Behavior of a patient-derived human fibroblast line (PD-NC4) containing a mutated chromosome 4 in which the normal centromere is epigenetically silenced and a neo-centromere has formed 25 Mbp down the q arm at a site that lacks centromere repeats. Shown is a z-series of an example of the PD-NC4 chromosome (upper, center) failing to align when the normal chromosomes have corrected syntelic attachments after monastrol withdrawal and are nearly aligned on the metaphase plate. Stained for DNA with DAPI (blue), CENP-C (green), and anti-centromere antibodies (ACA, red). See Fig 3C in Bassett et al. 2010. Epigenetic centromere specification directs aurora B accumulation but is insufficient to efficiently correct mitotic errors. J Cell Biol 190:177-185.
Cells were metaphase-arrested with nocodazole, swollen in 75 mM KCl, centrifuged onto glass slides, and fixed with formaldehyde before processing for indirect immunofluorescence. Digital z-series images were captured with an ORCA AG CCD mounted on DMI6000B Leica microscope using a 100X 1.4 NA objective lens and subsequently processed by deconvolution.
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