Byrne RD et al. (2012), Dynamics of PLCγ and Src family kinase 1 intera...

ECB-ART-42518

PLoS One 2012 Jan 01;77:e40669. doi: 10.1371/journal.pone.0040669.

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Dynamics of PLCγ and Src family kinase 1 interactions during nuclear envelope formation revealed by FRET-FLIM.

Byrne RD , Applebee C , Poccia DL , Larijani B .

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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.

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Species referenced: Echinodermata
Genes referenced: LOC100887844 LOC100889271 LOC115918707 LOC574936 SFK1

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	Figure 1. PLCÎ³ and SFK1 are co-recruited to the sperm nucleus in vivo.Unfertilised (T0) or fertilised (T+1 onwards) L. pictus eggs were fixed and stained with anti-PLCÎ³ (green) and with anti-SFK-Cy5 (red). The sperm nucleus was stained with Hoechst (blue). Arrows (white) indicate vesicles associated with the forming MPN that stain positive for both PLCÎ³ and SFK1. Data show the confocal midsections through nuclei, and are representative of those obtained from three independent experiments. Scale bar is 5 Âµm (T0) or 1 Âµm (T+1 onwards).
	Figure 2. PLCÎ³ and SFK1 interact during the early stages of male pronuclear envelope formation in vivo.(A) Unfertilised L. pictus eggs (T0) or five minutes post-fertilisation (T+5) were fixed and labelled with anti-PLCÎ³-OG488 alone or together with anti-SFK1-Cy5. Samples were subjected to two-photon time domain FLIM and the lifetime of the donor chromophore (OG488) determined in the absence and presence of acceptor (Cy5). Donor (PLCÎ³-OG488) two-photon images, donor lifetime â€˜heat mapsâ€™ and Hg-lamp epifluoresence acceptor (SFK-Cy5) images are shown. At T+5 the donor lifetimes in the whole egg (middle panel) and in the vicinity of the MPN only (bottom panel) were determined. In the donor two-photon image the MPN region of interest is indicated (dashed circle). The donor lifetime heat map and corresponding two-photon image are at the same scale. The images shown are from a donor alone labelled egg. The same process was repeated with eggs labelled with donor and acceptor. Data are representative of at least three independent experiments performed. (B) The FRET efficiency for each condition was calculated. Solid green boxes are the donor alone condition, green/red stripes for donor and acceptor conditions. Data are from at least three independent experiments, with a total of 6â€“18 eggs analysed per condition. Boxes display the median, upper and lower quartiles and whiskers the maximum and minimum values.
	Figure 3. PLCÎ³ is transiently phosphorylated on Y783 at the NE in vivo.(A) Fertilised (T1â€“12) L. pictus eggs were fixed and stained with anti-pY783 on PLCÎ³, (green) and Hoechst (red), and imaged by confocal microscopy. Arrows (white) indicate pY783 positive vesicles recruited to the forming MPN. Data are representative of those obtained in three independent experiments. Scale bar is 20 Âµm (whole egg) or 1 Âµm (20Ã— zoom). (B) Z-series from (A) were manipulated in Imaris (see Methods) to form a 3D reconstruction. Membrane vesicles in contact with the nucleus surface were scored (solid arrow). Those in close proximity were not (open arrow). (C) Quantification of the data in A, B. Vesicles were scored in three independent experiments. Data are expressed as mean+s.e.m.
	Figure 4. Interaction of PLCÎ³ and SFK1 on the MPN surface in vitro declines after GTP addition.(A) Condensed demembranated sperm nuclei were fixed and labelled with anti-PLCÎ³ or anti-SFK1 (red), Hoechst (blue) and DiOC6 (green). Nuclei were imaged by confocal microscopy. (B) Demembranated sperm nuclei were decondensed in fertilised egg cytoplasmic extract and ATP-GS. Nuclei were treated with 1 mM GTP as indicated (minutes), fixed and labelled with anti-PLCÎ³ (green) and anti-SFK1 (red) directly conjugated to OG488 and Cy5 respectively. Arrows denote PLCÎ³ and SFK1 co-localisation. All images are representative of those obtained in three independent experiments. Scale bar is 1 Âµm. (C) Decondensed nuclei were prepared as in B, and labelled with anti-PLCÎ³ and anti-SFK directly conjugated to OG488 (green, donor) and Alexa 546 (red, acceptor) respectively. The Ï„p and Ï„m of the donor chromophore were determined for each condition by frequency domain FLIM in the absence (green) and presence (green and red bars) of acceptor and used to calculate the FRET efficiency. 10â€“18 nuclei were analysed for each condition. Boxes display the median, upper and lower quartiles and whiskers the maximum and minimum values.
	Figure 5. PLCÎ³ is transiently phosphorylated on Y783 at the NE in vitro.Demembranated sperm nuclei were fixed alone (top row) or decondensed in fertilised egg cytoplasmic extract and ATP-GS (lower panels). Nuclei were additionally treated with 1 mM GTP for the times indicated (minutes) and were fixed and stained with Hoechst (blue), DiOC6 (green) and anti-pY783 of PLCÎ³ (red). Nuclei were imaged by confocal microscopy. Note the staining of the NERs with anti-pY783 is retained in nuclei decondensed in ATP-GS (2nd row). Data are representative of those obtained in three independent experiments. Scale bar is 1 Âµm.
	Figure 6. PLCÎ³ Y783 phosphorylation requires SFK catalytic activity and GTP hydrolysis.(A) Experiment was performed as in Figure. 5 with nuclei treated for 5 minutes with 1 mM GTP or 2 mM GTPÎ³S. Nuclei were also pre-incubated with the Src inhibitor PP2 or its inactive analogue PP3 (both 10 ÂµM). After this time nuclei were fixed and stained with Hoechst (blue), DiOC6 (green) and anti-pY783 (red), and imaged by confocal microscopy. (B) Nuclei in A were scored for multiple pY783-positive sites on the nucleus surface. Data are expressed as mean+s.e.m from three independent experiments. Scale bar is 1 Âµm. (C) Experiments performed as in (B) with NE proteins subjected to an anti-pY783 western blot. pY783 bands were normalised to total PLCÎ³ and expressed as a fold-change compared to nuclei assembled in the presence of ATP only. Data are expressed as mean+s.e.m from at least three independent experiments.
	Figure 7. NE formation in vitro is dependent upon SFK kinase activity.(A) Experiments were performed as in Figure 6 with incubation of 1 mM GTP for 2 hours. After this time nuclear membranes were stained with DiOC6, and the nuclei scored as having bound vesicles (punctate signal) or a complete NE (continuous rim). Epifluoresence images and quantification (mean+s.e.m) are for three independent experiments. (B) Experiment performed as in A with GTP replaced with either normal IgY or anti-SFK1 serum (both at 1 Âµg/ml). Nuclei were scored as above. Scale bar is 5 Âµm.
	Figure 8. Schematic model and data summary of molecular events leading to NE formation.Once the sperm has entered the egg in vivo, or upon the sperm nucleus mixing with egg cytoplasmic extract and ATP-GS in vitro, the sperm nucleus decondenses (not shown), with concomitant membrane vesicle binding to the nucleus surface (grey). Left: MV1 vesicles enriched in SFK1, PLCÎ³ and PtdIns(4,5)P2 bind in a polar fashion near the two sperm NERs. SFK1 and PLCÎ³ directly interact. The premature phosphorylation of PLCÎ³ by SFK1 is prevented by Csk inhibition of SFK1. Middle: Upon addition of GTP, Csk inhibition of SFK1 is removed, and SFK1 phosphorylates PLCÎ³ on its Y783 residue, a pre-requisite for full PLCÎ³ activation. Once active, PLCÎ³ hydrolyses PtdIns(4,5)P2 to the fusogenic lipid DAG, which initially promotes membrane fusion events between MV1 vesicles and possibly NERs. Right: Fusion events subsequently spread over the surface of the sperm nucleus [19] until it is enclosed by a continuous double bilayer perforated with nuclear pore complexes (not shown). After fusion is initiated, PLCÎ³ and SFK1 dissociate but remain on the NE. L and C denote the luminal and cytoplasmic faces of vesicles respectively. ER denotes endoplasmic reticulum derived membranes.

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