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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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The centromere is assembled during prophase. Nuclei from wild-type (A–C), hcp-3(RNAi) (D–F), and hcp-4 (RNAi) (G–I) embryos were fixed and stained with DAPI (blue), α–HCP-3 pAb (red), and α–HCP-1 (green). A P0 hcp-3(RNAi) embryo, postpronuclei fusion, stained with DAPI (J), α–HCP-3 pAb (K), and α–HCP-4 pAb (L). (M) Diagram summarizing the order of assembly for the centromere–kinetochore complex. Bar, 5 μm.
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Figure 5: The centromere is assembled during prophase. Nuclei from wild-type (A–C), hcp-3(RNAi) (D–F), and hcp-4 (RNAi) (G–I) embryos were fixed and stained with DAPI (blue), α–HCP-3 pAb (red), and α–HCP-1 (green). A P0 hcp-3(RNAi) embryo, postpronuclei fusion, stained with DAPI (J), α–HCP-3 pAb (K), and α–HCP-4 pAb (L). (M) Diagram summarizing the order of assembly for the centromere–kinetochore complex. Bar, 5 μm.

Mentions: HCP-4 is the third protein that we have localized to the centromere. Because of differences in the timing of association of these proteins with the centromere, we wanted to determine if there is an ordered assembly pathway. We reduced the expression of HCP-3 by RNAi and observed that HCP-1 and HCP-4 were not localized to chromosomes (Fig. 5E and Fig. L, respectively). HCP-1 was present as dots distributed throughout the nucleus (Fig. 5 F) and the mitotic spindle (Fig. 4 F). HCP-4, although not chromosomally associated, was diffusely present in the nucleus (Fig. 5 L). Reduction of HCP-4 expression did not eliminate HCP-3 chromosome association, but did eliminate chromosomal association of HCP-1, which was again present as dots distributed throughout the nucleus (Fig. 5, G–I). These results, combined with the dynamic changes in localization during mitosis (Fig. 3), suggest that HCP-3 is necessary for HCP-4 centromere localization, which in turn is necessary for localization of the kinetochore protein, HCP-1 (Fig. 5 M).


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)

The centromere is assembled during prophase. Nuclei from wild-type (A–C), hcp-3(RNAi) (D–F), and hcp-4 (RNAi) (G–I) embryos were fixed and stained with DAPI (blue), α–HCP-3 pAb (red), and α–HCP-1 (green). A P0 hcp-3(RNAi) embryo, postpronuclei fusion, stained with DAPI (J), α–HCP-3 pAb (K), and α–HCP-4 pAb (L). (M) Diagram summarizing the order of assembly for the centromere–kinetochore complex. Bar, 5 μm.
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Related In: Results  -  Collection

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Figure 5: The centromere is assembled during prophase. Nuclei from wild-type (A–C), hcp-3(RNAi) (D–F), and hcp-4 (RNAi) (G–I) embryos were fixed and stained with DAPI (blue), α–HCP-3 pAb (red), and α–HCP-1 (green). A P0 hcp-3(RNAi) embryo, postpronuclei fusion, stained with DAPI (J), α–HCP-3 pAb (K), and α–HCP-4 pAb (L). (M) Diagram summarizing the order of assembly for the centromere–kinetochore complex. Bar, 5 μm.
Mentions: HCP-4 is the third protein that we have localized to the centromere. Because of differences in the timing of association of these proteins with the centromere, we wanted to determine if there is an ordered assembly pathway. We reduced the expression of HCP-3 by RNAi and observed that HCP-1 and HCP-4 were not localized to chromosomes (Fig. 5E and Fig. L, respectively). HCP-1 was present as dots distributed throughout the nucleus (Fig. 5 F) and the mitotic spindle (Fig. 4 F). HCP-4, although not chromosomally associated, was diffusely present in the nucleus (Fig. 5 L). Reduction of HCP-4 expression did not eliminate HCP-3 chromosome association, but did eliminate chromosomal association of HCP-1, which was again present as dots distributed throughout the nucleus (Fig. 5, G–I). These results, combined with the dynamic changes in localization during mitosis (Fig. 3), suggest that HCP-3 is necessary for HCP-4 centromere localization, which in turn is necessary for localization of the kinetochore protein, HCP-1 (Fig. 5 M).

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