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First published online 20 July 2004
doi: 10.1242/jcs.01249

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Nucleolar localization of the human telomeric repeat binding factor 2 (TRF2)

¹ Biochemistry, Institute of Molecular Biotechnology, PO Box 100 813, 07708 Jena, Germany
² Molecular Biology, Institute of Molecular Biotechnology, PO Box 100 813, 07708 Jena, Germany

View larger version (57K): [in a new window]   Fig. 1. Immunofluorescence with a rabbit polyclonal antibody against TRF2 (H-300) revealed a nucleolar localization of TRF2 in human MCF-7 cells (A-B) but not in mouse NIH 3T3 cells (C-D). The same antibody was affinity purified with immobilized recombinant TRF2 and used for western blotting of MCF-7 or 3T3 whole cell lysate (E). The affinity purified TRF2 antibody also revealed a nucleolar staining together with several nucleoplasmic dots that are supposed to be telomeres (F-G). Arrows indicate the positions of the nucleolus.

View larger version (57K): [in a new window]   Fig. 2. Comparison of nucleolar TRF2 with UBF and B23 in human MCF-7 cells. The colocalization of these proteins was probed by double-immunofluorescence of TRF2 and UBF (A-C) or B23 (F-H). The reorientation of TRF2, UBF, and B23 was measured after applying 0.05 µg/ml actinomycin D for 3 hours and following the signals of TRF2 and UBF (D-E) or TRF2 and B23 (I-J). The colocalization of TRF2 with UBF in C-C' or B23 in F-H was observed by laser scanning confocal microscopy. Red and green arrows in C' and H represent TRF2 and UBF or B23, respectively. The frames presented in A-B and D-E are the magnification of the arrow-indicated nucleoli of the same picture. Smaller arrows in the frames of D-E indicate the relocalization of UBF after actinomycin D treatment. Fluorescence measurements along the dashed lines in C' and H are presented in C'' and H'.

View larger version (100K): [in a new window]   Fig. 3. Nucleolar localization of human TRF2 in interphase. The colocalization of TRF2 in human MCF-7 cells with Ki-67 protein (A-C), BrdU (D-F), and cyclin B1 (G-I) are shown at different stages of interphase. Arrows in A-C indicate a G0 phase cell that lacks a positive staining of Ki-67. Arrows in D-F indicate S-phase cells displayed by BrdU incorporation. Arrows in G-I indicate G2 phase cells with a high expression of cyclin B1.

View larger version (75K): [in a new window]   Fig. 4. Localization of TRF2, B23 and UBF in prophase and metaphase. The immunofluorescence of TRF2 and B23 or UBF is shown in mitotic human MCF-7 cells during prophase (A-C and H-K) and metaphase (D-G and L-O). Panels A-C were observed by light microscopy and panels D-O were observed by laser-scanning confocal microscopy. Panels G', K' and O' depict the fluorescent measurements along the indicated lines in G, K and O, respectively.

View larger version (42K): [in a new window]   Fig. 5. Nucleolar relocalization of TRF2, B23 and Ku86 during telophase of MCF-7 cells. (A) Comparison of TRF2 with B23 during early telophase (a-c), middle telophase (d-f) and late telophase (g-i). In (e) the larger arrows indicate the positions of the nucleoli or NOR, whereas the small arrow indicates the prenucleolar body (PNB). (B) Comparison of TRF2 with Ku86 in early prophase (a-c) and late telophase (d-f). Nucleoli are indicated by arrows.

View larger version (86K): [in a new window]   Fig. 6. Actinomycin D delayed the nucleolar release of TRF2 in G2 phase and mitosis. The effect of actinomycin D on the nucleolar localization of human TRF2 was examined at G2 phase, as determined by the expression of cyclin B1 (A-C), or at early prophase and compared to the localizations of B23 (D-F) and Ku86 (G-I). Arrows indicate the position of the nucleoli.

View larger version (77K): [in a new window]   Fig. 7. After treatment with actinomycin D, TRF2 co-localized with UBF in metaphase. The actinomycin D-delayed release of TRF2 from the nucleolus offered the opportunity to visualize its co-localization with UBF. Laser scanning confocal microscopy revealed an adjacent localization between TRF2 and UBF in prophase (A-C), and a complete overlap of both proteins in metaphase (D-F). Arrows indicate NORs where UBF or TRF2 were localized. Images with overlaid DNA stains are presented in A'-F'. C'' and F'' represent magnifications of the framed parts shown in C and F. Fluorescence measurements in C'' and F'' show the co-localization of TRF2 and UBF along the arrowed red lines.

View larger version (26K): [in a new window]   Fig. 8. Sucrose gradient centrifugation revealed high molecular weight TRF2-containing complexes in nucleolar extracts of human cells. Western blotting was performed to detect the presence of TRF2, Ku70 and B23 in the fractions of HeLa nucleolar extracts after ultracentrifugation through a 10 to 40% sucrose density gradient. MNase-sensitive and Ku-containing complexes occurred in fractions 8-16 and MNase-sensitive and B23-complexes showed up in fractions 10-17.

View larger version (76K): [in a new window]   Fig. 9. Actinomycin D treatment led to chromosome end-to-end fusions in mitotic cells. In response to actinomycin D treatment some telophase MCF-7 cells displayed chromosome end-to-end fusions (A) as well as the presence of TRF2 at the junctions of the observed chromosomal arcs (B). B' represents the magnified area framed in B. Chromosome fusion from cells undergoing cytokinesis is also shown (C-E). Co-localization of TRF2 with the telomere-binding protein Tin2 is shown in untreated (F-I) and actinomycin D-treated (J-M) MCF-7 cells. H', H'' and L' present profiles of fluorescence measurements along the indicated red lines shown in the same panel. Large arrows (F-M) indicate nucleoli and small arrows chromosome fusions (A-E) or telomeres (F-I and J-M). Total amounts of TRF2 were determined by western blotting of lysates of untreated and actinomycin D-treated MCF-7 cells (N). The lysates were prepared by directly dissolving cells on culture dishes into equal volumes of SDS-PAGE loading buffer.

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