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HIM-10 is required for kinetochore structure and function on Caenorhabditis elegans holocentric chromosomes.

Howe M, McDonald KL, Albertson DG, Meyer BJ - J. Cell Biol. (2001)

Bottom Line: Depletion of HIM-10 disrupts kinetochore structure, causes a failure of bipolar spindle attachment, and results in chromosome nondisjunction.HIM-10 is related to the Nuf2 kinetochore proteins conserved from yeast to humans.Thus, the extended kinetochores characteristic of C. elegans holocentric chromosomes provide a guide to the structure, molecular architecture, and function of conventional kinetochores.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Department of Molecular and Cell Biology, University of California, Berkeley 94720, USA.

ABSTRACT
Macromolecular structures called kinetochores attach and move chromosomes within the spindle during chromosome segregation. Using electron microscopy, we identified a structure on the holocentric mitotic and meiotic chromosomes of Caenorhabditis elegans that resembles the mammalian kinetochore. This structure faces the poles on mitotic chromosomes but encircles meiotic chromosomes. Worm kinetochores require the evolutionarily conserved HIM-10 protein for their structure and function. HIM-10 localizes to the kinetochores and mediates attachment of chromosomes to the spindle. Depletion of HIM-10 disrupts kinetochore structure, causes a failure of bipolar spindle attachment, and results in chromosome nondisjunction. HIM-10 is related to the Nuf2 kinetochore proteins conserved from yeast to humans. Thus, the extended kinetochores characteristic of C. elegans holocentric chromosomes provide a guide to the structure, molecular architecture, and function of conventional kinetochores.

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HIM-10 localizes to the kinetochore region of the kinetochore–centromere complex. All panels are false-color confocal images. Interpretative cartoons (right) represent images from D, G, K, and O. (A, H, and L) Wild-type embryos co-stained with DAPI (red) and anti-HIM-10 antibodies (green). (A) HIM-10 associates with prophase (arrows) but not interphase (arrowhead) chromosomes. (B–D) Enlargement of nucleus in A. In cross section, HIM-10 appears as paired dots flanking opposite sides of a prophase chromosome. (E–G) Enlargement of nucleus in A. HIM-10 forms two tracks flanking a prophase chromosome. (H) HIM-10 appears depleted from the cytoplasm of cells in metaphase. (I–K) Enlargement of metaphase cell (arrow) in H. HIM-10 appears as stripes associated with each poleward face of the metaphase plate. (L) An embryo with HIM-10 localized to the poleward face of anaphase chromosomes. (M–O) Enlargement of anaphase figure (arrow) in L. HIM-10 appears as many individual dots along the chromatin. These dots may represent the kinetochores associated with chromosomes moving at slightly different rates to the spindle pole. (P–X) Wild-type animals costained with DAPI (blue) and antibodies to both the centromeric marker HCP-3 (red) and HIM-10 (green). (P–R) Prophase nucleus from the mitotic region of the hermaphrodite gonad. (P and Q) Both proteins localize in two parallel tracks flanking the replicated chromosomes. (R) The HIM-10 signal overlaps with (yellow) and extends more distal to (green) the HCP-3 signal. In metaphase (S–U) and anaphase (V–X) of embryos, both proteins localize to the poleward face of the chromatin. In the merged images (U and X) HIM-10 overlaps with (yellow) and extends closer to the pole than (green) HCP-3. Bars, 2 μm.
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Figure 4: HIM-10 localizes to the kinetochore region of the kinetochore–centromere complex. All panels are false-color confocal images. Interpretative cartoons (right) represent images from D, G, K, and O. (A, H, and L) Wild-type embryos co-stained with DAPI (red) and anti-HIM-10 antibodies (green). (A) HIM-10 associates with prophase (arrows) but not interphase (arrowhead) chromosomes. (B–D) Enlargement of nucleus in A. In cross section, HIM-10 appears as paired dots flanking opposite sides of a prophase chromosome. (E–G) Enlargement of nucleus in A. HIM-10 forms two tracks flanking a prophase chromosome. (H) HIM-10 appears depleted from the cytoplasm of cells in metaphase. (I–K) Enlargement of metaphase cell (arrow) in H. HIM-10 appears as stripes associated with each poleward face of the metaphase plate. (L) An embryo with HIM-10 localized to the poleward face of anaphase chromosomes. (M–O) Enlargement of anaphase figure (arrow) in L. HIM-10 appears as many individual dots along the chromatin. These dots may represent the kinetochores associated with chromosomes moving at slightly different rates to the spindle pole. (P–X) Wild-type animals costained with DAPI (blue) and antibodies to both the centromeric marker HCP-3 (red) and HIM-10 (green). (P–R) Prophase nucleus from the mitotic region of the hermaphrodite gonad. (P and Q) Both proteins localize in two parallel tracks flanking the replicated chromosomes. (R) The HIM-10 signal overlaps with (yellow) and extends more distal to (green) the HCP-3 signal. In metaphase (S–U) and anaphase (V–X) of embryos, both proteins localize to the poleward face of the chromatin. In the merged images (U and X) HIM-10 overlaps with (yellow) and extends closer to the pole than (green) HCP-3. Bars, 2 μm.

Mentions: Confocal microscopy of animals stained with HIM-10 antibodies showed HIM-10 to be localized in a pattern expected for kinetochore proteins on holocentric chromosomes. Such chromosomes are distinguished by having their kinetochores and sites of microtubule attachment dispersed along the length of the chromosome. HIM-10 antibody staining was not detected on chromosomes of interphase nuclei. During prophase, HIM-10 was visualized as parallel tracks (in longitudinal sections) or paired dots (in cross sections) flanking each mitotic prophase chromosome (Fig. 4, A–G). At metaphase, HIM-10 was distributed in stripes along the poleward faces of the chromatin at the metaphase plate (Fig. 4, H–K). During anaphase, one poleward-facing stripe was associated with each set of sister chromatids (Fig. 4, L–O). Antibody staining was not detected during telophase. These staining patterns were most evident in the large cells of the early embryo but were also seen in the mitotic germline. HIM-10 antibody staining was not consistently seen on other structures in embryos or the mitotic germline, but a recurrent suggestion of spindle and centrosome staining was found with some antibody preparations. Both the specificity of the HIM-10 antibody and the dependence of the staining pattern on the presence of HIM-10 were confirmed by the severe reduction of HIM-10 antibody staining in him-10(RNAi) embryos (Fig. 3N and Fig. O) compared with wild-type embryos (Fig. 3L and Fig. M).


HIM-10 is required for kinetochore structure and function on Caenorhabditis elegans holocentric chromosomes.

Howe M, McDonald KL, Albertson DG, Meyer BJ - J. Cell Biol. (2001)

HIM-10 localizes to the kinetochore region of the kinetochore–centromere complex. All panels are false-color confocal images. Interpretative cartoons (right) represent images from D, G, K, and O. (A, H, and L) Wild-type embryos co-stained with DAPI (red) and anti-HIM-10 antibodies (green). (A) HIM-10 associates with prophase (arrows) but not interphase (arrowhead) chromosomes. (B–D) Enlargement of nucleus in A. In cross section, HIM-10 appears as paired dots flanking opposite sides of a prophase chromosome. (E–G) Enlargement of nucleus in A. HIM-10 forms two tracks flanking a prophase chromosome. (H) HIM-10 appears depleted from the cytoplasm of cells in metaphase. (I–K) Enlargement of metaphase cell (arrow) in H. HIM-10 appears as stripes associated with each poleward face of the metaphase plate. (L) An embryo with HIM-10 localized to the poleward face of anaphase chromosomes. (M–O) Enlargement of anaphase figure (arrow) in L. HIM-10 appears as many individual dots along the chromatin. These dots may represent the kinetochores associated with chromosomes moving at slightly different rates to the spindle pole. (P–X) Wild-type animals costained with DAPI (blue) and antibodies to both the centromeric marker HCP-3 (red) and HIM-10 (green). (P–R) Prophase nucleus from the mitotic region of the hermaphrodite gonad. (P and Q) Both proteins localize in two parallel tracks flanking the replicated chromosomes. (R) The HIM-10 signal overlaps with (yellow) and extends more distal to (green) the HCP-3 signal. In metaphase (S–U) and anaphase (V–X) of embryos, both proteins localize to the poleward face of the chromatin. In the merged images (U and X) HIM-10 overlaps with (yellow) and extends closer to the pole than (green) HCP-3. Bars, 2 μm.
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Related In: Results  -  Collection

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Figure 4: HIM-10 localizes to the kinetochore region of the kinetochore–centromere complex. All panels are false-color confocal images. Interpretative cartoons (right) represent images from D, G, K, and O. (A, H, and L) Wild-type embryos co-stained with DAPI (red) and anti-HIM-10 antibodies (green). (A) HIM-10 associates with prophase (arrows) but not interphase (arrowhead) chromosomes. (B–D) Enlargement of nucleus in A. In cross section, HIM-10 appears as paired dots flanking opposite sides of a prophase chromosome. (E–G) Enlargement of nucleus in A. HIM-10 forms two tracks flanking a prophase chromosome. (H) HIM-10 appears depleted from the cytoplasm of cells in metaphase. (I–K) Enlargement of metaphase cell (arrow) in H. HIM-10 appears as stripes associated with each poleward face of the metaphase plate. (L) An embryo with HIM-10 localized to the poleward face of anaphase chromosomes. (M–O) Enlargement of anaphase figure (arrow) in L. HIM-10 appears as many individual dots along the chromatin. These dots may represent the kinetochores associated with chromosomes moving at slightly different rates to the spindle pole. (P–X) Wild-type animals costained with DAPI (blue) and antibodies to both the centromeric marker HCP-3 (red) and HIM-10 (green). (P–R) Prophase nucleus from the mitotic region of the hermaphrodite gonad. (P and Q) Both proteins localize in two parallel tracks flanking the replicated chromosomes. (R) The HIM-10 signal overlaps with (yellow) and extends more distal to (green) the HCP-3 signal. In metaphase (S–U) and anaphase (V–X) of embryos, both proteins localize to the poleward face of the chromatin. In the merged images (U and X) HIM-10 overlaps with (yellow) and extends closer to the pole than (green) HCP-3. Bars, 2 μm.
Mentions: Confocal microscopy of animals stained with HIM-10 antibodies showed HIM-10 to be localized in a pattern expected for kinetochore proteins on holocentric chromosomes. Such chromosomes are distinguished by having their kinetochores and sites of microtubule attachment dispersed along the length of the chromosome. HIM-10 antibody staining was not detected on chromosomes of interphase nuclei. During prophase, HIM-10 was visualized as parallel tracks (in longitudinal sections) or paired dots (in cross sections) flanking each mitotic prophase chromosome (Fig. 4, A–G). At metaphase, HIM-10 was distributed in stripes along the poleward faces of the chromatin at the metaphase plate (Fig. 4, H–K). During anaphase, one poleward-facing stripe was associated with each set of sister chromatids (Fig. 4, L–O). Antibody staining was not detected during telophase. These staining patterns were most evident in the large cells of the early embryo but were also seen in the mitotic germline. HIM-10 antibody staining was not consistently seen on other structures in embryos or the mitotic germline, but a recurrent suggestion of spindle and centrosome staining was found with some antibody preparations. Both the specificity of the HIM-10 antibody and the dependence of the staining pattern on the presence of HIM-10 were confirmed by the severe reduction of HIM-10 antibody staining in him-10(RNAi) embryos (Fig. 3N and Fig. O) compared with wild-type embryos (Fig. 3L and Fig. M).

Bottom Line: Depletion of HIM-10 disrupts kinetochore structure, causes a failure of bipolar spindle attachment, and results in chromosome nondisjunction.HIM-10 is related to the Nuf2 kinetochore proteins conserved from yeast to humans.Thus, the extended kinetochores characteristic of C. elegans holocentric chromosomes provide a guide to the structure, molecular architecture, and function of conventional kinetochores.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Department of Molecular and Cell Biology, University of California, Berkeley 94720, USA.

ABSTRACT
Macromolecular structures called kinetochores attach and move chromosomes within the spindle during chromosome segregation. Using electron microscopy, we identified a structure on the holocentric mitotic and meiotic chromosomes of Caenorhabditis elegans that resembles the mammalian kinetochore. This structure faces the poles on mitotic chromosomes but encircles meiotic chromosomes. Worm kinetochores require the evolutionarily conserved HIM-10 protein for their structure and function. HIM-10 localizes to the kinetochores and mediates attachment of chromosomes to the spindle. Depletion of HIM-10 disrupts kinetochore structure, causes a failure of bipolar spindle attachment, and results in chromosome nondisjunction. HIM-10 is related to the Nuf2 kinetochore proteins conserved from yeast to humans. Thus, the extended kinetochores characteristic of C. elegans holocentric chromosomes provide a guide to the structure, molecular architecture, and function of conventional kinetochores.

Show MeSH
Related in: MedlinePlus