Figure 5.
Figure 5. PKC nuclear localization and activity increase during X. laevis development, and altering PKC activity in vivo affects nuclear size. (A) Western blots of whole embryo lysates (0.6 embryo equivalents per lane) from stages 2–12 were probed with a pan-PKC or Ran antibody. n = 2 different sets of whole embryo preparations. One representative gel is shown. (B and C) Nuclei assembled de novo in egg extract and endogenous stage 12 nuclei were stained with a phospho-cPKC antibody (green). Confocal images were acquired, and PKC staining intensity was quantified as described in Fig. 3. n = 2 different extracts. Bar, 10 µm. (D) PKC activity was detected in extracts using a colorimetric PKC-specific peptide substrate (see Materials and methods). Unphosphorylated substrate migrates toward the anode (above the loading well) while phosphorylated substrate migrates toward the cathode (below the loading well). Results for three different egg extracts and LEEs are shown. Where indicated, chelerythrine was added at 390 µM. (E) One-cell embryos were microinjected with morpholinos against PKC α, PKC β, or a scrambled control and allowed to develop to stage 12. Nuclei isolated from embryos were visualized with mAb414 and quantified. n = 3 different fertilizations, with at least 20 embryos each. (F) Stage 8 embryos were arrested in interphase by incubating for 30 min in cycloheximide (0.5 mM), followed by a 60-min incubation with chelerythrine or PMA as indicated. Nuclei were quantified as in E. n = 2 different fertilizations, with at least 15 embryos each. (G) Stage 8 embryos were incubated for 90 min with chelerythrine or PMA as indicated. Nuclei were quantified as in E. n = 2–3 different fertilizations, with at least 20 embryos each. (H) Single-cell embryos were microinjected with GFP-NLS mRNA to visualize nuclei, and allowed to develop to stage 12. Embryos were treated with 24 µM chelerythrine or 80 nM PMA, as indicated, and confocal time-lapse images were acquired every minute on the surface of live embryos for 45 min (see Videos 2–10). For individual nuclei, cross-sectional nuclear area was quantified every 5 min and normalized to the size at t = 0. For each treatment condition, n = 12 nuclei from three movies of different embryos. Thicker lines denote averages for a given treatment, and stars refer to specific nuclei highlighted in Videos 2, 5, and 8. **, P < 0.01; *, P < 0.05. Error bars represent SD.

PKC nuclear localization and activity increase during X. laevis development, and altering PKC activity in vivo affects nuclear size. (A) Western blots of whole embryo lysates (0.6 embryo equivalents per lane) from stages 2–12 were probed with a pan-PKC or Ran antibody. n = 2 different sets of whole embryo preparations. One representative gel is shown. (B and C) Nuclei assembled de novo in egg extract and endogenous stage 12 nuclei were stained with a phospho-cPKC antibody (green). Confocal images were acquired, and PKC staining intensity was quantified as described in Fig. 3. n = 2 different extracts. Bar, 10 µm. (D) PKC activity was detected in extracts using a colorimetric PKC-specific peptide substrate (see Materials and methods). Unphosphorylated substrate migrates toward the anode (above the loading well) while phosphorylated substrate migrates toward the cathode (below the loading well). Results for three different egg extracts and LEEs are shown. Where indicated, chelerythrine was added at 390 µM. (E) One-cell embryos were microinjected with morpholinos against PKC α, PKC β, or a scrambled control and allowed to develop to stage 12. Nuclei isolated from embryos were visualized with mAb414 and quantified. n = 3 different fertilizations, with at least 20 embryos each. (F) Stage 8 embryos were arrested in interphase by incubating for 30 min in cycloheximide (0.5 mM), followed by a 60-min incubation with chelerythrine or PMA as indicated. Nuclei were quantified as in E. n = 2 different fertilizations, with at least 15 embryos each. (G) Stage 8 embryos were incubated for 90 min with chelerythrine or PMA as indicated. Nuclei were quantified as in E. n = 2–3 different fertilizations, with at least 20 embryos each. (H) Single-cell embryos were microinjected with GFP-NLS mRNA to visualize nuclei, and allowed to develop to stage 12. Embryos were treated with 24 µM chelerythrine or 80 nM PMA, as indicated, and confocal time-lapse images were acquired every minute on the surface of live embryos for 45 min (see Videos 2–10). For individual nuclei, cross-sectional nuclear area was quantified every 5 min and normalized to the size at t = 0. For each treatment condition, n = 12 nuclei from three movies of different embryos. Thicker lines denote averages for a given treatment, and stars refer to specific nuclei highlighted in Videos 2, 5, and 8. **, P < 0.01; *, P < 0.05. Error bars represent SD.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal