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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 - PLoS ONE (2010)

Bottom Line: The first increase was quickly followed by a decrease about 40 seconds after sperm-egg contact.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.Our results suggest that PIP2 plays comprehensive roles in shaping Ca(2+) waves and guiding structural and functional changes required for successful fertilization.

View Article: PubMed Central - PubMed

Affiliation: Stazione Zoologica Anton Dohrn, Villa Comunale, Napoli, Italy. chun@szn.it

ABSTRACT

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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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.
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pone-0014100-g001: 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.

Mentions: To visualize PIP2 in the presence of other fluorescent probes for cytosolic Ca2+ (Calcium Green) and F-actin (Alexa Fluor 488-phalloidin), the PH domain of PLC-δ1 was tagged with either RFP or GFP. Within 5–10 minutes after microinjection into Astropecten aranciacus oocytes, both PH-GFP and RFP-PH were predominantly localized at the plasma membrane (Fig. 1A,E). The preferential distribution most likely represents their specific binding to PIP2, as the mutant probes (R40A) unable to bind PIP2 [29] failed to yield signals at the plasma membrane (Fig. 1B,F). Curiously, PH-GFP and RFP-PH also accumulated in the germinal vesicles (GV) (Fig. 1A,D,E), as was observed in the nucleus of mammalian cells [37]. However, whether these signals represent specific binding to PIP2 was questionable because the R40A probes also displayed similar patterns of distribution in the GV (Fig. 1B,F). Taken together, these results assure that both PH-GFP and RFP-PH probes specifically bind PIP2 at the plasma membrane of starfish oocytes whether the fluorescent tag was fused to the N-terminal (RFP-PH) or C-terminal (PH-GFP) side of the PH domain.


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 - PLoS ONE (2010)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2990714&req=5

pone-0014100-g001: 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.
Mentions: To visualize PIP2 in the presence of other fluorescent probes for cytosolic Ca2+ (Calcium Green) and F-actin (Alexa Fluor 488-phalloidin), the PH domain of PLC-δ1 was tagged with either RFP or GFP. Within 5–10 minutes after microinjection into Astropecten aranciacus oocytes, both PH-GFP and RFP-PH were predominantly localized at the plasma membrane (Fig. 1A,E). The preferential distribution most likely represents their specific binding to PIP2, as the mutant probes (R40A) unable to bind PIP2 [29] failed to yield signals at the plasma membrane (Fig. 1B,F). Curiously, PH-GFP and RFP-PH also accumulated in the germinal vesicles (GV) (Fig. 1A,D,E), as was observed in the nucleus of mammalian cells [37]. However, whether these signals represent specific binding to PIP2 was questionable because the R40A probes also displayed similar patterns of distribution in the GV (Fig. 1B,F). Taken together, these results assure that both PH-GFP and RFP-PH probes specifically bind PIP2 at the plasma membrane of starfish oocytes whether the fluorescent tag was fused to the N-terminal (RFP-PH) or C-terminal (PH-GFP) side of the PH domain.

Bottom Line: The first increase was quickly followed by a decrease about 40 seconds after sperm-egg contact.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.Our results suggest that PIP2 plays comprehensive roles in shaping Ca(2+) waves and guiding structural and functional changes required for successful fertilization.

View Article: PubMed Central - PubMed

Affiliation: Stazione Zoologica Anton Dohrn, Villa Comunale, Napoli, Italy. chun@szn.it

ABSTRACT

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.

Show MeSH
Related in: MedlinePlus