Fig. 11. Mislocalized CENP-A does not support the mitotic movement of non-centromeric chromosome fragments. (A) CHO cells expressing HA-tagged CENP-A were arrested at the G1/S-phase boundary with hydroxyurea, then induced to prematurely enter mitosis and fragment their genome by caffeine treatment. Centromere-kinetochore subunits, which stain intensely with SH-CREST antiserum, were observed to congress at the metaphase plate (arrow). Whereas non-centromeric genome fragments containing mistargeted CENP-A, detected using anti-HA antibody, stained less intensely with SH-CREST antiserum, were immotile and lay in clusters around the cell periphery. DNA was counterstained with DAPI. (B) CENP-A was transiently transfected and overexpressed in COLO 320DM cells, which contain numerous acentric double-minute (DM) chromosomes (arrows). Targeting of CENP-A to the DMs was not sufficient to assemble kinetochores, as demonstrated by an apparent lack of anti-ZW10 antibody reaction. (C) hSMC1, detected with a specific antibody, was observed to be recruited to the DMs (arrows) following the mistargeting of CENP-A, detected with anti-HA antibody, to these acentric chromosomes. Bars, 10 µm.
