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First published online 15 March 2005
doi: 10.1242/jcs.01716

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Embryonic-stage-dependent changes in the level of eIF4E-binding proteins during early development of sea urchin embryos

¹ Station Biologique de Roscoff, Cycle Cellulaire et Développement, Unité Mer et Santé (UMR 7150), Université Pierre et Marie Curie (EI 37), Centre National de la Recherche Scientifique (CNRS), Institut National des Sciences de l'Univers (INSU), BP 74, 29682 Roscoff Cedex, France
² Department of Biochemistry and McGill Cancer Center, McGill University, Montreal, Quebec H3G1Y6, Canada

View larger version (15K): [in a new window]   Fig. 1. 4E-BP peptide affects eIF4E–4E-BP association and the first mitotic division of sea urchin embryos. (A) Treatment of extracts obtained from unfertilized eggs with the 4E-BP peptide leads to dissociation of eIF4E from 4E-BP. Control extract (lane 1) or extract treated with 10 µM variant eIF4E-binding peptide (lane 2) or 10 µM eIF4E-binding peptide (wild type) (lane 3) were incubated for 1 hour at 4°C. After incubation, eIF4E was purified from each extract using 50 µl m7GTP columns as described in the Materials and Methods. Proteins bound to the beads were separated by SDS-PAGE and subjected to western blotting using antibodies to eIF4E (top panels) or 4E-BP (bottom panels). Data are representative of three independent experiments. (B) Microinjection of 4E-BP peptide into unfertilized eggs impedes the first mitotic division triggered by fertilization. The eIF4E-binding peptide (wild type; white bar) or variant eIF4E-binding peptide (light grey bar) were injected at a final intracellular concentration of 10 µM. Control corresponds to unfertilized eggs microinjected with buffer (dark grey bar). Cleavage was scored by observation under a light microscope at 180 minutes after fertilization. An average of 100 unfertilized eggs were injected for each compound in each experiment and vertical bars represent the standard deviation of three independent experiments. Significance was assessed using Fisher's F-test and Student's t-test. An asterisk indicates a significant difference between eggs microinjected with eIF4E-binding peptide (wild type) and eggs microinjected with variant eIF4E-binding peptide or control (* corresponds to P<0.01).

View larger version (23K): [in a new window]   Fig. 2. 4E-BP is present in an 80 kDa complex in unfertilized eggs. (A) Proteins (5 mg) from extracts prepared from unfertilized or from extracts from 30 minutes post-fertilization eggs were separated by chromatography on a Sephacryl-200 column and were monitored by absorbance at 280 nm. The bars show the position of 4E-BP from unfertilized (open bar) and from 30 minutes post-fertilization (filled bar) eggs as determined by western blot analysis of each fraction using an anti-human 4E-BP2 antibody. The column was calibrated with the molecular mass standards indicated at the top. (B,C) Aliquots of each eluted fraction were resolved by electrophoresis and analysed by western blotting using 4E-BP2 antibodies. The position of the molecular mass standards (in kDa) used to calibrate the gel filtration column is indicated by arrows at the top of the figure. Data are representative of two independent experiments.

View larger version (19K): [in a new window]   Fig. 3. eIF4E shifts from a 80 kDa complex in unfertilized eggs to a high-molecular mass complex following fertilization. (A) Eluted fractions from the Sephacryl-200 gel filtration purification of unfertilized (top) or 30 minutes post-fertilization (bottom) extracts were applied to a m7GTP column and bound proteins were subjected to western blotting using anti-human eIF4E and anti-human 4E-BP2 antibodies. Arrows at the top of the figure correspond to the position of molecular mass standards (in kDa) used to calibrate the gel filtration column and the numbers at the bottom correspond to the fraction from the gel filtration purification showed in Fig. 2A. (B) Quantification of the results obtained from unfertilized (open circle) or 30 minutes post-fertilization (filled square) extracts. eIF4E amounts in each fraction were expressed as a percentage of the total amount of eIF4E contained in unfertilized or 30 minutes post-fertilization extracts, respectively. Arrows at the top of the figure correspond to the position of the molecular mass standards (in kDa) used to calibrate the gel filtration column showed in Fig. 2A. These data are representative of more than two independent experiments.

View larger version (25K): [in a new window]   Fig. 4. The 40 kDa protein associates with eIF4E. (A) Total extract obtained from unfertilized eggs was subjected either to western blotting using 4E-BP2 antibody (lane 1) or to far-western analysis (see Materials and Methods) using 32P-labelled mouse recombinant HMK-eIF4E as a probe (lane 2). (B) Western blot and far-western analyses of the purified 40 kDa protein. Proteins from unfertilized egg extracts were immunopurified using 4E-BP2 antibodies coupled to Sepharose beads (1) or using a m7GTP column (3). The proteins fixed on the beads were resolved either by western blotting using 4E-BP2 antibodies (left panel) or by far-western analysis using an eIF4E-HMK probe (right panel). 4E-BP2-coupled Sepharose beads alone (lane 2) or m7GTP-column beads alone (lane 4) were used as controls. Arrows point to the positions of 4E-BP and the 40 kDa protein. Data are representative of at least three independent experiments.

View larger version (28K): [in a new window]   Fig. 5. The new 40 kDa eIF4E-binding protein presents behaviour similar to that of the sea urchin 4E-BP already identified. (A) Rapamycin inhibits the disappearance of the 40 kDa protein following fertilization. The total amount of the 40 kDa protein from unfertilized eggs (lane 1) and from untreated (lane 2) or rapamycin-treated (lane 3) eggs obtained 60 minutes following fertilization was analysed by western blotting using 4E-BP2 antibodies (top panel). A western blot anti-actin was also performed (bottom panel) as loading control. (B) Rapamycin inhibits the dissociation of the 40 kDa protein from eIF4E. Proteins affinity-purified using m7GTP columns prior to fertilization or at the indicated times following fertilization of untreated (left) or rapamycin-treated (right) eggs were subjected to western blotting using anti-4E-BP2 (top) or anti-eIF4E (bottom) antibodies. The data are representative of at least five independent experiments. (C) Treatment of extracts obtained from unfertilized eggs with the 4E-BP peptide also leads to the dissociation between eIF4E and the 40 kDa protein. Control extract (lane 1) or extract treated with 10 µM variant eIF4E-binding peptide (lane 2) or 10 µM eIF4E-binding peptide (lane 3) were incubated for 1 hour at 4°C. After incubation, eIF4E was purified using a m7GTP column. Proteins bound to the beads were separated by SDS-PAGE and subjected to western blotting using 4E-BP (top panels) or eIF4E (bottom panels) antibodies.

View larger version (46K): [in a new window]   Fig. 6. 4E-BP and the 40 kDa proteins re-appear during late embryogenesis of sea urchin. (A) Total extracts of unfertilized eggs or eggs at the indicated times following fertilization were subjected to western blotting using 4E-BP2 or eIF4E antibodies. Tubulin antibody was used as a loading control. Data are representative of at least five independent experiments. (B) Total extracts prior to fertilization or at the indicated times following fertilization were subjected to far-western blotting using an eIF4E-HMK probe. Data are representative of two independent experiments. (C) Proteins affinity purified using an m7GTP column prior to fertilization or at the indicated times following fertilization were subjected to western blotting using 4E-BP or anti-eIF4E antibodies. Data are representative of at least two independent experiments.