
Chun JT
,
Puppo A
,
Vasilev F
,
Gragnaniello G
,
Garante E
,
Santella L
.
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BACKGROUND: Fertilization of echinoderm eggs is accompanied by dynamic changes of the actin cytoskeleton and by a drastic increase of cytosolic Ca(2+). Since the plasma membrane-enriched phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2) serves as the precursor of inositol 1,4,5 trisphosphate (InsP(3)) and also regulates actin-binding proteins, PIP2 might be involved in these two processes.
METHODOLOGY/PRINCIPAL FINDINGS: In this report, we have studied the roles of PIP2 at fertilization of starfish eggs by using fluorescently tagged pleckstrin homology (PH) domain of PLC-δ1, which has specific binding affinity to PIP2, in combination with Ca(2+) and F-actin imaging techniques and transmission electron microscopy. During fertilization, PIP2 increased at the plasma membrane in two phases rather than continually decreasing. The first increase was quickly followed by a decrease about 40 seconds after sperm-egg contact. However, these changes took place only after the Ca(2+) wave had already initiated and propagated. The fertilized eggs then displayed a prolonged increase of PIP2 that was accompanied by the appearance of numerous spikes in the perivitelline space during the elevation of the fertilization envelope (FE). These spikes, protruding from the plasma membrane, were filled with microfilaments. Sequestration of PIP2 by RFP-PH at higher doses resulted in changes of subplasmalemmal actin networks which significantly delayed the intracellular Ca(2+) signaling, impaired elevation of FE, and increased occurrences of polyspermic fertilization.
CONCLUSIONS/SIGNIFICANCE: Our results suggest that PIP2 plays comprehensive roles in shaping Ca(2+) waves and guiding structural and functional changes required for successful fertilization. We propose that the PIP2 increase and the subsequent formation of actin spikes not only provide the mechanical supports for the elevating FE, but also accommodate increased membrane surfaces during cortical granule exocytosis.
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21124897
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Figure 1. The PH domain of PLC-δ1 specifically bind to plasma membrane PI(4,5)P2 in starfish oocytes.Immature oocytes of A. aranciacus were microinjected with PH-GFP (A) or RFP-PH (E) fusion proteins (150 µM, pipette concentration), and the equatorial plane was monitored with confocal microscopy. The control proteins (R40A mutants) without the capability of PIP2-binding were not localized to the plasma membrane (B, F). (C) Bright field view of the same oocyte microinjected with RFP-PH in panel A. (D) The line intensity profile of the PH-GFP signals corresponding to the interrupted line in panel A. Abbreviation: PM, plasma membrane; GV, germinal vesicle. Scale bar, 50 µm.
Figure 2. During fertilization, the intracellular Ca2+ release is followed by the biphasic increase of PIP2 at the plasma membrane.Mature eggs loaded with Calcium Green were microinjected with RFP-PH (150 µM, pipette concentration) to monitor the changes of PIP2 and sperm-induced Ca2+ signals. A representative result from 15 independent experiments was presented. (A) Changes of RFP-PH signals at the site of fertilization. The moment the spermatozoon is stopped at the egg surface was set to t = 0:00 (min:sec). The onset of PIP2 increment is evident by 0:36 (arrows). (B) Images of instantaneous increment (Finst = [(Ft-Ft−1)/Ft−1]) in Ca2+ release (green) and PIP2 (red) were merged with the light transmission photomicrographs of the fertilized egg at the corresponding time points. The position of the fertilizing sperm was indicated with an arrow. (C) Temporal relationship between sperm-induced Ca2+ signaling (Frel = [F−F0]/F0; green curve) and the fluctuation of the relative fluorescence (Frel = [Fpm−Fct]/Fct; red curve) for plasma membrane PIP2 in the region of the framed area of the sperm-egg interaction.
Figure 3. The increase of PIP2 at the fertilization site is accompanied by formation of numerous spikes that protrude the plasma membrane concomitantly with the fertilization envelope elevation.Mature eggs were microinjected either with RFP-PH (150 µM, pipette concentration) or with its control protein (R40A mutant) prior to fertilization. (A) Confocal microscopic images of RFP-PH fluorescence (left column) and the corresponding bright field views. The moment of sperm-egg contact was set to t = 0 (top panels). At 2 min, as the fertilization envelope started to be elevated, RFP-PH began to visualize spikes formation (white arrows). Those spikes perfectly laid on the confocal plane spanned the entire depth of the perivitelline space, and their length grew as the vitelline layer was elevated (arrow at 4 and 20 min). (B) The control probe R40A did not visualize spikes formation, but labeled the actin-rich fertilization cone (arrow at 5 min). Abbreviation: SP, sperm; FE, fertilization envelope; FC, fertilization cone.
Figure 4. The spikes protruding into the perivitelline space during fertilization are composed of actin filaments.Mature eggs of A. aranciacus were microinjected with Alexa Fluor 488-phalloidin (50 µM, pipette concentration) prior to fertilization. (A) Transmission photomicrograph of the fertilized egg 10 min after insemination. (B) The corresponding confocal image of Alexa Fluor 488 phalloidin-stained F-actin. In addition to the strong staining of the actin bundles associated with the penetrating sperm (arrow), fluorescent phalloidin disclosed numerous spikes in the perivitelline space (arrowheads). Abbreviation: FE (fertilization envelope), PV (perivitelline space), PM (plasma membrane). Scale bar, 50 µm.
Figure 5. Sequestration of PIP2 by RFP-PH causes structural changes in the subplasmalemmal actin network.Mature eggs of A. aranciacus were microinjected with either RFP-PH or the R40A mutant proteins (330 µM, pipette concentration) and incubated for 25 min. The actin cytoskeleton was visualized by Alexa Fluor 488-conjugated phalloidin in three representative eggs for each treatment. Scale bar, 20 µm.
Figure 7. Sequestration of PIP2 by RFP-PH significantly delays the InsP3-induced Ca2+ release.
A. aranciacus oocytes microinjected with Ca2+ dye and caged InsP3 were exposed to 1-MA for 1 h. Mature eggs were microinjected with either RFP-PH or the R40A mutant proteins (330 µM, pipette concentration), and were incubated for 20 min. (A) After photoactivation of the caged InsP3 (t = 0), the changes of cytosolic free Ca2+ were monitored by the relative fluorescence of Ca2+ signals. (B) Ca2+ signals quantified over the entire cytoplasmic field of the oocytes injected with RFP-PH and the R40A mutant proteins were represented in brown and green curves, respectively. (C and D) Bright field views of the eggs 12 min after InsP3 uncaging. Elevation of the vitelline layer after Ca2+ signaling is severely affected by RFP-PH.
Figure 8. Sequestration of PIP2 by RFP-PH affects translocation of cortical granules during meiotic maturation.Transmission EM exhibited the ultrastructure of the eggs matured in the presence of RFP-PH or the R40A proteins (330 µM, pipette concentration). (A) In the eggs microinjected with R40A, cortical granules are intimately apposed to the plasma membrane, often perpendicular to the cell surface (arrows). (B) In the eggs microinjected with RFP-PH at the GV stage, cortical granules failed to be stacked underneath the plasma membrane, and were often located at a considerable distance from the plasma membrane (arrows). Scale bar, 10 µm.
Figure 9. Perivitelline spike formation is not limited to starfish eggs, but is also present in sea urchin eggs at fertilization.Starfish (A. pectinifera) (A) and sea urchin (P. lividus) eggs (B) were stained with FM 1-43 as described in Materials and Methods. After immediate rinse with FSW, sperm were added (t = 0), and the eggs were imaged with confocal microscopy to monitor the spike formation and the elevation of the fertilization envelope.
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