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 protein aligns along a subset of midzone microtubules at anaphase. (A) Immunostaining of hRif1 in early anaphase and late anaphase LOX cells using hRif1 antibody (red), β-tubulin antibody (green), and DAPI (blue). Images were acquired with a Deltavision microscopy system as described in Fig. 8. (B) Enlarged images of early anaphase cells in A to show the alignment of hRif1 along microtubules. (C) Schematic representation of hRif1 deletion constructs used for analyses of telophase chromosome association and anaphase midzone microtubule colocalization. GFP-hRif1 deletion constructs were transfected into LOX cells and immunostaining was performed to determine their intracellular localization across the cell cycle. Bars, 10 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172464&req=5

fig9: hRif1 protein aligns along a subset of midzone microtubules at anaphase. (A) Immunostaining of hRif1 in early anaphase and late anaphase LOX cells using hRif1 antibody (red), β-tubulin antibody (green), and DAPI (blue). Images were acquired with a Deltavision microscopy system as described in Fig. 8. (B) Enlarged images of early anaphase cells in A to show the alignment of hRif1 along microtubules. (C) Schematic representation of hRif1 deletion constructs used for analyses of telophase chromosome association and anaphase midzone microtubule colocalization. GFP-hRif1 deletion constructs were transfected into LOX cells and immunostaining was performed to determine their intracellular localization across the cell cycle. Bars, 10 μm.

Mentions: Interestingly, during early anaphase, a significant amount of hRif1 was observed to align along fiber-like structures that were coincident with (or minimally, overlapped with) a subset of the midzone microtubules located in between the two sets of separating chromosomes (Fig. 9, A and B). Furthermore, hRif1 was on more fibers in early anaphase cells than in late anaphase cells, indicating that this localization is a dynamic and regulated process. Inspection of the separating chromosomes by deconvolution microscopy excluded the possibility that any anaphase bridges were present and that the hRif1 might have been associated with them. In addition, staining of anaphase cells with antibodies against 53BP1 does not show the same pattern (unpublished data). Hence, the hRif1 staining in this region was not attributable to association with chromosome damage sites. No localization to the midbody in cytokinesis was observed. The same pattern of cell cycle–dependent association of hRif1 with chromatin and midzone microtubules was corroborated by immunofluorescence staining of LOX cells and HeLa cells with two independent affinity-purified rabbit pAbs raised against two different hRif1 peptide epitopes using either PFA or methanol as fixation reagent, and also by transfecting LOX cells with a GFP-hRif1 fusion protein (Fig. S5, available at http://www.jcb.org/cgi/content/full/jcb.200408181/DC1). These different methods of hRif1 detection all gave identical results, confirming that this dynamic subcellular pattern was attributable to hRif1.


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

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

hRif1 protein aligns along a subset of midzone microtubules at anaphase. (A) Immunostaining of hRif1 in early anaphase and late anaphase LOX cells using hRif1 antibody (red), β-tubulin antibody (green), and DAPI (blue). Images were acquired with a Deltavision microscopy system as described in Fig. 8. (B) Enlarged images of early anaphase cells in A to show the alignment of hRif1 along microtubules. (C) Schematic representation of hRif1 deletion constructs used for analyses of telophase chromosome association and anaphase midzone microtubule colocalization. GFP-hRif1 deletion constructs were transfected into LOX cells and immunostaining was performed to determine their intracellular localization across the cell cycle. Bars, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

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

fig9: hRif1 protein aligns along a subset of midzone microtubules at anaphase. (A) Immunostaining of hRif1 in early anaphase and late anaphase LOX cells using hRif1 antibody (red), β-tubulin antibody (green), and DAPI (blue). Images were acquired with a Deltavision microscopy system as described in Fig. 8. (B) Enlarged images of early anaphase cells in A to show the alignment of hRif1 along microtubules. (C) Schematic representation of hRif1 deletion constructs used for analyses of telophase chromosome association and anaphase midzone microtubule colocalization. GFP-hRif1 deletion constructs were transfected into LOX cells and immunostaining was performed to determine their intracellular localization across the cell cycle. Bars, 10 μm.
Mentions: Interestingly, during early anaphase, a significant amount of hRif1 was observed to align along fiber-like structures that were coincident with (or minimally, overlapped with) a subset of the midzone microtubules located in between the two sets of separating chromosomes (Fig. 9, A and B). Furthermore, hRif1 was on more fibers in early anaphase cells than in late anaphase cells, indicating that this localization is a dynamic and regulated process. Inspection of the separating chromosomes by deconvolution microscopy excluded the possibility that any anaphase bridges were present and that the hRif1 might have been associated with them. In addition, staining of anaphase cells with antibodies against 53BP1 does not show the same pattern (unpublished data). Hence, the hRif1 staining in this region was not attributable to association with chromosome damage sites. No localization to the midbody in cytokinesis was observed. The same pattern of cell cycle–dependent association of hRif1 with chromatin and midzone microtubules was corroborated by immunofluorescence staining of LOX cells and HeLa cells with two independent affinity-purified rabbit pAbs raised against two different hRif1 peptide epitopes using either PFA or methanol as fixation reagent, and also by transfecting LOX cells with a GFP-hRif1 fusion protein (Fig. S5, available at http://www.jcb.org/cgi/content/full/jcb.200408181/DC1). These different methods of hRif1 detection all gave identical results, confirming that this dynamic subcellular pattern was attributable to hRif1.

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