Figure 2.
Figure 2. Postmeiotic upshift of a temperature-sensitive Aurora BAIR-2 mutant can be used to bypass the meiotic defects resulting from CPC inhibition. (A) Schematic illustrating the postmeiotic upshift conditions. Embryos are maintained at the permissive temperature (16°C) after fertilization until both rounds of meiotic chromosome segregation are complete. Embryos are upshifted to the nonpermissive temperature (26.5°C) during migration of the resulting maternal pronucleus toward the sperm pronucleus, which occurs during a ∼350-s interval that precedes nuclear envelope breakdown (NEBD), mitosis, and cytokinesis. (B) Schematic showing that air-2(RNAi) embryos or air-2(or207ts) mutant embryos upshifted before meiosis fail to segregate their chromosomes during the two meiotic divisions, resulting in a maternal pronucleus that contains four times the normal amount of DNA. air-2(or207ts) mutant embryos upshifted after meiosis have a normal amount of DNA in the maternal pronucleus. (C) Maximum intensity projections of 5 × 2–µm z series of embryos expressing GFP:tubulin and GFP:histone H2B. Time is given in seconds after NEBD. All embryos were imaged at the restrictive temperature for the mutant. The maternal chromatin resulting from meiotic failure in the air-2(RNAi) embryo is indicated (red arrows). Bar, 10 µm. (D) Immunofluorescence of air-2(or207ts) embryos fixed at the permissive or restrictive temperature. Images are maximum intensity projections of the midzone region of the cell. At the permissive temperature, the mutant Aurora BAIR-2 (P265L) localized to midzone microtubule bundles like the wild-type protein, whereas at the restrictive temperature, Aurora BAIR-2 P265L remained on the chromosomes. A few strands of Aurora BAIR-2 P265L were observed in the midzone in the anaphase embryos at the restrictive temperature, but closer inspection revealed that these strands corresponded to Aurora BAIR-2 P265L localized along chromatin bridges that extended into the region between the chromatin masses (yellow arrows; Severson et al., 2000). Consistent with prior work, the density of midzone microtubules was reduced in the upshifted embryos (white arrows; Severson et al., 2000). In addition, the levels of midzone-localized MKLP1ZEN-4 were significantly reduced, and its localization was less focused than that in mutant embryos fixed at the permissive temperature. Bars, 5 µm.

Postmeiotic upshift of a temperature-sensitive Aurora BAIR-2 mutant can be used to bypass the meiotic defects resulting from CPC inhibition. (A) Schematic illustrating the postmeiotic upshift conditions. Embryos are maintained at the permissive temperature (16°C) after fertilization until both rounds of meiotic chromosome segregation are complete. Embryos are upshifted to the nonpermissive temperature (26.5°C) during migration of the resulting maternal pronucleus toward the sperm pronucleus, which occurs during a ∼350-s interval that precedes nuclear envelope breakdown (NEBD), mitosis, and cytokinesis. (B) Schematic showing that air-2(RNAi) embryos or air-2(or207ts) mutant embryos upshifted before meiosis fail to segregate their chromosomes during the two meiotic divisions, resulting in a maternal pronucleus that contains four times the normal amount of DNA. air-2(or207ts) mutant embryos upshifted after meiosis have a normal amount of DNA in the maternal pronucleus. (C) Maximum intensity projections of 5 × 2–µm z series of embryos expressing GFP:tubulin and GFP:histone H2B. Time is given in seconds after NEBD. All embryos were imaged at the restrictive temperature for the mutant. The maternal chromatin resulting from meiotic failure in the air-2(RNAi) embryo is indicated (red arrows). Bar, 10 µm. (D) Immunofluorescence of air-2(or207ts) embryos fixed at the permissive or restrictive temperature. Images are maximum intensity projections of the midzone region of the cell. At the permissive temperature, the mutant Aurora BAIR-2 (P265L) localized to midzone microtubule bundles like the wild-type protein, whereas at the restrictive temperature, Aurora BAIR-2 P265L remained on the chromosomes. A few strands of Aurora BAIR-2 P265L were observed in the midzone in the anaphase embryos at the restrictive temperature, but closer inspection revealed that these strands corresponded to Aurora BAIR-2 P265L localized along chromatin bridges that extended into the region between the chromatin masses (yellow arrows; Severson et al., 2000). Consistent with prior work, the density of midzone microtubules was reduced in the upshifted embryos (white arrows; Severson et al., 2000). In addition, the levels of midzone-localized MKLP1ZEN-4 were significantly reduced, and its localization was less focused than that in mutant embryos fixed at the permissive temperature. Bars, 5 µm.

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