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First published online May 24, 2004
doi: 10.1242/10.1242/jcs.01109

Journal of Cell Science 117, 2513-2521 (2004)
Published by The Company of Biologists 2004
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Cell cycle-dependent Ca2+ oscillations in mouse embryos are regulated by nuclear targeting of PLC{zeta}

Mark G. Larman1,2, Christopher M. Saunders2, John Carroll3, F. Anthony Lai2 and Karl Swann1,*

1 Department of Anatomy and Developmental Biology, University College London, London, WC1E 6BT, UK
2 Cell Signalling Laboratory, Wales Heart Research Institute, University of Wales College of Medicine, Cardiff, CF14 4XN, UK
3 Department of Physiology, University College London, London, WC1E 6BT, UK



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  		Fig. 1. PLC{zeta}-induced Ca2+ oscillations are cell cycle-dependent. PLC{zeta} cRNA was injected into MII-arrested mouse eggs and at two different stages of the first cell cycle following parthenogenetic activation: interphase G1 and late G2. Eggs were microinjected with Fura-dextran and PLC{zeta} cRNA. Intracellular Ca2+ was monitored by changes in fluorescence excitation ratio. Time zero indicates approximately the point at which PLC{zeta} was microinjected. (a) A series of Ca2+ oscillations occurs during MII that ceases as the egg enters interphase (n=20). (b) MII eggs were activated with Sr2+ in the presence of cytochalsin D. 2 hours after pronuclear formation the eggs were injected with Fura-dextran and PLC{zeta}. No Ca2+ transients were detected in these parthenotes (n=20). (c) Parthenogentically activated eggs were cultured until interphase (G2), then 2-6 hours before NEB was due to occur embryos were injected with Fura-dextran and PLC{zeta} cRNA (n=15). Ca2+ oscillations again occur.

 


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  		Fig. 2. PLC{zeta}-induced Ca2+ oscillations at mitosis are controlled by nuclear membrane integrity. As in Fig. 1c, MII eggs were parthenogentically activated in the presence of cytochalsin D and cultured until interphase (G2). To monitor the exact timing of NEB and NER, embryos were injected (2-6 hours before NEB) with a fluorescent probe for nuclear membrane integrity (FITC-BSA-NLS) in addition to Fura-dextran and PLC{zeta} cRNA. (a) Two pronuclei can be seen with FITC-BSA-NLS. At approximately 416 minutes (415.5) after injection of PLC{zeta} cRNA the first pronucleus begins to break down. By 430 minutes both pronuclei have broken down and the FITC-BSA-NLS can no longer be detected. Measuring the decrease in nuclear FITC-BSA-NLS intensity of both pronuclei (red and blue lines) it was found that NEB of the first pronucleus occurred 6.3±4.1 minutes before the initial Ca 2+ transient (n=12) that was indicated by the Fura-dextran fluorescence ratio (black line). (b) NER was also monitored with FITC-BSA-NLS. Measuring the accumulation of FITC-BSA-NLS into the reforming nuclei showed that the first indication of NER occurred just prior to the last Ca2+ transient (14.3±4.3 minutes). As in a, the red and blue lines represent fluorescence from the FITC-BSA-NLS regions and the black line is the Fura-dextran fluorescence ratio.

 


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  		Fig. 3. PLC{zeta} localises to the pronuclei. MII eggs were injected with PLC{zeta} tagged with a c-Myc epitope and cultured in the presence of cytochalsin D until pronuclear formation, then they were fixed and stained using the c-Myc (9E10) monoclonal antibody and the anti-mouse Alexa Fluor® 488 secondary antibody. Control eggs were activated with untagged PLC{zeta}. Confocal and brightfield images were simultaneously obtained. The black arrows in brightfield images indicate the nucleoli. Though not easily visible, the nuclear membrane surrounds the nucleolus. The control embryo shows non-specific binding. Eggs activated with Myc-PLC{zeta} show a striking localisation to the nucleoplasm of each pronuclei. Scale bar: 10 µm.

 


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  		Fig. 4. PLC{zeta}-induced Ca2+ oscillations are regulated by nuclear import. MII-arrested mouse eggs were microinjected with Fura-dextran and PLC{zeta} cRNA. Intracellular Ca2+ was monitored by changes in fluorescence excitation ratio. Time zero indicates approximately the point at which PLC{zeta} was microinjected. The trace in a shows a series of Ca2+ oscillations in a control egg injected with PLC{zeta} cRNA. In b, parallel treated eggs were also injected with 10 mg/ml (pipette concentration) WGA, which significantly (P<0.0001) prolonged the oscillations (control: mean 444.2±142.0 minutes, n=14; WGA: mean 826.5±104.3 minutes, n=13). MII eggs were also injected with Fura-dextran and Myc-tagged versions of either control PLC{zeta} or PLC{zeta}K377E to monitor the nuclear localisation. Trace ci shows a series of Ca2+ oscillations in a control egg injected with Myc-PLC{zeta} cRNA. In di, parallel eggs were injected with Myc-PLC{zeta}K377E. Oscillations were significantly (P<0.0001) prolonged with the NLS mutant (control: mean 335.9±93.2 minutes, n=20; K377E: mean 702.2±162.8 minutes, n=22). Embryos were fixed and stained (see Fig. 3). Confocal and brightfield images were simultaneously obtained (cii and dii) showing the absence of Myc-PLC{zeta}K377E in the nucleoplasm.

 


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  		Fig. 5. Preventing PLC{zeta} nuclear localisation permits Ca2+ oscillations during interphase. MII eggs were activated with Sr2+ in the presence of cytochalsin D. 2 hours after pronuclear formation the eggs were injected with Fura-dextran and Myc-PLC{zeta}. (a) No Ca2+ transients were detected in these parthenotes (n=20). (b) Parallel parthenotes were also injected with WGA to block nuclear transport. In these embryos Myc-PLC{zeta} triggered Ca2+ oscillations within 1 hour (n=20). Embryos were fixed and stained at the end of the experiment (as in Fig. 2) demonstrating that blocking nuclear pores with WGA, prevents import of PLC{zeta} into the pronuclei (aii,bii). (c) Parthenotes were also injected with Myc-PLC{zeta}K377E (n=10). As with inhibiting nuclear import, preventing nuclear localisation of PLC{zeta} through mutation of the NLS permitted Ca2+ oscillations during interphase. Such Ca2+ oscillations were not observed in parallel controls (n=10).

 


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  		Fig. 6. Model for the regulation of Ca2+ oscillations during the first cell cycle. Model whereby PLC{zeta} (green) is introduced from the sperm into the egg upon fusion. Long-lasting Ca2+ oscillations are maintained while PLC{zeta} remains in the cytosol. During the initial phase of Ca2+ oscillations there is downregulation of InsP3 receptors and a change in the endoplasmic reticulum (ER): a decrease in cortical clusters. Both of these changes may lead to a change in the early phase of Ca2+ oscillations. When the pronuclei form, indicating the start of interphase, PLC{zeta} is transported into the nucleoplasm of the pronuclei terminating the Ca2+ oscillations. No further increases in Ca2+ are observed until the nuclear membranes breakdown upon mitosis entry, releasing PLC{zeta} back into the cytosol. Again, Ca2+ oscillations are sustained until the nuclear membranes reform in the two daughter cells and PLC{zeta} is imported into the nuclei.

 




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