Limits...
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.

Show MeSH

Related in: MedlinePlus

hRif1 accumulation on uncapped telomeres synthesized by hTER template mutant 47A. 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, images were viewed with the Quick Projection option. (A) hRif1 protein translocates to telomeres upon expression of hTER template mutant 47A (47A-hTer). LOX cells were infected with lentiviruses expressing wild-type hTER (WT-hTER) or 47A-hTer. 3 d after infection, cells were fixed and stained with TRF2 antibody (green), hRif1 antibody PAB2852 (red), and DAPI (blue). (B) hRif1 foci overlap with 53BP1 foci in LOX cells expressing 47A-hTer. Lentiviral infections were performed as described in A. Cells were stained with 53BP1 antibody (green), hRif1 antibody (red), and DAPI (blue). Bars, 10 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172464&req=5

fig4: hRif1 accumulation on uncapped telomeres synthesized by hTER template mutant 47A. 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, images were viewed with the Quick Projection option. (A) hRif1 protein translocates to telomeres upon expression of hTER template mutant 47A (47A-hTer). LOX cells were infected with lentiviruses expressing wild-type hTER (WT-hTER) or 47A-hTer. 3 d after infection, cells were fixed and stained with TRF2 antibody (green), hRif1 antibody PAB2852 (red), and DAPI (blue). (B) hRif1 foci overlap with 53BP1 foci in LOX cells expressing 47A-hTer. Lentiviral infections were performed as described in A. Cells were stained with 53BP1 antibody (green), hRif1 antibody (red), and DAPI (blue). Bars, 10 μm.

Mentions: As an independent way of uncapping telomeres, we introduced a lentiviral vector expressing a telomerase template mutant, MT-hTer-47A (Li et al., 2004), in LOX and HeLa cells. This mutant telomerase RNA synthesizes mutant telomeric repeats (Marusic et al., 1997; Li et al., 2004) that are predicted to have lost binding affinity for TRF1 and -2. The control was cells infected in parallel with a lentiviral expression vector expressing wild-type telomerase RNA (WT-hTER). hRif1 localization was then examined via deconvolution microscopy. In these control cells, the hRif1 staining pattern was indistinguishable from that of the mock-infected cells (unpublished data). In contrast, in cells expressing 47A-hTer, hRif1 accumulated in numerous distinct nuclear foci (mean number 34.4 foci per cell; Fig. 3 C and Fig. 4 A). To investigate the nature of the foci, we first performed coimmunostaining of hRif1 with antibody against TRF2. TRF2 protein specifically localizes at human telomeres (Broccoli et al., 1997). As shown in Fig. 4 A, the majority of hRif1 foci localized at or near telomeres. Because uncapped telomeres can elicit cellular responses similar to damaged DNA (Takai et al., 2003; Li et al., 2004), we then performed dual staining of hRif1 with the DNA damage response protein 53BP1. 53BP1 is known as a mediator of DNA damage signaling; upon DNA damage, 53BP1 is recruited rapidly to sites of DNA double-strand breaks and forms discrete nuclear foci (Schultz et al., 2000; Rappold et al., 2001). As shown in Fig. 4 B, expression of hTER template mutant 47A induced numerous 53BP1 foci in the nucleus. Almost all the 53BP1 foci overlapped with the hRif1 foci in these cells, indicating that hRif1 localized at aberrant telomeres to produce foci like those known to accumulate at DNA damage sites. Dual staining with antibody against phosphorylated ATM protein (Bakkenist and Kastan, 2003), another early DNA damage response sensor, and with antibody against hRif1 showed similar colocalization results (unpublished data).


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

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

hRif1 accumulation on uncapped telomeres synthesized by hTER template mutant 47A. 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, images were viewed with the Quick Projection option. (A) hRif1 protein translocates to telomeres upon expression of hTER template mutant 47A (47A-hTer). LOX cells were infected with lentiviruses expressing wild-type hTER (WT-hTER) or 47A-hTer. 3 d after infection, cells were fixed and stained with TRF2 antibody (green), hRif1 antibody PAB2852 (red), and DAPI (blue). (B) hRif1 foci overlap with 53BP1 foci in LOX cells expressing 47A-hTer. Lentiviral infections were performed as described in A. Cells were stained with 53BP1 antibody (green), hRif1 antibody (red), and DAPI (blue). Bars, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172464&req=5

fig4: hRif1 accumulation on uncapped telomeres synthesized by hTER template mutant 47A. 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, images were viewed with the Quick Projection option. (A) hRif1 protein translocates to telomeres upon expression of hTER template mutant 47A (47A-hTer). LOX cells were infected with lentiviruses expressing wild-type hTER (WT-hTER) or 47A-hTer. 3 d after infection, cells were fixed and stained with TRF2 antibody (green), hRif1 antibody PAB2852 (red), and DAPI (blue). (B) hRif1 foci overlap with 53BP1 foci in LOX cells expressing 47A-hTer. Lentiviral infections were performed as described in A. Cells were stained with 53BP1 antibody (green), hRif1 antibody (red), and DAPI (blue). Bars, 10 μm.
Mentions: As an independent way of uncapping telomeres, we introduced a lentiviral vector expressing a telomerase template mutant, MT-hTer-47A (Li et al., 2004), in LOX and HeLa cells. This mutant telomerase RNA synthesizes mutant telomeric repeats (Marusic et al., 1997; Li et al., 2004) that are predicted to have lost binding affinity for TRF1 and -2. The control was cells infected in parallel with a lentiviral expression vector expressing wild-type telomerase RNA (WT-hTER). hRif1 localization was then examined via deconvolution microscopy. In these control cells, the hRif1 staining pattern was indistinguishable from that of the mock-infected cells (unpublished data). In contrast, in cells expressing 47A-hTer, hRif1 accumulated in numerous distinct nuclear foci (mean number 34.4 foci per cell; Fig. 3 C and Fig. 4 A). To investigate the nature of the foci, we first performed coimmunostaining of hRif1 with antibody against TRF2. TRF2 protein specifically localizes at human telomeres (Broccoli et al., 1997). As shown in Fig. 4 A, the majority of hRif1 foci localized at or near telomeres. Because uncapped telomeres can elicit cellular responses similar to damaged DNA (Takai et al., 2003; Li et al., 2004), we then performed dual staining of hRif1 with the DNA damage response protein 53BP1. 53BP1 is known as a mediator of DNA damage signaling; upon DNA damage, 53BP1 is recruited rapidly to sites of DNA double-strand breaks and forms discrete nuclear foci (Schultz et al., 2000; Rappold et al., 2001). As shown in Fig. 4 B, expression of hTER template mutant 47A induced numerous 53BP1 foci in the nucleus. Almost all the 53BP1 foci overlapped with the hRif1 foci in these cells, indicating that hRif1 localized at aberrant telomeres to produce foci like those known to accumulate at DNA damage sites. Dual staining with antibody against phosphorylated ATM protein (Bakkenist and Kastan, 2003), another early DNA damage response sensor, and with antibody against hRif1 showed similar colocalization results (unpublished data).

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