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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 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.
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pone-0014100-g004: 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.

Mentions: The physiological significance of the slow-rising second phase of the PIP2 increase at the plasma membrane (Fig. 2C) was studied further by confocal microscopy. By 2 min after sperm-egg contact, when the vitelline layer was visibly elevated to form the fertilization envelope, labeling of PIP2 by RFP-PH exhibited striking morphological changes at the egg plasma membrane (Fig. 3A). The intensity of RFP-PH signals at the plasma membrane was substantially increased (10.4% higher) in gray scale compared with the level at 0:00, suggesting that the second phase of RFP-PH increase at the plasma membrane was temporally correlated with the elevation process of the vitelline layer. Surprisingly, the labeling of membrane PIP2 with RFP-PH clearly delineated numerous spike-like structures (approximately 0.5 µm in diameter) protruding from the plasma membrane (Fig. 3A, arrows at 2 and 4 min). Concurrent with the elevation of the fertilization envelope, the spikes elongated and traversed the entire depth of perivitelline space (up to 30–50 µm), with their tips reaching the elevating fertilization envelope (arrows at 2, 4, and 20 min). To test if the spikes were filled with microfilaments, F-actin was stained with fluorescent phalloidin (Fig. 4). Phalloidin has been known to stabilize actin polymers and thereby interfere with some actin-based biological processes such as cell locomotion and chromatin ‘fetching’ during meiotic maturation [40], [41]. Since our goal is to visualize the spikes to demonstrate that they are made of F-actin, starfish eggs were microinjected with low doses of fluorescent phalloidin that do not have any inhibitory effect on the fertilization process of starfish eggs [14]. As expected, starfish eggs preinjected with Alexa Fluor 488- phalloidin (50 µM, pipette concentration) were able to undergo normal fertilization process and displayed numerous microfilament-filled spikes in the perivitelline space (Fig. 4B, arrowheads), as well as the thick actin bundles that were formed at the head of the penetrating sperm (Fig. 4B, arrow). Taken together, these observations suggest that the PIP2 increase at the plasma membrane of the fertilized egg is closely linked to the formation of microfilament-filled spikes in the perivitelline space.


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 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.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2990714&req=5

pone-0014100-g004: 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.
Mentions: The physiological significance of the slow-rising second phase of the PIP2 increase at the plasma membrane (Fig. 2C) was studied further by confocal microscopy. By 2 min after sperm-egg contact, when the vitelline layer was visibly elevated to form the fertilization envelope, labeling of PIP2 by RFP-PH exhibited striking morphological changes at the egg plasma membrane (Fig. 3A). The intensity of RFP-PH signals at the plasma membrane was substantially increased (10.4% higher) in gray scale compared with the level at 0:00, suggesting that the second phase of RFP-PH increase at the plasma membrane was temporally correlated with the elevation process of the vitelline layer. Surprisingly, the labeling of membrane PIP2 with RFP-PH clearly delineated numerous spike-like structures (approximately 0.5 µm in diameter) protruding from the plasma membrane (Fig. 3A, arrows at 2 and 4 min). Concurrent with the elevation of the fertilization envelope, the spikes elongated and traversed the entire depth of perivitelline space (up to 30–50 µm), with their tips reaching the elevating fertilization envelope (arrows at 2, 4, and 20 min). To test if the spikes were filled with microfilaments, F-actin was stained with fluorescent phalloidin (Fig. 4). Phalloidin has been known to stabilize actin polymers and thereby interfere with some actin-based biological processes such as cell locomotion and chromatin ‘fetching’ during meiotic maturation [40], [41]. Since our goal is to visualize the spikes to demonstrate that they are made of F-actin, starfish eggs were microinjected with low doses of fluorescent phalloidin that do not have any inhibitory effect on the fertilization process of starfish eggs [14]. As expected, starfish eggs preinjected with Alexa Fluor 488- phalloidin (50 µM, pipette concentration) were able to undergo normal fertilization process and displayed numerous microfilament-filled spikes in the perivitelline space (Fig. 4B, arrowheads), as well as the thick actin bundles that were formed at the head of the penetrating sperm (Fig. 4B, arrow). Taken together, these observations suggest that the PIP2 increase at the plasma membrane of the fertilized egg is closely linked to the formation of microfilament-filled spikes in the perivitelline space.

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