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HCP-4, a CENP-C-like protein in Caenorhabditis elegans, is required for resolution of sister centromeres.

Moore LL, Roth MB - J. Cell Biol. (2001)

Bottom Line: HCP-4 is found in the cytoplasm during interphase, but is nuclear localized in mitosis, where it localizes specifically to the centromere.The localization of HCP-4 to the centromere is dependent on the centromeric histone HCP-3; in addition, HCP-3 and HCP-4 are both required for localization of a CENP-F-like protein, HCP-1, indicating an ordered assembly pathway.These chromosomes also failed to form a functional kinetochore.

View Article: PubMed Central - PubMed

Affiliation: Division of Basic Sciences, Molecular and Cellular Biology Program, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, USA.

ABSTRACT
The centromere plays a critical role in the segregation of chromosomes during mitosis. In mammals, sister centromeres are resolved from one another in the G2 phase of the cell cycle. During prophase, chromosomes condense with sister centromeres oriented in a back to back configuration enabling only one chromatid to be captured by each half spindle. To study this process, we identified a centromere protein (CENP)-C-like protein, holocentric protein (HCP)-4, in Caenorhabditis elegans based on sequence identity, loss of function phenotype, and centromeric localization. HCP-4 is found in the cytoplasm during interphase, but is nuclear localized in mitosis, where it localizes specifically to the centromere. The localization of HCP-4 to the centromere is dependent on the centromeric histone HCP-3; in addition, HCP-3 and HCP-4 are both required for localization of a CENP-F-like protein, HCP-1, indicating an ordered assembly pathway. Loss of HCP-4 expression by RNA-mediated interference resulted in a failure to generate resolution of sister centromeres on chromosomes, suggesting that HCP-4 is required for sister centromere resolution. These chromosomes also failed to form a functional kinetochore. Thus, the CENP-C-like protein HCP-4 is essential for both resolution sister centromeres and attachment to the mitotic spindle.

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Sister centromere resolution. Individual chromosomes from prophase nuclei were stained with DAPI (blue), α–HCP-1 antibody (not shown), and α–HCP-3 antibody (red). Nuclei containing little or no HCP-1 staining were optically sectioned and a three-dimensional projection generated using the volume viewer function. The resulting images were rotated around the Y-axis. (A) Chromosomes showing different degrees of sister centromere separation. Cartoon of the Z-axis of each chromosome depicting the sister centromeres migration from a juxtaposed position to a maximally resolved orientation is shown adjacent to each image. (B) A single chromosome showing an intermediate “splitting” of sister centromeres. (C and D) Sister centromere resolution is a microtubule-independent process. (C) P1 blastomere from a wild-type embryo stained with DAPI (blue), antitubulin antibody (green), and α–HCP-3 antibody (red). (D) P1 blastomere, from an embryo incubated in the presence of 30 μg/ml nocodazole at room temperature for 30 min, stained as in C. The division time for a two-cell embryo at room temperature is ∼20–30 min, so this embryo had probably just finished cell division when treated with nocodazole. Furthermore, the centrosomes are duplicated (not shown) but have not migrated, indicating that loss of spindle microtubules occurred before centrosome migration, an event that occurs concurrent or before sister centromere resolution (C). Bar, 5 μM.
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Figure 6: Sister centromere resolution. Individual chromosomes from prophase nuclei were stained with DAPI (blue), α–HCP-1 antibody (not shown), and α–HCP-3 antibody (red). Nuclei containing little or no HCP-1 staining were optically sectioned and a three-dimensional projection generated using the volume viewer function. The resulting images were rotated around the Y-axis. (A) Chromosomes showing different degrees of sister centromere separation. Cartoon of the Z-axis of each chromosome depicting the sister centromeres migration from a juxtaposed position to a maximally resolved orientation is shown adjacent to each image. (B) A single chromosome showing an intermediate “splitting” of sister centromeres. (C and D) Sister centromere resolution is a microtubule-independent process. (C) P1 blastomere from a wild-type embryo stained with DAPI (blue), antitubulin antibody (green), and α–HCP-3 antibody (red). (D) P1 blastomere, from an embryo incubated in the presence of 30 μg/ml nocodazole at room temperature for 30 min, stained as in C. The division time for a two-cell embryo at room temperature is ∼20–30 min, so this embryo had probably just finished cell division when treated with nocodazole. Furthermore, the centrosomes are duplicated (not shown) but have not migrated, indicating that loss of spindle microtubules occurred before centrosome migration, an event that occurs concurrent or before sister centromere resolution (C). Bar, 5 μM.

Mentions: We were interested in whether HCP-4 was required at an early step in centromere maturation. An early aspect of centromere maturation is sister centromere resolution. In C. elegans, only a single centromere of α–HCP-3 staining is observed early in prophase; later in prophase two sister centromeres are observed on opposite sides of the chromosome (Buchwitz et al. 1999). Further examination of prophase nuclei showed chromosomes with two sister centromeres in various degrees of separation from one another (Fig. 6 A). Initiation of separation was sometimes seen as a “Y”-shaped intermediate (Fig. 6 B). This resolution of sister centromeres was not dependent on microtubules, because microtubules are likely to be absent from prophase nuclei (Fig. 6 C) and because resolution of sister centromeres occurred in the presence of the microtubule destabilizing drug nocodazole (Fig. 6 D).


HCP-4, a CENP-C-like protein in Caenorhabditis elegans, is required for resolution of sister centromeres.

Moore LL, Roth MB - J. Cell Biol. (2001)

Sister centromere resolution. Individual chromosomes from prophase nuclei were stained with DAPI (blue), α–HCP-1 antibody (not shown), and α–HCP-3 antibody (red). Nuclei containing little or no HCP-1 staining were optically sectioned and a three-dimensional projection generated using the volume viewer function. The resulting images were rotated around the Y-axis. (A) Chromosomes showing different degrees of sister centromere separation. Cartoon of the Z-axis of each chromosome depicting the sister centromeres migration from a juxtaposed position to a maximally resolved orientation is shown adjacent to each image. (B) A single chromosome showing an intermediate “splitting” of sister centromeres. (C and D) Sister centromere resolution is a microtubule-independent process. (C) P1 blastomere from a wild-type embryo stained with DAPI (blue), antitubulin antibody (green), and α–HCP-3 antibody (red). (D) P1 blastomere, from an embryo incubated in the presence of 30 μg/ml nocodazole at room temperature for 30 min, stained as in C. The division time for a two-cell embryo at room temperature is ∼20–30 min, so this embryo had probably just finished cell division when treated with nocodazole. Furthermore, the centrosomes are duplicated (not shown) but have not migrated, indicating that loss of spindle microtubules occurred before centrosome migration, an event that occurs concurrent or before sister centromere resolution (C). Bar, 5 μM.
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Related In: Results  -  Collection

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Figure 6: Sister centromere resolution. Individual chromosomes from prophase nuclei were stained with DAPI (blue), α–HCP-1 antibody (not shown), and α–HCP-3 antibody (red). Nuclei containing little or no HCP-1 staining were optically sectioned and a three-dimensional projection generated using the volume viewer function. The resulting images were rotated around the Y-axis. (A) Chromosomes showing different degrees of sister centromere separation. Cartoon of the Z-axis of each chromosome depicting the sister centromeres migration from a juxtaposed position to a maximally resolved orientation is shown adjacent to each image. (B) A single chromosome showing an intermediate “splitting” of sister centromeres. (C and D) Sister centromere resolution is a microtubule-independent process. (C) P1 blastomere from a wild-type embryo stained with DAPI (blue), antitubulin antibody (green), and α–HCP-3 antibody (red). (D) P1 blastomere, from an embryo incubated in the presence of 30 μg/ml nocodazole at room temperature for 30 min, stained as in C. The division time for a two-cell embryo at room temperature is ∼20–30 min, so this embryo had probably just finished cell division when treated with nocodazole. Furthermore, the centrosomes are duplicated (not shown) but have not migrated, indicating that loss of spindle microtubules occurred before centrosome migration, an event that occurs concurrent or before sister centromere resolution (C). Bar, 5 μM.
Mentions: We were interested in whether HCP-4 was required at an early step in centromere maturation. An early aspect of centromere maturation is sister centromere resolution. In C. elegans, only a single centromere of α–HCP-3 staining is observed early in prophase; later in prophase two sister centromeres are observed on opposite sides of the chromosome (Buchwitz et al. 1999). Further examination of prophase nuclei showed chromosomes with two sister centromeres in various degrees of separation from one another (Fig. 6 A). Initiation of separation was sometimes seen as a “Y”-shaped intermediate (Fig. 6 B). This resolution of sister centromeres was not dependent on microtubules, because microtubules are likely to be absent from prophase nuclei (Fig. 6 C) and because resolution of sister centromeres occurred in the presence of the microtubule destabilizing drug nocodazole (Fig. 6 D).

Bottom Line: HCP-4 is found in the cytoplasm during interphase, but is nuclear localized in mitosis, where it localizes specifically to the centromere.The localization of HCP-4 to the centromere is dependent on the centromeric histone HCP-3; in addition, HCP-3 and HCP-4 are both required for localization of a CENP-F-like protein, HCP-1, indicating an ordered assembly pathway.These chromosomes also failed to form a functional kinetochore.

View Article: PubMed Central - PubMed

Affiliation: Division of Basic Sciences, Molecular and Cellular Biology Program, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, USA.

ABSTRACT
The centromere plays a critical role in the segregation of chromosomes during mitosis. In mammals, sister centromeres are resolved from one another in the G2 phase of the cell cycle. During prophase, chromosomes condense with sister centromeres oriented in a back to back configuration enabling only one chromatid to be captured by each half spindle. To study this process, we identified a centromere protein (CENP)-C-like protein, holocentric protein (HCP)-4, in Caenorhabditis elegans based on sequence identity, loss of function phenotype, and centromeric localization. HCP-4 is found in the cytoplasm during interphase, but is nuclear localized in mitosis, where it localizes specifically to the centromere. The localization of HCP-4 to the centromere is dependent on the centromeric histone HCP-3; in addition, HCP-3 and HCP-4 are both required for localization of a CENP-F-like protein, HCP-1, indicating an ordered assembly pathway. Loss of HCP-4 expression by RNA-mediated interference resulted in a failure to generate resolution of sister centromeres on chromosomes, suggesting that HCP-4 is required for sister centromere resolution. These chromosomes also failed to form a functional kinetochore. Thus, the CENP-C-like protein HCP-4 is essential for both resolution sister centromeres and attachment to the mitotic spindle.

Show MeSH
Related in: MedlinePlus