Abstract
The nuclear envelope (NE) breaks down and reforms during each mitotic cycle. A similar process happens to the sperm NE following fertilisation. The formation of the NE in both these circumstances involves endoplasmic reticulum membranes enveloping the chromatin, but PLCγ-dependent membrane fusion events are also essential. Here we demonstrate the activation of PLCγ by a Src family kinase (SFK1) during NE assembly. We show by time-resolved FRET for the first time the direct in vivo interaction and temporal regulation of PLCγ and SFK1 in sea urchins. As a prerequisite for protein activation, there is a rapid phosphorylation of PLCγ on its Y783 residue in response to GTP in vitro. This phosphorylation is dependent upon SFK activity; thus Y783 phosphorylation and NE assembly are susceptible to SFK inhibition. Y783 phosphorylation is also observed on the surface of the male pronucleus (MPN) in vivo during NE formation. Together the corroborative in vivo and in vitro data demonstrate the phosphorylation and activation of PLCγ by SFK1 during NE assembly. We discuss the potential generality of such a mechanism.
Highlights
Following fertilisation, the male and female genomes come to occupy a common nuclear compartment, the zygote nucleus [1]
To test whether the phospholipase Cc (PLCc)/SFK1 enriched vesicles participate in nuclear envelope (NE) assembly in vivo, eggs were fertilised and fixed at various times up to 15 minutes post- fertilisation
While the localisation of PLCc and SFK1 appeared to be unchanged during NE formation, it was possible that as in vivo, the two proteins were undergoing a reversible interaction not detectable by confocal microscopy. To test this we examined the putative interaction of PLCc and SFK1 in the cell-free assay by Forster resonance energy transfer (FRET) detected by multiple-frequency domain fluorescence lifetime imaging microscopy (FLIM)
Summary
The male and female genomes come to occupy a common nuclear compartment, the zygote nucleus [1] This is accomplished either by fusion of the two pronuclear membranes prior to mitosis or mixture of the parental chromosomes following disassembly of the male and female pronuclear envelopes at prophase and reformation of a common nuclear envelope (NE) after mitosis. These strategies represent respectively the so-called ‘‘sea urchin type’’ and ‘‘Ascaris type’’ distinguished by Wilson [2]. Migration of the MPN and fusion of its outer and inner nuclear membranes with the female pronucleus result in a zygote nucleus
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