Relationships between DNA synthesis and mitotic events in fertilized sea urchin eggs: aphidicolin inhibits DNA synthesis, nuclear breakdown and proliferation of microtubule organizing centers, but not cycles of microtubule assembly.
The synthesis of DNA in fertilized eggs of the American Gulf Coast sea urchin Lytechinus variegatus is 90% inhibited in the presence of 5.0 micrograms/ml aphidicolin. This inhibition may be imposed immediately upon addition of aphidicolin to the external medium when embryos are in "S" phase. Observations of living embryos with Nomarski optics and time-lapse video microscopy reveal that when eggs are fertilized and cultured in the continuous presence of aphidicolin, nuclear envelope breakdown, chromosome condensation, and cytokinesis are inhibited. All other post-fertilization events observable with this technique, including the assembly and disassembly of a bipolar spindle, proceed in the presence of aphidicolin. Antitubulin immunofluorescence microscopy of aphidicolin-arrested embryos demonstrates that microtubules attempt to assemble a mitotic apparatus at the first cell cycle; the arrested intact zygote nucleus is embedded within this bipolar structure. Subsequent cycles of microtubule assembly and disassembly proceed roughly on schedule with later division cycles, but the microtubule organizing centers (MTOC's) are unable to duplicate properly and irregular monasters are observed. If aphidicolin is added to embryos after the first DNA synthetic period, nuclear envelope breakdown, chromosome condensation, and cytokinesis proceed for that cycle and the embryos arrest at the two-cell stage. These results suggest that the direct inhibitory effects of aphidicolin may well be limited to the synthesis of DNA, which itself regulates nuclear cycles independently from the subsequent generation of mitotic poles, and that cytoplasmic clocks regulate microtubule assembly cycles but not the configuration of microtubule arrays.