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.