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Human Rif1 protein binds aberrant telomeres and aligns along anaphase midzone microtubules.

Xu L, Blackburn EH - J. Cell Biol. (2004)

Bottom Line: The hRif1 level rose during late S/G2 but hRif1 was not visible on chromosomes in metaphase and anaphase; however, notably, specifically during early anaphase, hRif1 aligned along a subset of the midzone microtubules between the separating chromosomes.In telophase, hRif1 localized to chromosomes, and in interphase, it was intranuclear.These results define a novel subcellular localization behavior for hRif1 during the cell cycle.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Biophysics, University of California, San Francisco, CA 94143, USA.

ABSTRACT
We identified and characterized a human orthologue of Rif1 protein, which in budding yeast interacts in vivo with the major duplex telomeric DNA binding protein Rap1p and negatively regulates telomere length. Depletion of hRif1 by RNA interference in human cancer cells impaired cell growth but had no detectable effect on telomere length, although hRif1 overexpression in S. cerevisiae interfered with telomere length control, in a manner specifically dependent on the presence of yeast Rif1p. No localization of hRif1 on normal human telomeres, or interaction with the human telomeric proteins TRF1, TRF2, or hRap1, was detectable. However, hRif1 efficiently translocated to telomerically located DNA damage foci in response to the synthesis of aberrant telomeres directed by mutant-template telomerase RNA. The hRif1 level rose during late S/G2 but hRif1 was not visible on chromosomes in metaphase and anaphase; however, notably, specifically during early anaphase, hRif1 aligned along a subset of the midzone microtubules between the separating chromosomes. In telophase, hRif1 localized to chromosomes, and in interphase, it was intranuclear. These results define a novel subcellular localization behavior for hRif1 during the cell cycle.

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Cell cycle–regulated expression and intracellular distribution of hRif1 protein. (A) hRif1 protein levels are regulated across the cell cycle. Contact-inhibited T24 cells were released into the cell cycle for 0–33 h, as indicated above the lanes. Whole cell extracts were probed with hRif1 antibody PAB2857 or α-tubulin antibody. DNA contents of synchronized cells at different time points were determined by FACS analysis. Noc., cells were released into the cell cycle for 28 h and then treated with 0.3 μg/ml nocodazole for 10 h for a M phase block. (B) hRif1 dynamically associates with chromatin during the cell cycle. LOX cells were fixed and immunostained with β-tubulin antibody (green), hRif1 antibody (red), and DAPI (blue). Images were analyzed with a Deltavision microscopy system using the Deltavision SoftWorx resolve3D capture program and collected as a stack of 0.2-μm increments in the z axis. After deconvolution, an image of a representative single section on the z axis saved as a Photoshop file is presented. Bars, 10 μm.
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fig8: Cell cycle–regulated expression and intracellular distribution of hRif1 protein. (A) hRif1 protein levels are regulated across the cell cycle. Contact-inhibited T24 cells were released into the cell cycle for 0–33 h, as indicated above the lanes. Whole cell extracts were probed with hRif1 antibody PAB2857 or α-tubulin antibody. DNA contents of synchronized cells at different time points were determined by FACS analysis. Noc., cells were released into the cell cycle for 28 h and then treated with 0.3 μg/ml nocodazole for 10 h for a M phase block. (B) hRif1 dynamically associates with chromatin during the cell cycle. LOX cells were fixed and immunostained with β-tubulin antibody (green), hRif1 antibody (red), and DAPI (blue). Images were analyzed with a Deltavision microscopy system using the Deltavision SoftWorx resolve3D capture program and collected as a stack of 0.2-μm increments in the z axis. After deconvolution, an image of a representative single section on the z axis saved as a Photoshop file is presented. Bars, 10 μm.

Mentions: To investigate whether hRif1 protein expression varies during the cell cycle, we measured hRif1 protein steady-state levels by Western blot analysis using synchronized populations of T24 human bladder carcinoma cells. Parallel cultures of T24 cells were arrested at G0/G1 by contact inhibition. Cells were then released from arrest by replating them at low density in fresh medium (Chen et al., 1996). The cell-cycle distribution profile of the synchronized culture at each time point was determined by FACS analysis of DNA content. As shown in Fig. 8 A, hRif1 protein levels were low in G0, G1, and S phase, but rose in G2/M phase. In cells arrested in M phase by nocodazole treatment for 10 h, hRif1 accumulated at intermediate levels (Fig. 8 A). The periodic variations of hRif1 protein expression at different cell cycle stages were further analyzed with dual immunostaining of hRif1 with the G2/M phase-specific protein cyclin B. Cyclin B synthesis starts in the last one third of S phase and reaches a maximum level in G2 and M phases (Gong et al., 1993). As shown in Fig. S4 (available at http://www.jcb.org/cgi/content/full/jcb.200408181/DC1), interphase cells that showed no staining of cyclin B, and which were judged on that basis to be in G1 or early- to mid-S phase, had much lower levels of hRif1 staining compared with interphase cells that had abundant cyclin B, which were in late S or G2 phase. Therefore, we conclude that the rise of hRif1 protein levels in the cell cycle occurs in late S or G2 phase.


Human Rif1 protein binds aberrant telomeres and aligns along anaphase midzone microtubules.

Xu L, Blackburn EH - J. Cell Biol. (2004)

Cell cycle–regulated expression and intracellular distribution of hRif1 protein. (A) hRif1 protein levels are regulated across the cell cycle. Contact-inhibited T24 cells were released into the cell cycle for 0–33 h, as indicated above the lanes. Whole cell extracts were probed with hRif1 antibody PAB2857 or α-tubulin antibody. DNA contents of synchronized cells at different time points were determined by FACS analysis. Noc., cells were released into the cell cycle for 28 h and then treated with 0.3 μg/ml nocodazole for 10 h for a M phase block. (B) hRif1 dynamically associates with chromatin during the cell cycle. LOX cells were fixed and immunostained with β-tubulin antibody (green), hRif1 antibody (red), and DAPI (blue). Images were analyzed with a Deltavision microscopy system using the Deltavision SoftWorx resolve3D capture program and collected as a stack of 0.2-μm increments in the z axis. After deconvolution, an image of a representative single section on the z axis saved as a Photoshop file is presented. Bars, 10 μm.
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Related In: Results  -  Collection

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fig8: Cell cycle–regulated expression and intracellular distribution of hRif1 protein. (A) hRif1 protein levels are regulated across the cell cycle. Contact-inhibited T24 cells were released into the cell cycle for 0–33 h, as indicated above the lanes. Whole cell extracts were probed with hRif1 antibody PAB2857 or α-tubulin antibody. DNA contents of synchronized cells at different time points were determined by FACS analysis. Noc., cells were released into the cell cycle for 28 h and then treated with 0.3 μg/ml nocodazole for 10 h for a M phase block. (B) hRif1 dynamically associates with chromatin during the cell cycle. LOX cells were fixed and immunostained with β-tubulin antibody (green), hRif1 antibody (red), and DAPI (blue). Images were analyzed with a Deltavision microscopy system using the Deltavision SoftWorx resolve3D capture program and collected as a stack of 0.2-μm increments in the z axis. After deconvolution, an image of a representative single section on the z axis saved as a Photoshop file is presented. Bars, 10 μm.
Mentions: To investigate whether hRif1 protein expression varies during the cell cycle, we measured hRif1 protein steady-state levels by Western blot analysis using synchronized populations of T24 human bladder carcinoma cells. Parallel cultures of T24 cells were arrested at G0/G1 by contact inhibition. Cells were then released from arrest by replating them at low density in fresh medium (Chen et al., 1996). The cell-cycle distribution profile of the synchronized culture at each time point was determined by FACS analysis of DNA content. As shown in Fig. 8 A, hRif1 protein levels were low in G0, G1, and S phase, but rose in G2/M phase. In cells arrested in M phase by nocodazole treatment for 10 h, hRif1 accumulated at intermediate levels (Fig. 8 A). The periodic variations of hRif1 protein expression at different cell cycle stages were further analyzed with dual immunostaining of hRif1 with the G2/M phase-specific protein cyclin B. Cyclin B synthesis starts in the last one third of S phase and reaches a maximum level in G2 and M phases (Gong et al., 1993). As shown in Fig. S4 (available at http://www.jcb.org/cgi/content/full/jcb.200408181/DC1), interphase cells that showed no staining of cyclin B, and which were judged on that basis to be in G1 or early- to mid-S phase, had much lower levels of hRif1 staining compared with interphase cells that had abundant cyclin B, which were in late S or G2 phase. Therefore, we conclude that the rise of hRif1 protein levels in the cell cycle occurs in late S or G2 phase.

Bottom Line: The hRif1 level rose during late S/G2 but hRif1 was not visible on chromosomes in metaphase and anaphase; however, notably, specifically during early anaphase, hRif1 aligned along a subset of the midzone microtubules between the separating chromosomes.In telophase, hRif1 localized to chromosomes, and in interphase, it was intranuclear.These results define a novel subcellular localization behavior for hRif1 during the cell cycle.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Biophysics, University of California, San Francisco, CA 94143, USA.

ABSTRACT
We identified and characterized a human orthologue of Rif1 protein, which in budding yeast interacts in vivo with the major duplex telomeric DNA binding protein Rap1p and negatively regulates telomere length. Depletion of hRif1 by RNA interference in human cancer cells impaired cell growth but had no detectable effect on telomere length, although hRif1 overexpression in S. cerevisiae interfered with telomere length control, in a manner specifically dependent on the presence of yeast Rif1p. No localization of hRif1 on normal human telomeres, or interaction with the human telomeric proteins TRF1, TRF2, or hRap1, was detectable. However, hRif1 efficiently translocated to telomerically located DNA damage foci in response to the synthesis of aberrant telomeres directed by mutant-template telomerase RNA. The hRif1 level rose during late S/G2 but hRif1 was not visible on chromosomes in metaphase and anaphase; however, notably, specifically during early anaphase, hRif1 aligned along a subset of the midzone microtubules between the separating chromosomes. In telophase, hRif1 localized to chromosomes, and in interphase, it was intranuclear. These results define a novel subcellular localization behavior for hRif1 during the cell cycle.

Show MeSH
Related in: MedlinePlus