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. 1998 Jun 1;141(5):1159-68.
doi: 10.1083/jcb.141.5.1159.

ZYG-9, a Caenorhabditis elegans protein required for microtubule organization and function, is a component of meiotic and mitotic spindle poles

L R Matthews  1 , P Carter, D Thierry-Mieg, K Kemphues
Affiliations

ZYG-9, a Caenorhabditis elegans protein required for microtubule organization and function, is a component of meiotic and mitotic spindle poles

L R Matthews et al. J Cell Biol. .

Abstract

We describe the molecular characterization of zyg-9, a maternally acting gene essential for microtubule organization and function in early Caenorhabditis elegans embryos. Defects in zyg-9 mutants suggest that the zyg-9 product functions in the organization of the meiotic spindle and the formation of long microtubules. One-cell zyg-9 embryos exhibit both meiotic and mitotic spindle defects. Meiotic spindles are disorganized, pronuclear migration fails, and the mitotic apparatus forms at the posterior, orients incorrectly, and contains unusually short microtubules. We find that zyg-9 encodes a component of the meiotic and mitotic spindle poles. In addition to the strong staining of spindle poles, we consistently detect staining in the region of the kinetochore microtubules at metaphase and early anaphase in mitotic spindles. The ZYG-9 signal at the mitotic centrosomes is not reduced by nocodazole treatment, indicating that ZYG-9 localization to the mitotic centrosomes is not dependent upon long astral microtubules. Interestingly, in embryos lacking an organized meiotic spindle, produced either by nocodazole treatment or mutations in the mei-1 gene, ZYG-9 forms a halo around the meiotic chromosomes. The protein sequence shows partial similarity to a small set of proteins that also localize to spindle poles, suggesting a common activity of the proteins.

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Figures

Figure 1
Figure 1
Map position of zyg-9. The top line shows a portion of the genetic map of linkage group II giving relative positions of the relevant markers. zyg-9 maps 0.009 m.u. or ∼24 kb to the right of the fP1 insertion site which is cloned in cosmid K07D6. The positions of K07D6, C45G11, and C28G11 and the right end of the deletion mnDf104 are indicated. The locations of the zyg-9 gene and partial cDNA yk28d8 on C28G11 is indicated below with relevant HindIII and XhoI sites marked.
Figure 2
Figure 2
(a) Genomic Southern blot analysis of wild-type and zyg-9 mutants. Genomic DNA from wild type (N2) and heterozygous zyg-9 it63, it64, and it152 animals was resolved by electrophoresis after digestion with HindIII (four leftmost lanes) or XhoI (four rightmost lanes), then blotted and probed with yk28d8 cDNA. Novel allele-specific hybridizing bands correlate with the presence of the zyg-9 mutations. (b) Northern blot analysis of mRNA from wild-type (N2) adults. Poly A mRNA was isolated, separated by electrophoresis, blotted, and then probed with the yk28d8 cDNA clone. A single band of ∼4.9 kb was detected.
Figure 3
Figure 3
Results of RNA interference experiments. Nomarski micrographs of the first cell cycle of a wild-type embryo (a, c, and e) and embryos from wild-type hermaphrodites injected with yk28d8 antisense RNA (b, d, and f). Embryos from the hermaphrodites injected with antisense yk28d8 RNA exhibit defects characteristic of zyg-9 mutations. These include defects in meiosis II indicated by multiple female pronuclei (b, arrows), failure of pronuclear migration (d, white arrow; location of female pronuclei undergoing nuclear envelope breakdown), abnormal size and positioning of the first cleavage spindle (d, black arrows; centrosomes), and furrows (f, arrowheads; positions of cleavage furrows). Bar, ∼10 μm.
Figure 4
Figure 4
(a) Comparison of sequences of ZYG-9, Homo sapiens ch-TOG, S. pombe p93Dis1, and S. cerevisiae Stu2p. One domain of ∼220 amino acids is conserved in all four proteins (region 1, black boxes). This region is present in two divergent copies separated by 65 amino acids in the ZYG-9 protein. A second region of similarity between ZYG-9 and ch-TOG spans ∼280 amino acids (region 2, shaded boxes). The degree of identity between ZYG-9 and each protein for each domain is indicated in boldface and the degree of similarity (identities + similarities) is indicated in parentheses. Sequence alignments are shown below, with region 1 shown in b and region 2 shown in c.
Figure 5
Figure 5
Detection of endogenous ZYG-9 protein on Western blots. Extracts from wild-type or zyg-9 mutant embryos were resolved by SDS-PAGE, blotted, and then probed with affinity- purified anti–ZYG-9 antibodies. A band of 155 kD, the predicted size of the ZYG-9 protein, was detected in wild-type embryos (N2) and in the temperature-sensitive zyg-9 allele b244, but was not detected in the strong alleles b279, b301, it3, and it152 or in the temperature-sensitive allele b288. Antibodies against α-tubulin provide a loading control.
Figure 6
Figure 6
Distribution of ZYG-9 during meiosis. Immunofluorescence micrographs of wild-type (a–f) and zyg-9 (it3) mutant embryos (g–h) fixed and labeled with anti–α-tubulin antibodies (left column), anti-ZYG-9 (middle column), and DAPI (right column). (a–c) Early metaphase I. (d–f) Late metaphase I. (a and d) Tubulin is detected throughout the spindle but is not noticeably concentrated at the poles. (b and e) ZYG-9 is detected throughout the spindle and at polar regions not obviously occupied by microtubules (arrows). (g–i) Meiotic stage embryos of the strong zyg-9 mutant it3. Note the disorganized spindle (g), absence of ZYG-9 staining (h), and abnormal chromosome configuration (i). Bar, 10 μm.
Figure 7
Figure 7
ZYG-9 distribution during the first mitosis. Immunofluorescence micrographs of wild-type (a–o) and zyg-9 (it3) mutant embryos (p–r) fixed and labeled with anti–α-tubulin antibodies (left column), anti-ZYG-9 antibodies (middle column), and DAPI (right column). ZYG-9 is first detected as two brightly staining dots adjacent to the male pronucleus (b). The location of these spots corresponds to the location of the newly duplicated centrosomes (a). Protein is detected at the spindle poles through anaphase (e, h, and k), but becomes diffuse at late telophase (n). During metaphase (e and h; two different embryos) and early anaphase (data not shown), ZYG-9 is also detected in the central spindle region (arrows). At the central spindle of a metaphase stage embryo (h; enlarged and contrast-enhanced to show detail), ZYG-9 is detected in the kinetochore region. (g–i) ZYG-9 protein is not detected in mitotic cells of zyg-9 (it3) embryos (q). The DAPI-stained bodies at the right in panel r are polar bodies and an abnormal female pronucleus. Bar, 10 μm.
Figure 8
Figure 8
ZYG-9 distribution in later stage embryos (a and b) and in adult gonads (c and d) stained with ZYG-9 antibodies (left column) and DAPI (right column). ZYG-9 distributes to the centrosome and central spindle region in mitotic cells throughout embryogenesis and in mitotic cells of the gonad. (a) Arrowheads; location of a metaphase and an interphase cell for comparison. Note the relatively high concentration of ZYG-9 in the cytoplasm of interphase cells relative to mitotic cells. (c) Arrows; ZYG-9 staining in centrosomes of a cell just entering mitosis in an adult gonad. Bar, 10 μm.
Figure 9
Figure 9
Distribution of ZYG-9 protein after the disruption of microtubules by nocodazole. Immunofluorescence micrographs of wild-type embryos treated with nocodazole, fixed, and then labeled with anti–α-tubulin antibodies (left column), anti–ZYG-9 antibodies (middle column), and DAPI (right column). (a–c) In mitotic stage embryonic cells, spindle microtubules are disrupted although weak tubulin staining is still detected at the centrosome (see a, arrowheads). ZYG-9 is detected at the centrosomes (b) and in the region surrounding the chromosomes. (d–f) A one-cell embryo treated with nocodazole before pronuclear migration. ZYG-9 accumulates at the sperm-derived centrosomes (e, right, bright dots) and around the paternal chromosomes (small arrow) and condensed chromosomes of the female pronucleus (large arrow). (g–i) Meiotic-stage embryos treated with nocodazole. Spindle microtubules are undetectable (g). ZYG-9 localizes around individual meiotic chromosomes (h). (j and k) ZYG-9 localizes around the widely dispersed meiotic chromosomes in the disorganized meiotic spindles of mei-1 mutants. Bar, 10 μm.
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