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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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Related in: MedlinePlus

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.
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pone-0014100-g005: 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.

Mentions: As PIP2 binds to an array of actin-binding proteins in cells [27], [28], [43], the fine structure of the actin cytoskeleton can be modified by altering the local levels of PIP2 [44]. Indeed, sequestering the plasma membrane PIP2 by over-expressing PH-GFP led to substantial reduction of the actin filaments in mammalian cells [38]. In line with these observations, microinjection of starfish eggs with RFP-PH (330 µM, pipette concentration) led to subtle changes in the organization of the actin cytoskeleton underneath the plasma membrane, as judged by Alexa Fluor 488-phalloidin. Whereas R40A-microinjected eggs displayed clustered actin bundles nearly perpendicular to the plasma membrane, as normally observed in mature eggs, the orderly organization of the actin filaments in the subplasmalemmal region was largely diminished in the eggs microinjected with RFP-PH (Fig. 5). Instead, the actin bundles resembling ‘pine spines’ were concomitantly enhanced in the inner cytoplasm.


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)

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.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0014100-g005: 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.
Mentions: As PIP2 binds to an array of actin-binding proteins in cells [27], [28], [43], the fine structure of the actin cytoskeleton can be modified by altering the local levels of PIP2 [44]. Indeed, sequestering the plasma membrane PIP2 by over-expressing PH-GFP led to substantial reduction of the actin filaments in mammalian cells [38]. In line with these observations, microinjection of starfish eggs with RFP-PH (330 µM, pipette concentration) led to subtle changes in the organization of the actin cytoskeleton underneath the plasma membrane, as judged by Alexa Fluor 488-phalloidin. Whereas R40A-microinjected eggs displayed clustered actin bundles nearly perpendicular to the plasma membrane, as normally observed in mature eggs, the orderly organization of the actin filaments in the subplasmalemmal region was largely diminished in the eggs microinjected with RFP-PH (Fig. 5). Instead, the actin bundles resembling ‘pine spines’ were concomitantly enhanced in the inner cytoplasm.

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