Figure 4.
INCENP-GFP localization, chromosome segregation, and cytokinesis in living Survivin1-142, Survivin89-142, and SurvivinC84A complemented cells. U2OS cells were grown in glass-bottom wells and cotransfected with Survivin siRNA and Survivin1-142 (A) Survivin89-142 (B and D), empty plasmid (C), or SurvivinC84A (E). To visualize CPC behavior, INCENP-GFP and H2B-diHcRed were cotransfected (A–C); in the other panels, only H2B-GFP was cotransfected (D and E). Cells were mounted on a time-lapse microscope 10 h after release from a thymidine block. Fluorescent and DIC images were captured every 1–5 min. Imaging was started at the onset of nuclear envelope breakdown in D and in prometaphase for A–C and E. To better visualize the localization of INCENP-GFP, enlargements of the images are shown in A–C. (D and E) Both the Survivin89-142 (3/8) and SurvivinC84A (3/9) reconstituted cells partially align their chromosomes. However, even in the situation where chromosome alignment was incomplete, cells start to segregate their sister chromatids, enter anaphase, and undergo cytokinesis (D and E). Note that in two or eight Survivin89-142- and five of nine SurvivinC84A-reconstituted cells alignment was normal. Time is hours:minutes.