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Kinetochore localisation of the DNA damage response component 53BP1 during mitosis

Denis Jullien, Paola Vagnarelli, William C. Earnshaw and Yasuhisa Adachi*

The Wellcome Trust Centre for Cell Biology, Institute of Cell and Molecular Biology, The University of Edinburgh, King’s Buildings, Edinburgh EH9 3JR, UK



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  		Fig. 1. Western blotting showing the specificity of two anti-m53BP1 antibodies. Extracts prepared from mouse NIH3T3 cells were probed with affinity-purified antibodies (I) against N-m53BP1 (lane 2) and those against C-m53BP1 (lane 4). Both antibodies specifically recognised a single protein band migrating at about 300 kDa. The pre-immune sera (P) from the same rabbits gave no or very little signal (lanes 1,3).

 


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  		Fig. 2. Kinetochore localisation of 53BP1 in mitosis. Prometaphase HeLa cells were immunostained doubly with anti-human-53BP1 and anti-centromeric antibodies (ACA) (a-d, 53BP1 in red, ACA in green), anti-CENP-B (i-l, 53BP1 in green, CENP-B in red), anti-CENP-E antibodies (m-p, 53BP1 in green, CENP-E in red). Prometaphase NIH3T3 cells were co-stained with anti-N-m53BP1 and ACA (e-h, m53BP1 in red, ACA in green). In a,e,i,m, DAPI staining is in blue. Human and mouse 53BP1 are detected as foci (c,g k,o) on the condensed mitotic chromatin (a,e,i,m) that are adjacent to the ACA and CENP-B dots (b,f,j). and partially overlap with the kinetochore protein CENP-E (panel n). Insets in b,f,j,n,o and p are magnified views of a kinetochores pairs (indicated with the arrow). Note that the cells are in natural mitosis without colcemid treatment. Images were recorded with a Delta Vision microscope.

 


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  		Fig. 3. Identification of kinetochore-targeting domain of 53BP1 by GFP-fusion analysis. (A) The various portion of m53BP1 that were fused to GFP and tested for their ability to be recruited (+) or not (–) to kinetochores (K.L.), and to localise (+) or not (–) to the nucleus during interphase (N.L.). (B) Prometaphase HeLa cells expressing GFP fused to the indicated m53BP1 amino acid position were stained with monoclonal antibody against CENP-E to visualise kinetochores. CENP-E was detected with Texas Red-conjugated anti-mouse secondary antibodies. Chromosomes were stained with DAPI. GFP:m53BP11-1957 (a), GFP:m53BP1753-1957 (b), GFP:m53BP11-1601 (d), GFP:m53BP1753-1601 (f), GFP:m53BP11140-1703 (g), GFP:m53BP11140-1601 (h), GFP:m53BP11169-1601 (i), GFP:m53BP11220-1601 (j) properly generated a GFP signal colocalising with CENP-E, whereas GFP:m53BP11294-1957 (c), GFP:m53BP11-1139 (e), GFP:m53BP11294-1601 (k), GFP:m53BP11220-1515 (l) failed to do so. Images were recorded with a conventional microscope.

 


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  		Fig. 4. 53BP1 association to kinetochores during mitosis. Double immunofluorescence staining of HeLa cells at various stages of mitosis with anti-53BP1 antibodies (second column) and a monoclonal antibody against CENP-B (rows a,b, e-h; centromere staining), or CENP-E (rows c,d). DNA was labeled with DAPI (far-left column). Merged images are shown in the far-right column (53BP1 in green and CENP-B or CENP-E in red). (a) In interphase HeLa cells, 53BP1 foci and centromere (CENP-B) do not share significant spatial relationships. (b) 53BP1 and centromeres are first observed adjacent to cells presumably in prophase as judged by the chromatin condensation. (c) When 53BP1 was clearly detected as paired foci in late prophase (see chromosome condensation in c, left panel), overlapping CENP-E signal was hardly detectable. (d) 53BP1 highly accumulated at kinetochores by prometaphase and a clear coincident CENP-E signal was observed. Then, although the 53BP1 signal gradually diminished, 53BP1 was still detected at kinetochores in metaphase (e) and early anaphase (f), but was completely released by mid-anaphase (g). During telophase, 53BP1 started to re-associate with chromatin (h). Note the diffuse cytoplasmic 53BP1 signal from nuclear envelope breakdown to telophase. Images were recorded with a conventional microscope.

 


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  		Fig. 5. NIH3T3 cell in late prometaphase co-stained for m53BP1 (red) and ACA (green) showed a stronger 53BP1 signal on the kinetochores of unaligned chromosomes (indicated with arrows) than those on aligned chromosomes (a,b,c), whereas the level of ACA signal was more homogeneous on all kinetochores (b,d). DNA stained with DAPI is shown in blue (a). Images were recorded with a Delta Vision microscope.

 


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  		Fig. 6. 53BP1 is hyperphosphorylated in mitosis. 53BP1 was immunoprecipitated from protein extracts prepared from asynchronous (A, lane 1), mitotic (M, lane 2 and 3), and colcemid blocked (M+C, lane 4,5) HeLa cells. The precipitates were incubated in the presence (lanes 3,5) or in the absence (lanes 1,2,4) of {lambda} protein phosphatase ({lambda}PPase). The proteins were run on a gel and the electrophoretic mobility of 53BP1 was examined by western blotting. In mitotic cells (lane 2), 53BP1 exhibited a lower electrophoretic mobility compared with that in interphase cells (lane 1). The retardation was even more pronounced in mitotically blocked cells with colcemid (compare lane 2 versus 4). The 53BP1 electrophoretic retardation was eliminated by the protein phosphatase treatment (lanes 3,5). (B) Mitotically blocked HeLa cells exhibits a strong crescent shaped 53BP1 signal on kinetochores. Colcemid treated HeLa cells were double-stained for 53BP1 (green) and CENP-E (red) as in Fig. 2, bottom row. Inset in panels b, c, and d are a magnified view of the kinetochore pair shown by the arrow. The identity of the signal(s) displayed in each panel is written above the pictures. DNA stained with DAPI is shown in blue (a). Images were recorded with a Delta Vision microscope.

 

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