The Biphasic Increase of PIP2 in the Fertilized Eggs of Starfish: New Roles in Actin Polymerization and Ca2+ Signaling

Abstract

BackgroundFertilization of echinoderm eggs is accompanied by dynamic changes of the actin cytoskeleton and by a drastic increase of cytosolic Ca2+. Since the plasma membrane-enriched phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2) serves as the precursor of inositol 1,4,5 trisphosphate (InsP3) and also regulates actin-binding proteins, PIP2 might be involved in these two processes.Methodology/Principal FindingsIn 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 Ca2+ 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 Ca2+ 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 Ca2+ signaling, impaired elevation of FE, and increased occurrences of polyspermic fertilization.Conclusions/SignificanceOur results suggest that PIP2 plays comprehensive roles in shaping Ca2+ 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.

Actin, more than just a housekeeping protein at the scene of fertilization

“…As aforementioned, nearly all experimental paradigms that rearranged cortical actin networks resulted in alteration of either amplitude or spatiotemporal dynamics of Ca 2+ signals. Interestingly, when the structure of the cortical actin was modified, the starfish eggs were prone to polyspermy as was observed with heparin, jasplakinolide, and sequestration of PIP2 [105,106]. These eggs also failed to undergo a normal elevation of the fertilization envelope although they were contacted by multiple sperm and produced comparable level of Ca 2+ signals at fertilization.…”

“…However, direct measurements of cGMP, cADPr and InsP 3 at fertilization have shown that the increase in the level of cGMP is followed by those of cADPr and InsP 3 , and the major rise of InsP 3 takes place only after the Ca 2+ wave has already initiated [110]. Likewise, in the experiment where the real time changes of the PIP2 levels at the plasma membrane were monitored in a living cell by the use of the RFP-fused marker that contained the PH domain of PLC-δ1 RFP, the starfish eggs at fertilization displayed the initial Ca 2+ rise nearly 10 sec before the plasma membrane PIP2 level began to decline as a result of PLC activation [106]. Hence, these results cast a doubt on the role of InsP 3 as the very first triggering message of Ca 2+ signaling at fertilization.…”

“…The level of plasma membrane PIP2 eventually increases 1-5 min after fertilization of starfish eggs [106], which is in line with the findings in mouse eggs [111]. This late increase of PIP2 at the plasma membrane of the fertilized eggs of starfish is spatiotemporally linked to the formation of numerous spikes that traverse the perivitelline space, which is thought to aid the elevation of the fertilization envelope and the accommodation of the increased lipid surface as the cortical granules are being fused with the plasma membrane during exocytosis [106].…”

“…This late increase of PIP2 at the plasma membrane of the fertilized eggs of starfish is spatiotemporally linked to the formation of numerous spikes that traverse the perivitelline space, which is thought to aid the elevation of the fertilization envelope and the accommodation of the increased lipid surface as the cortical granules are being fused with the plasma membrane during exocytosis [106]. Since many actin-binding proteins have a stretch of basic amino acid residues that interact with the negatively charged head group of PIP2 at the plasma membrane, sequestration of PIP2 could be used as a method to shift the actin dynamics in cells (reviewed in [112−114]).…”

The eggshell in the C. elegans oocyte‐to‐embryo transition

“…During the ''slow'' block the extracellular ''vitelline'' layer surrounding the oocyte is lifted and converted into a fertilization envelope. In starfish (echinoderm), lifting of the VL has been shown to depend on actin remodeling beginning at the site of sperm entry (Chun and Santella, 2009;Chun et al, 2010;Kyozuka et al, 2008;Puppo et al, 2008).…”