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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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Centromeric association of HCP-4 during mitosis. Two-cell C. elegans embryos at different stages of the mitotic cell cycle were fixed and stained with α-HCP3 antibody (A, D, G, J, and M, green), α-HCP4 (B, E, H, K, and N, red), and DAPI (blue). Colocalization of HCP-3 and HCP-4 was visualized in the merged images as yellow (C, F, I, L, and O). All images, except metaphase, are a projection of the Z-stack of the entire nucleus. (A–C) Early prophase; (D–F) late prophase; (G–I) metaphase; (J–L) anaphase; (M–O) telophase. Bars, 5 μm.
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Figure 3: Centromeric association of HCP-4 during mitosis. Two-cell C. elegans embryos at different stages of the mitotic cell cycle were fixed and stained with α-HCP3 antibody (A, D, G, J, and M, green), α-HCP4 (B, E, H, K, and N, red), and DAPI (blue). Colocalization of HCP-3 and HCP-4 was visualized in the merged images as yellow (C, F, I, L, and O). All images, except metaphase, are a projection of the Z-stack of the entire nucleus. (A–C) Early prophase; (D–F) late prophase; (G–I) metaphase; (J–L) anaphase; (M–O) telophase. Bars, 5 μm.

Mentions: The localization of HCP-4 in mitotic cells suggested that HCP-4 might be a centromere protein (Fig. 2 D). To address this possibility, we used multiple wavelength fluorescence microscopy to perform a colocalization experiment between HCP-4 and the centromeric histone, HCP-3. HCP-3 is present during interphase as discrete dots or units and as a continuous line of reactivity along the axis of prophase chromosome (Fig. 3 A; Buchwitz et al. 1999). HCP-4 was also found to be present in a single line of reactivity along the axis of the chromosome (Fig. 3 B). HCP-4, like HCP-3, appeared as two lines of reactivity later in prophase (Fig. 3D and Fig. E). These two lines are oriented towards the two half spindles at metaphase and at anaphase (Fig. 3G and Fig. H, and Fig. J and Fig. K, respectively). When we merged the HCP-3 and HCP-4 images we observed that both colocalized nearly completely beginning in prophase and persisting through metaphase and anaphase (Fig. 3C, Fig. F, Fig. I, and Fig. L, yellow). This colocalization of HCP-4 and HCP-3 showed that HCP-4 was centromere localized during mitosis. During telophase as chromosome decondensation occurs, HCP-3 was again observed as discrete units throughout the nucleoplasm (Fig. 3 M). We observed HCP-4 staining also became punctate after mitosis and did not overlap with HCP-3 staining (Fig. 3N and Fig. O).


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)

Centromeric association of HCP-4 during mitosis. Two-cell C. elegans embryos at different stages of the mitotic cell cycle were fixed and stained with α-HCP3 antibody (A, D, G, J, and M, green), α-HCP4 (B, E, H, K, and N, red), and DAPI (blue). Colocalization of HCP-3 and HCP-4 was visualized in the merged images as yellow (C, F, I, L, and O). All images, except metaphase, are a projection of the Z-stack of the entire nucleus. (A–C) Early prophase; (D–F) late prophase; (G–I) metaphase; (J–L) anaphase; (M–O) telophase. Bars, 5 μm.
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Figure 3: Centromeric association of HCP-4 during mitosis. Two-cell C. elegans embryos at different stages of the mitotic cell cycle were fixed and stained with α-HCP3 antibody (A, D, G, J, and M, green), α-HCP4 (B, E, H, K, and N, red), and DAPI (blue). Colocalization of HCP-3 and HCP-4 was visualized in the merged images as yellow (C, F, I, L, and O). All images, except metaphase, are a projection of the Z-stack of the entire nucleus. (A–C) Early prophase; (D–F) late prophase; (G–I) metaphase; (J–L) anaphase; (M–O) telophase. Bars, 5 μm.
Mentions: The localization of HCP-4 in mitotic cells suggested that HCP-4 might be a centromere protein (Fig. 2 D). To address this possibility, we used multiple wavelength fluorescence microscopy to perform a colocalization experiment between HCP-4 and the centromeric histone, HCP-3. HCP-3 is present during interphase as discrete dots or units and as a continuous line of reactivity along the axis of prophase chromosome (Fig. 3 A; Buchwitz et al. 1999). HCP-4 was also found to be present in a single line of reactivity along the axis of the chromosome (Fig. 3 B). HCP-4, like HCP-3, appeared as two lines of reactivity later in prophase (Fig. 3D and Fig. E). These two lines are oriented towards the two half spindles at metaphase and at anaphase (Fig. 3G and Fig. H, and Fig. J and Fig. K, respectively). When we merged the HCP-3 and HCP-4 images we observed that both colocalized nearly completely beginning in prophase and persisting through metaphase and anaphase (Fig. 3C, Fig. F, Fig. I, and Fig. L, yellow). This colocalization of HCP-4 and HCP-3 showed that HCP-4 was centromere localized during mitosis. During telophase as chromosome decondensation occurs, HCP-3 was again observed as discrete units throughout the nucleoplasm (Fig. 3 M). We observed HCP-4 staining also became punctate after mitosis and did not overlap with HCP-3 staining (Fig. 3N and Fig. O).

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