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First published online 25 April 2006
doi: 10.1242/jcs.02907

Journal of Cell Science 119, 2025-2034 (2006)
Published by The Company of Biologists 2006
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Tau protein binds to pericentromeric DNA: a putative role for nuclear tau in nucleolar organization

Marcela K. Sjöberg1,*, Elena Shestakova2, Zeyni Mansuroglu2, Ricardo B. Maccioni1,3,{ddagger} and Eliette Bonnefoy2,{ddagger}

1 Laboratory of Cellular, Molecular Biology and Neurosciences, Millennium Institute for Advanced Studies in Cell Biology and Biotechnology (CBB), Faculty of Sciences, University of Chile, Las Encinas 3370, Ñuñoa, Santiago, Chile
2 Régulation de la Transcription et Maladies Génétiques, CNRS UPR2228, UFR Biomédicale, Université René Descartes, 45 rue des Saints-Pères, 75270 Paris CEDEX 06, France
3 Department of Neurological Sciences, Faculty of Medicine, University of Chile, Av. Salvador 486, Santiago, Chile


Figure 1
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  		Fig. 1. Nuclear tau localizes at the border of the nucleolus. Colocalization of endogenous tau with heterochromatin was analyzed by immunofluorescent technique and confocal microscopy in human skin fibroblasts and HeLa cells. Each row represents a single optical section of the same nucleus. Left panels (A,D) correspond to diMeH3K9 distribution revealed with rabbit anti-diMeH3K9. Middle panels (B,E) show subnuclear tau distribution detected with the Tau-1 monoclonal antibody. Merged images of tau with diMeH3K9 are shown on right panels for human fibroblasts (C) and HeLa cells (F) with double-labeled pixels displayed in white. (C',F') Enlarged views of C and F showing the overlap between tau and diMeH3K9 at the nucleolus in white. Scale bar, 5 µm.

 

Figure 2
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  		Fig. 2. Nuclear tau partially colocalizes with nucleolin. Colocalization of endogenous tau with nucleolar protein nucleolin was analyzed by immunofluorescence and confocal microscopy in human skin fibroblasts and HeLa cells. Each row represents a single optical section of the same nucleus. Upper panels (A,B), show phase-contrast image (A) and fluorescent nucleolin distribution (B) in human fibroblasts. Lower panels (C,D), show phase-contrast image (C) and fluorescent nucleolin distribution (D) in HeLa cells. (E,H) Tau distribution revealed with Tau-1 monoclonal antibody. (F,I) Subnuclear nucleolin distribution detected with nucleolin C23 polyclonal antibody. Merged images of tau with nucleolin are shown on the right for human fibroblasts (G) and HeLa cells (J). Bars, 10 µm (A-D); 5 µm (E-J).

 

Figure 3
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  		Fig. 3. Tau partially colocalizes with pericentromeric {alpha}-satellite DNA. Colocalization of endogenous tau with pericentromeric {alpha}-satellite DNA was studied by immuno-FISH and confocal microscopy in human skin fibroblasts, human lymphoblasts and HeLa cells. Each row represents a single optical section of the same nucleus. Left panels (A,D,G) correspond to subnuclear tau distribution detected with the Tau-1 monoclonal antibody. Middle panels (B,E,H) show {alpha}-satellite DNA repeats distribution revealed by FISH using fluoRED labeled {alpha}-satellite p82H plasmid as a probe. Merged images of tau with pericentromeric {alpha}-satellite DNA are shown on right panels for human skin fibroblasts (C), human lymphoblasts (F) and HeLa cells (I) with double-labeled pixels displayed in white. C',F',I' are enlarged views of C,F,I respectively. Bars, 5 µm.

 

Figure 4
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  		Fig. 4. Tau localizes inside the nucleolus and partially colocalizes with pericentromeric {alpha}-satellite DNA. Non-confocal conventional fluorescence microscopy was used to analyze total DNA distribution and colocalization of tau with {alpha}-satellite DNA in human skin fibroblasts and HeLa cells. (A and F) Total DNA distribution in cells revealed with Hoechst 33258. (B,G) Subnuclear tau distribution detected with the Tau-1 monoclonal antibody. (C,H) Distribution of {alpha}-satellite DNA repeats revealed by FISH using {alpha}-satellite p82H plasmid as a probe. Merged images of tau with Hoechst 33258 are shown in panels D (human skin fibroblasts) and I (HeLa cells) and merged images of tau with pericentromeric {alpha}-satellite DNA are shown in panels E (human skin fibroblasts) and J (HeLa cells). Bars, 10 µm.

 

Figure 5
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  		Fig. 5. Tau protein associates with {alpha}-satellite DNA. The association of tau protein with {alpha}-satellite DNA was analyzed by electrophoretic mobility shift assay (EMSA). (A) Purified tau protein from bovine brain was resolved by 12% SDS-PAGE and then stained with Coomassie Blue (CB) or immunodetected with Tau-5 monoclonal antibody to confirm the absence of contaminants. (B) Different concentrations of purified tau protein (100 ng, 200 ng, 300 ng and 400 ng) were incubated with the 32P-labeled {alpha}-satellite DNA probe of 700 bp. The incubations were performed in the presence of 0.5 µg unlabeled sonicated salmon sperm DNA (ssDNA) used as random non-sequence specific competitor DNA in 50 mM NaCl buffer. The EMSA gels were dried and radioactivity was visualized using PhosphorImager and the computer program ImageQuant. The arrows indicate the shift observed for the labeled probe owing to the interaction of the probe with tau and the free probe.

 

Figure 6
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  		Fig. 6. Association of tau protein with murine {gamma}-satellite DNA. Electrophoretic mobility shift assays (EMSA) were performed to analyze the interaction of purified tau protein with murine {gamma}-satellite DNA sequences. (A) Different concentrations of purified tau protein (100 ng, 200 ng, 300 ng and 400 ng) were incubated with the 32P-labeled {gamma}-satellite DNA probe of 936 bp in the presence of 0.5 µg ssDNA as random non-sequence specific competitor DNA. The gels were dried and the radioactivity was visualized using PhosphorImager and the computer program ImageQuant. (B) Indicated amounts of tau protein or HMGI protein were incubated with the {gamma}-satellite DNA probe, either labeled (lanes 1-5) or unlabeled (lanes 6-13) in the presence of 0.5 µg ssDNA unlabeled competitor. No satellite DNA was added in lanes 9 and 13. The complexes were resolved in a non-denaturing TBE-polyacrylamide gel. The gel was either, dried and autoradiographed (lanes 1-5) or transferred to a nitrocellulose membrane and immunoblotted using either the Tau-5 monoclonal antibody (lanes 6-9) or an anti-HMGI polyclonal antibody (lanes 10-13). The arrows indicate the shift observed for the complexes owing to the interaction with tau and the free-labeled probe.

 

Figure 7
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  		Fig. 7. Tau protein specifically interacts with {gamma} or {alpha}-satellite DNA sequences. (A) 400 ng purified tau protein were incubated with 32P-labeled {gamma}-satellite DNA probe in the absence (lane 2) or presence of an excess of unlabeled fECFD DNA fragment (lanes 3,4), unlabeled sonicated poly dI/dC (lanes 5-7), unlabeled sonicated poly dG/dC (lanes 8-10) or unlabeled {gamma}-satellite DNA (lanes 11,12). (B) 400 ng purified tau protein were incubated with 32P-labeled {alpha}-satellite DNA probe in the absence (lane 2) or presence of an excess of unlabeled fECFD DNA fragment (lanes 3,4), unlabeled sonicated poly dI/dC (lanes 5-7), unlabeled sonicated poly dG/dC (lanes 8-10) or unlabeled {alpha}-satellite DNA (lanes 11,12). The arrows indicate the shift observed for the complexes between the satellite probe and tau and the free-labeled probe at the bottom of the gel. The EMSA gels were dried and radioactivity was visualized using PhosphorImager and the computer program ImageQuant.

 

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© The Company of Biologists Ltd 2006