Materials

P. lividus sea urchins were purchased from Macrae and company, or collected from the coast of Armintza (Basque Country). 3′3-Dihexyloxacarbocyanine iodide (DiOC₆), Hoechst 33342, ProLong antifade reagent, and chemically competent Escherichia coli MAX efficiency DH5α, peroxidase suppressor and tyramide signal amplification (TSA) kit with Alexa-Fluor-594 tyramide were from Invitrogen Life Technologies. Anti-PI3K p85 antibody (# 86714; 1:100), anti-Rab7 antibody (# 50533; 1:100), donkey anti-mouse IgG conjugated to Alexa-Fluor-488 (# 150105; 1:200), goat anti-chicken IgY conjugated to Alexa-Fluor-647 (# 150171; 1:500), goat polyclonal antibody to GST conjugated with DyLight650 (# 117497; 1:500) and anti-GST antibody (# 9085; 1:500) were from Abcam. AffiniPure F (ab′) 2 IgG anti-mouse (# 715-006-150; 1:500) and peroxidase AffiniPure F(ab′)₂ fragment donkey anti-rabbit IgG (# 711-036-152; 1:500) were purchased from Jackson ImmunoResearch. Inhibitors used were: LY 294002 from Cayman Chemicals and class-I-specific PIK-75 and BYL719 from Selleck Chemicals. Membrane lipid strips and PI(4,5)P₂ Grip (PLC-δ1-PH) were purchased from Echelon. 3-Amino-1,2,4-triazole (ATA), Triton X-100 (TX-100), paraformaldehyde (PFA), adenosine 5′-triphosphate disodium salt hydrate (ATP), guanosine 5′-triphosphate sodium salt hydrate (GTP), phosphocreatine disodium salt hydrate (CP), phosphocreatine kinase (CPK), poly-L-lysine solution, saponin and fatty-acid-free bovine serum albumin (FAF-BSA) were from Sigma-Aldrich. Poly-L-lysine-coated coverslips were from BD Biosciences. PD-10 dye removal columns and PD MiniTrap G-25 were purchased from GE Healthcare Life Sciences. inositol 1,3,4,5-tetrakisphosphate (IP₄) was purchased from Cell Signals. All other materials (salts and organic solvents) were of analytical grade.

Recombinant protein purification and specificity testing

E. coli cells were transformed with pGEX plasmid containing GST–Grp1-PH. A resulting colony was used to inoculate a Luria broth (LB) culture, which was grown at 37°C, with shaking at 200 rpm. Protein expression was induced at OD₆₀₀ 0.6 with 1 mM IPTG at 18°C overnight. Cells were harvested by centrifugation at 11,000 g for 20 min at 4°C, resuspended in distilled water and treated with 10 mg of lysozyme. The suspensions were frozen overnight at −20°C and, the following day, resuspended in 10× lysis buffer [500 mM Tris-HCl, pH 7.4, 10% (v/v) Triton X-100, 20 mM EDTA, 10 mM DTT, 10 mM AEBSF] before probe sonication for 5 min. After centrifugation at 12,000 g for 20 min at 4°C to remove cell debris, the supernatant was loaded onto 0.5 ml of slurry of glutathione Sepharose beads that had been pre-equilibrated in 50 mM Tris-HCl, pH 7.4, 140 mM NaCl, 2.7 mM KCl. The sample was incubated for 15 min at 4°C. Two washes with equilibration buffer (pre-equilibration buffer supplemented with 2 mM DTT) and two further washes with equilibration buffer supplemented with 500 mM NaCl were performed. Recombinant GST-tagged protein was eluted by adding 0.5 ml of elution buffer (50 mM Tris-HCl, pH 7.4, 20 mM glutathione, 250 mM NaCl, 2 mM DTT, 20% glycerol), vortexing and incubating for 10 min on ice. Aliquots of the eluted fractions were analysed by SDS-PAGE on a 4–12% NuPAGE pre-cast gel (Invitrogen). The eluted protein was quantified by Bradford assay. To corroborate that the purified protein was the protein of interest, a western blot was performed with an anti-GST antibody.

The construct was also identified by protein mass spectrometry. For this, it was first detected by Coomassie Blue staining, and the band of the expected molecular mass (44 kDa) was excised, diced and sent for analysis (in collaboration with the Protein Analysis Facility, Clare Hall, Cancer Research UK). Excised protein bands were subjected to in-gel trypsin digestion using a Perkin-Elmer Janus Automated Workstation. Peptide mixtures were acidified to contain 0.1% formic acid and injected onto a nanoACQUITY UPLC instrument (Waters Corporation) coupled to an LTQ-Orbitrap XL (Thermo Fisher Scientific) through an Advion Biosciences Nanomate. Peptides were eluted over a 30 min gradient [5–40% (v/v) acetonitrile]. Peak lists were extracted using Mascot distiller and searched with Mascot v.2.4.1 (Matrix Science) against the UniProt reference database. Oxidation of methionine was entered as a variable modification and the search tolerances were 5 ppm and 0.8 Da for peptides and fragments, respectively. Searches were compiled in Scaffold 4.1.1.

GST–Grp1-PH probe specificity was demonstrated using dot blots, as described previously (Jethwa et al., 2015). Commercial lipid strips were incubated with blocking buffer (3% FAF-BSA, 0.1% Tween-20 PBS) for 1 h at room temperature. 1 µg/ml of GST–Grp1-PH in blocking buffer was added and incubated for 1 h at room temperature. Excess buffer was washed with PBS-T (five washes, 5 min each). Then, anti-GST antibody (1:2500) was added in blocking buffer, incubated for 1 h at room temperature and washed as before. Finally, anti-mouse HRP-conjugated antibody (1:5000) in blocking buffer was added and incubated for 1 h at room temperature. Washes were performed as above and rinsed with PBS. Blots were photo-activated with a 1:1 mixture of the Amersham ECL reagents and exposed to photographic AGFA Cronex 5 Medical X-Ray Film in a Kodak BioMax MS cassette for various periods of time. The films were developed using the IGP Compact2 Developer.

Nuclei and egg extracts

Gamete shedding and collection of P. lividus took place as described previously (Byrne et al., 2009b) through intracoelomic injection of 0.5 M KCl. Sea urchin sperm was permeabilised with 0.1% Triton X-100, and fertilised egg cytoplasmic extract (S10) was prepared by twice vigorously homogenising the egg suspension 10 min after fertilisation with a syringe with a 22-gauge, 1.5-inch needle and centrifugation to remove the yolk (upper layer) and the pigment pellet (Byrne et al., 2009b). The S10 fraction, which includes cytosol and cytoplasmic membrane vesicles, can be subsequently fractionated using a sucrose gradient to separate the different membrane vesicle populations (MV1, MV2, etc.) that contribute to nuclear envelope formation (Byrne et al., 2009b).

Cell-free assembly of the male pronucleus

De-membraned sperm nuclei were mixed with the S10 fraction and an ATP-generating system (1:1:1 ATP:creatine phosphate:creatine phosphate kinase ratio) and incubated for 1 h at room temperature. Nuclei were decondensed, and membrane vesicles were bound to chromatin after this time. Then, GTP (1 or 5 mM final concentration) was added and incubated for 2 h at room temperature to initiate nuclear envelope formation. The reaction was stopped by diluting the samples approximately sevenfold with cold lysis buffer (10 mM Hepes, pH 8.0, 250 mM NaCl, 5 mM MgCl₂, 110 mM glycine, 250 mM glycerol, 1 mM DTT, 1 mM AEBSF). For analysis, samples were settled in poly-L-lysine-coated coverslips and fixed with 3.7% PFA in isolation buffer.

For inhibitor studies of nuclear envelope assembly, LY 294002, PIK-75 or BYL719 were added before the GTP addition and incubated for 15 min.

Sea urchin egg time series

Eggs were collected in Millipore-filtered sea water (MPSW) and dejellied by passing them twice through a Nytex mesh of the appropriate pore size (210 µm) (Foltz et al., 2004). Subsequently, the eggs were fertilised and left on a rotator shaker. At various time points, post-fertilisation eggs were fixed by mixing 1 ml of the egg suspension with 1 ml of 7.4% PFA in cold isolation buffer (80 mM Pipes, pH 7.2, 5 mM EGTA, 5 mM MgCl₂, 1 M glycerol). Fixation was terminated after 1 h of gentle shaking. The eggs were centrifuged (150 g, 4°C, 1 min) and washed twice with PBS. Finally, eggs were resuspended in 0.05% azide in PBS and stored at 4°C.

For inhibitor studies, inhibitors were added to eggs 3–4 min after fertilisation. Aliquots were fixed and stored as described above.

Immunofluorescence

For confocal imaging of the cell-free assay, nuclei were labelled by adding Hoechst 33342 (2 µg/ml), and membranes were labelled with DiOC₆ (2 µM), both in TBS. If GST–Grp1-PH was added, the sample was blocked with 3% FAF-BSA in TBS for 15 min before the recombinant protein was added in 3% FAF-BSA in TBS, and incubated for 1 h. The excess was washed with TBS, followed by labelling with Dylight650-conjugated anti-GST antibody (1:500) for 1 h. Secondary antibody alone was incubated as a control with nuclei to check the absence of signal.

Intact eggs were blocked and permeabilised before antibody addition by incubating the cells with 3% FAF-BSA and 0.5–1% saponin in TBS for 15–50 min. Then, the required antibodies were introduced into the buffer (3% FAF-BSA 0.5% saponin TBS), and samples were incubated for 1 h on an overhead mixer and washed after each incubation.

After labelling and settling onto poly-L-lysine-coated coverslips, ProLong reagent was used to mount slides. Image acquisition was performed on a Zeiss 710 upright confocal microscope run by Zen software (2009), with a 63×1.4NA oil immersion objective and the custom filter set-up for the probes as indicated; or an inverted confocal fluorescence microscope Nikon D-ECLIPSE C1 (Nikon Inc., Melville, N.Y.) with a 60×1.4 NA oil immersion objective.

Inhibitors used in the cell-free assay

In the cell-free experiments, when inhibitors were added, the formation of the nuclear envelope of at least 100 nuclei was scored in three independent experiments using a wide-field inverted Eclipse-Ti microscope (Nikon) PL APO 60×1.4 NA oil immersion objective. Because inhibitors were added in DMSO, the corresponding vehicle controls were performed.

Quantification of colocalisation

The threshold for every image was subjected to the algorithm moments (ImageJ software). This method keeps the moments of the original image in each thresholded result (Tsai, 1985).

The regions of interest were selected, and the colocalisation was calculated in the following manner. The colocalisation of the various proteins and phospholipids under investigation were analysed only in a region of interest around the female pronucleus, in unfertilised eggs, or in female and male pronuclei in fertilised eggs. This was performed in order to mainly focus on processes occurring within the vicinity of the nuclear envelope. The colocalisation measured in these regions of interest was expressed as a percentage of the total number of pixels in each region of interest – i.e. the ratio of pixels in two different channels (corresponding to the two fluorescent labels) relative to the total number of pixels in the regions of interest. These were plotted as box and whisker plots, and the statistics were performed using a non-parametric Mann–Whitney test. This quantification was performed for at least ten eggs for each condition.

Data acquisition for the measurement of FRET

Fixed eggs were blocked and permeabilised as described above. Cell autofluorescence was quenched with 2 mg/ml of sodium borohydrate (NaBH₄) in PBS for 2 min, and endogenous peroxidase activity was suppressed by incubating the cells with the specific reagent for 20 min. Proteins were labelled with anti-Rab7 antibody followed by anti-chicken F(ab′)₂–Atto-488, and lipids were labelled with GST–Grp1-PH and then an anti-GST antibody followed by anti-rabbit F(ab′)₂ conjugated to HRP. All antibody incubations took place in 3% FAF-BSA with 0.5% saponin in PBS for 1 h. To create an enhanced acceptor signal, eggs were treated with TSA–Alexa-Fluor-594 for 10 min at room temperature (Byrne et al., 2014; Veeriah et al., 2014), as per the manufacturer's instructions, with the enhancement reaction being terminated by PBS washes (150 g, 1 min). Eggs were additionally labelled with 2 μg/ml Hoechst 33342 and mounted in ProLong Gold.

A multiple frequency domain FLIM lifetime imaging microscope from Lambert Instruments was used, and samples were excited with the 473-nm diode laser. Images were acquired on an Eclipse-Ti microscope (Nikon) PL APO 60×1.4 NA oil-immersion objective. FRET efficiency (E_f) was determined using the following formula: E_{f (%)}=[1−(t_DA/t_D)]×100; where t_D is donor lifetime and t_DA is the donor plus acceptor lifetime. Only samples with donor intensity at least two times higher than the autofluorescence intensity were included for FRET efficiency calculations.

Conjugation of Atto-488 to anti-chicken F(ab′)₂ fragments

100 μg of anti-chicken F(ab′)₂ antibody was conjugated to 25 μg of chromophore in a 100-μl volume adjusted to pH 8.5 with 50 mM sodium borate buffer. Atto-488 (donor chromophore) was conjugated to an anti-chick F(ab′)₂ fragment for 1 h in the dark at 20°C with shaking. Free chromophore was separated from antibody–dye conjugate on a PD Minitrap™ G-25 column (GE Healthcare). The dye-to-protein ratio of the chromophore–antibody conjugate was calculated as per the manufacturer's instructions. Conjugates had a dye-to-protein ratio of <3:1.

Statistical analyses

Statistical analysis for the box and whisker plots was performed using the Origin software. Boxes represent the 25–75% percentiles of the data set, and the whiskers represent 100% of the population. Individual data points are shown as diamonds. Statistical significance between the groups was calculated with Mann–Whitney test. For other graphs, statistical analysis was performed in SigmaPlot subjecting data to an unpaired t-test. Differences were considered statistically significant when P≤0.05.