Mutations of a redundant alpha-tubulin gene affect Caenorhabditis elegans early embryonic cleavage via MEI-1/katanin-dependent and -independent pathways

Abstract

The C. elegans zygote supports both meiosis and mitosis within a common cytoplasm. The meiotic spindle is small and is located anteriorly, whereas the first mitotic spindle fills the zygote. The C. elegans microtubule-severing complex, katanin, is encoded by the mei-1 and mei-2 genes and is solely required for oocyte meiotic spindle formation; ectopic mitotic katanin activity disrupts mitotic spindles. Here we characterize two mutations that rescue the lethality caused by ectopic MEI-1/MEI-2. Both mutations are gain-of-function alleles of tba-2 alpha-tubulin. These tba-2 alleles do not prevent MEI-1/MEI-2 microtubule localization but do interfere with its activity. TBA-1 and TBA-2 are redundant for viability, but when katanin activity is limiting, TBA-2 is preferred over TBA-1 by katanin. This is similar to what we previously reported for the beta-tubulins. Removing both preferred alpha- and beta-isoforms results in normal development, suggesting that the katanin isoform preferences are not absolute. We conclude that while the C. elegans embryo expresses redundant alpha- and beta-tubulin isoforms, they nevertheless have subtle functional specializations. Finally, we identified a dominant tba-2 allele that disrupts both meiotic and mitotic spindle formation independently of MEI-1/MEI-2 activity. Genetic studies suggest that this tba-2 mutation has a "poisonous" effect on microtubule function.

Figure 1.—

Meiotic and mitotic phenotypes of embryos from tba-2(sb51)/+ hermaphrodites at 25°. (A–D) α-Tubulin staining was used to visualize spindle structures. (E–H) Nomarski images of pronuclear fusion-stage embryos and embryos in their first mitosis. Meiotic spindle poles of mutant embryos (C) are not as well focused as wild type (A), and meiotic failure often leads to multiple maternal pronuclei (G) as compared to wild type (E). Mutant first mitotic spindles are always aligned on the transverse axis (D and H) and have short aster microtubules (D) as compared to wild type (B and F). Bars: 2 μm in A; 10 μm in H.

Figure 2.—

Alignment of TBA-1 and TBA-2. TBA-1 and TBA-2 sequences were aligned using the Clustal W program (EMBL-European Bioinformatics Institute). Dark shading indicates regions of identity between TBA-1 and TBA-2 sequences. The amino acid sequence change in each TBA-2 allele is indicated.

Figure 3.—

Mislocalization of MEI-1 and MEI-2 to mitotic centrosomes and chromosomes in mei-1(ct46); tba-2(sb25) and mei-1(ct46); tba-2(sb27) double-mutant embryos. Indirect immunofluorescence images of anti-MEI-1 staining show ectopic MEI-1 expression in (A) mei-1(ct46gf), (B) mei-1(ct46gf); tba-2(sb25), and (C) mei-1(ct46gf); tba-2(sb27). Similar results were seen with anti-MEI-2 staining in (D) mei-1(ct46gf), (E) mei-1(ct46gf); tba-2(sb25), and (F) mei-1(ct46gf); tba-2(sb27). Both MEI-1 and MEI-2 localize to mitotic spindle microtubules, centrosomes (open arrowheads), and chromosomes (open arrowheads). The photographs were digitally deconvolved. Bar, 10 μm.

Figure 4.—

tba-2(sb27); tbb-2(sb26) and wild-type microtubules behave similarly in a cold environment. Representative one-cell embryos of wild-type (A, C, and E) and tba-2(sb27); tbb-2(sb26) (B, D, and F) embryos (n > 20 for both genotypes) were placed on ice for 0 (A and B), 5 (C and D), and 8 (E and F) min, fixed, and stained with anti-α-tubulin antibody to visualize spindle microtubule structure. Bar, 10 μm.

Figure 5.—

Meiotic phenotype of the tba-2(sb25); tbb-2(sb26) double mutant. Shown are Nomarski images of the first cell division of a wild-type (A–C) and of a tba-2(sb25); tbb-2(sb26) embryo (D–F) at 25°. In tba-2(sb25); tbb-2(sb26) embryos, female and male pronuclei migrate normally to the center of the embryo and rotate 90° (D); then the first mitotic spindle is aligned longitudinally (E), and the cell divides asymmetrically into a larger anterior daughter and a smaller posterior daughter (F). All of these features resemble those of wild type (A–C). However, tba-2(sb25); tbb-2(sb26) embryos often have enlarged polar bodies (open arrow; compare D–F to A–C), indicating meiotic spindle defects. Bar, 10 μm.

Figure 6.—

Meiotic spindle defects of tba-2(sb27); tbb-2(sb26) mutant embryos. Anti-α-tubulin staining of metaphase or early anaphase meiotic spindles of (A) wild-type, (B) tba-2(sb27); tbb-2(sb26), (C) mei-2(ct98), and (D) mei-1(ct46ct101) embryos are shown to reveal morphological defects of the mutant spindles. Bar, 2 μm.