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Calcineurin dependent protein phosphorylation changes during egg activation in Drosophila melanogaster

ABSTRACT: In animals studied to date, the crucial process of egg activation, by which an arrested mature oocyte transits into an actively developing embryo, initiates with an increase of Ca2+ in the oocyte’s cytoplasm. This Ca2+ rise sets off a series of downstream events, including the completion of meiosis and the dynamic remodeling of the oocyte transcriptome and proteome, which prepare the oocyte to undertake embryogenesis. Calcineurin is a highly conserved phosphatase that is activated directly by Ca2+ upon egg activation and that is required for the resumption of meiosis in Xenopus, ascidians and Drosophila. The molecular mechanisms by which calcineurin transduces the calcium signal to regulate meiosis and other downstream events are still unclear. In this study, we investigate the regulatory role of calcineurin during egg activation in Drosophila melanogaster. Using mass spectrometry, we quantify the phosphoproteomic and proteomic changes that occur during egg activation, and we examine how these events are affected when calcineurin function is perturbed in female germ cells. Our results show that calcineurin regulates hundreds of phosphosites and also influences the abundance of numerous proteins during egg activation. We find calcineurin-dependent changes in cell cycle regulators including Fzy, Greatwall (Gwl) and Endosulfine (Endos), protein translation modulators including PNG, NAT, eIF4G and eIF4B, and important components of signaling pathways including GSK3β and Akt1. Our results help elucidate the events that occur during the transition from oocyte to embryo

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Drosophila Melanogaster (fruit Fly)

TISSUE(S): Oocyte

DISEASE(S): Rnai_reagent

SUBMITTER: Zijing Zhang

LAB HEAD: Mariana Wolfner

PROVIDER: PXD011930 | Pride | 2019-03-13

REPOSITORIES: Pride

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			Action	DRS
	Zhijing10393887_TMT10pex_10FracCombOneRatio.pdResult	Other
	Zhijing10393887_TMT10pex_10FracCombOneRatio.raw	Raw
	Zhijing10393887_TMT10plex_MSA_SPSMS3.raw	Raw
	Zhijing10393887_TMT10plex_MSA_SPSMS3_2repRuns4_9ratios.pdResult	Other
	Zhijing10393887_TMT10plex_MSA_SPSMS3rep.raw	Raw

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Project description:The Ca2+ oscillations initiated by the fertilizing sperm (but terminating concomitant with pronucleus formation) apparently ensure that the events constituting egg activation occur in the correct temporal order; early events (e.g., cortical granule exocytosis) require fewer oscillations than later events (e.g., recruitment of maternal mRNA). Whether the Ca2+ signaling events impact long-term development, in particular development to term, is unknown. Using fertilized eggs that have undergone the first few Ca2+ oscillations, we developed procedures that result either in inhibiting or stimulating the natural pattern of Ca2+ signaling of inseminated eggs. Although the incidence of development to the blastocyst stage is unaltered by these procedures, fewer offspring are born following embryo transfer, indicating that developmental competence of the blastocysts is reduced. Interestingly, embryo transfer experiments reveal that when the natural regime of Ca2+ oscillations is precociously interrupted, the incidence of implantation is compromised whereas hyper-stimulation of Ca2+ signaling events compromises post-implantation development. Moreover, although there was no major difference in the overall growth rates of the offspring, those obtained following hyper-stimulation exhibited a far greater variability in their weight. Analysis of global patterns of gene expression by microarray analysis revealed that approximately 20% of the transcripts are mis-regulated when too few oscillations are experienced by the embryo and EASE analysis indicates that genes preferentially involved in RNA processing and polymerase II transcription are differentially affected. In addition, a set of genes involved in cell adhesion is also mis-expressed and could thus be mechanistically linked to the observed reduced implantation. Only about 3% of the transcripts were mis-regulated following hyper-stimulation, and EASE analysis indicates that genes preferentially involved in metabolism are differentially affected. In toto, these results indicate that a range Ca2+ signaling events following fertilization (an excess or reduction) has long-term effects on both gene expression and development to term. Experiment Overall Design: We profiled the global gene expression in the blastocysts by treatment of Ca2+ oscillations, and identified the genes differentially expressed.

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Calcineurin dependent protein phosphorylation changes during egg activation in Drosophila melanogaster

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