Nuclear envelope assembly is promoted by phosphoinositide-specific phospholipase C with selective recruitment of phosphatidylinositol-enriched membranes

Abstract

Nuclear envelope (NE) formation in a cell-free egg extract proceeds by precursor membrane vesicle binding to chromatin in an ATP-dependent manner, followed by a GTP-induced NE assembly step. The requirement for GTP in the latter step of this process can be mimicked by addition of bacterial PI-PLC [phosphoinositide (PtdIns)-specific phospholipase C]. The NE assembly process is here dissected in relation to the requirement for endogenous phosphoinositide metabolism, employing recombinant eukaryotic PI-PLC, inhibitors and direct phospholipid analysis using ESI-MS (electrospray ionization mass spectrometry). PtdIns (phosphatidylinositol) species analysis by ESI-MS indicates that the chromatin-bound NE precursor vesicles are enriched for specific PtdIns species. Moreover, during GTP-induced precursor vesicle fusion, the membrane vesicles become partially depleted of the PtdIns 18:0/20:4 species. These data indicate that eukaryotic PI-PLC can support NE formation, and the sensitivity to exogenous recombinant PtdIns-5-phosphatases shows that the endogenous PLC hydrolyses a 5-phosphorylated species. It is shown further that the downstream target of this DAG (diacylglycerol) pathway does not involve PKC (protein kinase C) catalytic function, but is mimicked by phorbol esters, indicating a possible engagement of one of the non-PKC phorbol ester receptors. The results show that ESI-MS can be used as a sensitive means to measure the lipid composition of biological membranes and their changes during, for example, membrane fusogenic events. We have exploited this and the intervention studies to illustrate a pivotal role for PI-PLC and its product DAG in the formation of NEs.

Figure 1. Cytoplasmic MV lipid analysis by ESI-MS

Total MVs of cytoplasmic extracts (MV0) were analysed by ESI-MS. Molecular species of PtdIns were detected by diagnostic precursor scan of the dehydrated inositol phosphate fragment ion (m/z=−241), while molecular species of PtdCho were detected by equivalent scan of the PtdCho fragment ion (m/z=+184).

Figure 2. The molecular species composition of PtdIns changes upon binding of MVs to chromatin and after NE assembly

The quantities of PtdIns (A) and PtdCho (B) species in the S1 (black bars), ATP pellet (grey striped bars) and GTP pellet (grey solid bars) were determined as described in Table 1, and expressed as the mole% composition of total PtdIns or PtdCho. Data are expressed as means±S.D. from three independent experiments; * P<0.05 at the 95% confidence interval.

Figure 3. PI-PLC and PMA induce NE formation in vitro via the PtdIns(4,5)P₂-DAG pathway

(A) MVs from fertilized cytoplasmic extracts of sea urchin eggs were bound in vitro to sperm nuclei with ATP-GS (ATP). NE formation was induced by either GTP, eukaryotic PI-PLC (eukPI-PLC) or PMA. DiOC₆-labelled nuclei, after centrifugation through sucrose, were visualized by phase and fluorescence microscopy at 100× magnification. Images are representative of three independent experiments. ‘NE assembly’ is defined by a smooth fluorescent rim as opposed to patchy fluorescence of ‘bound’ membranes. Scale bar=5 μm. ATP and PMA images taken are of P. lividus nuclei. (B) Total lipid extracts from untreated MV0 and bacterial PI-PLC-treated MV0 were analysed by ESI-MS. Molecular species of PtdIns were detected by diagnostic precursor scan of the dehydrated inositol phosphate fragment ion (m/z=−241). Note the hydrolysis of several PtdIns species by bacterial PLC, as indicated by the arrows.

Figure 4. PMA induction results in recruitment of RanGTPase and a nuclear pore protein

NEs were assembled in the presence of ATP-GS and 50 nM PMA as described in the text (Decondensed). Nuclei were treated for an additional 30 min with 2 mM ATP-GS to induce nuclei swelling. (A) Decondensed and swollen nuclei were viewed by phase microscopy at 100× magnification. Scale bar=5 μm. (B) Swollen nuclei were collected by centrifugation, washed and resuspended in SDS sample buffer. The protein content of S10, 0.1% nuclei, swollen nuclei and unbound material was assessed by Western analysis with antibodies specific for RanGTPase, the nuclear pore complex protein Nup62 (mAb414) and α-tubulin. Data are representative of two independent experiments.