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First published online 8 March 2005
doi: 10.1242/jcs.01727

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Nuclear localization of HTLV-I bZIP factor (HBZ) is mediated by three distinct motifs

Laboratoire Infections Rétrovirales et Signalisation Cellulaire, CNRS/UM I UMR 5121/IFR 122, Institut de Biologie, 34960 Montpellier Cedex 2, France

View larger version (24K): [in a new window]   Fig. 1. Molecular structure of HBZ. (A) HBZ is composed of an N-terminal activation domain (AD), two basic regions (BR1 and BR2) and a DNA-binding domain (DBD) preceding its leucine zipper (ZIP). (B) Amino acid sequences of DBD, BR1 and BR2. The basic amino acids are indicated in bold.

View larger version (16K): [in a new window]   Fig. 2. A summary of subcellular locations of HBZ and its deletion mutants. The indicated HBZ-EGFP fusions were expressed in COS cells and their subcellular distribution was analyzed by fluorescence microscopy. , deletion.

View larger version (39K): [in a new window]   Fig. 3. Western blot analysis of COS cells transfected with the different vectors expressing HBZ-EGFP fusion proteins. Expression of the different fusion proteins was analyzed by SDS-PAGE and western blotting with a mouse anti-EGFP antibody. Molecular size markers (kDa) are shown on the left.

View larger version (39K): [in a new window]   Fig. 4. Subcellular localization of HBZ and its mutants in COS cells. HBZ and its mutants fused to EGFP were transiently transfected into COS cells. Cells were cultivated on glass sides, fixed and stained with Hoechst solution, and then green fluorescence was analyzed by fluorescence microscopy. The specificity of the fluorescent staining is indicated by the absence of signal in flanking untransfected cells. The blue fluorescence of the nuclei is visualized by UV illumination. Magnification, 1000 x.

View larger version (45K): [in a new window]   Fig. 5. Colocalization of HBZ mutants deleted of their N- and C-terminal domains with C23 in the nucleoli. COS cells transfected with the mutants HBZ-BR1/DBD, HBZ-BR2/DBD and HBZ-BR2/BR1 fused to EGFP (column EGFP) were labeled with a mouse anti-C23 antibody (column C23) and detected using goat anti-mouse IgG antibody coupled to TRITC. Analysis of the green, red and merged fluorescence was performed with a fluorescence microscope. Magnification, 1200 x.

View larger version (26K): [in a new window]   Fig. 6. In vivo expression of the Myc-tagged HBZ mutants. COS cells were transfected with pcDNA3.1(–)/Myc-His expressing HBZ and the mutants HBZ-ZIP, HBZ-BR2/BR1/DBD and HBZ-AD fused to the Myc epitope. After transfection in COS cells, expression of the different mutants was checked by western blotting (top) and their localization analyzed by fluorescence microscopy using the mouse anti-Myc antibody (bottom). For immunofluorescence analysis, the anti-Myc antibody was detected with goat anti-mouse IgG antibody coupled to FITC. Magnification, 1500 x.

View larger version (60K): [in a new window]   Fig. 7. Analysis of HBZ subnuclear localization. Localization of the transiently expressed HBZ-EGFP was analyzed by immunofluorescence microscopy. (A) COS cells cotransfected with HBZ-EGFP and PML protein expression vector were labeled with a mouse anti-PML protein antibody and detected using goat anti-mouse IgG antibody coupled to TRITC. Analysis of the green, red and merged fluorescence was performed with a fluorescence microscope. (B) The same approach was performed with HBZ-EGFP-transfected COS cells to localize endogenous SC35 labeled with a mouse anti-SC35 antibody, and (C) with COS cells cotransfected with pEGFP-HBZ and pSG-Tax. Tax wax labeled with a culture supernatant of the anti-Tax 168A51-42 hybridoma. Magnification, 1500 x.

View larger version (129K): [in a new window]   Fig. 8. HBZ localization in heterochromatin of transfected COS cells. COS cells transfected with pcDNA3.1(–)/Myc-His-HBZ (A-D) or pcDNA3.1(–)/Myc-His (E-F) for 36 hours were fixed with 2.5% formaldehyde and embedded in Lowicryl resin. Ultrathin sections were stained with the mouse anti-Myc antibody, then incubated with goat anti-mouse IgG antibody conjugated to 10 nm gold particles, and analyzed by electron microscopy. Bar, 0.5 µm. NU, nucleus; NO, nucleolus.

View larger version (48K): [in a new window]   Fig. 9. HBZ colocalizes with HP1. (A,B) Endogenous HP1 was localized in EGFP-HBZ-transfected COS cells with a mouse HP1 antibody detected using goat anti-mouse IgG antibody coupled to TRITC. Analysis of the green, red and merged fluorescence was performed with a fluorescence microscope. Magnification, 2000 x.

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