Figure 4
KIF4 and PRC1 repress chromosome congression when Cdk1 is partially active.
(A) Experimental setup of a nocodazole washout assay. Thymidine blocked cells are released, nocodazole-arrested and washed free from nocodazole to allow spindle assembly in presence of the proteasome inhibitor MG132, together with RO-3306. (B) Representative immunofluorescence images of mitotic cells, before and after nocodazole washout. Examples of different alignment defects are shown. Scale bar, 10 μm. (C) PRC1 and KIF4 prevent chromosome alignment when Cdk1 activity is compromised. Bar graph shows a quantification of the experimental setup shown in (A). (Mean ± s.e.m, n = 3, at least 150 cells/experiment). (D) Western blot analysis of the experiment in (C) shows the knockdown efficiency of the shRNAs targeting KIF4 or PRC1. (E) PRC1 present at microtubule overlaps partly localizes to astral microtubules after suppression of Cdk1 activity. U2OS cells were treated with or without 3 μM RO-3306 for 16 hours, fixed and processed for immunofluorescence. Insets demonstrate PRC1 localization to astral microtubules in the vicinity of unaligned chromosomes when Cdk1 is inhibited. Scale bar, 10 μm. (F) PRC1 localization to astral microtubules is dependent on the presence of KIF4. U2OS WT or KIF4 KO cells were treated and processed as in (E). Prometaphase cells were scored for the presence of PRC1 on astral microtubules (MTs). Scale bar, 10 μm. (Mean ± s.e.m, n = 3, at least 30 cells/experiment; ****p < 0.0001, Student’s t test). (G) Schematic overview of the progression from prometaphase to metaphase with normal or compromised cyclin B-Cdk1 activity. Maximal Cdk1 activation directs chromosome alignment and the establishment of stable k-MT attachments in metaphase. Partial reduction in Cdk1 activity by RO-3306 treatment results in a prometaphase arrest that is dependent on KIF4 and PRC1. Due to a weakened spindle checkpoint, cells will exit from mitosis with lagging chromosomes leading to the formation of micronuclei. Western blots in panel (D) have been cropped and full-length gels can be viewed in Supplementary Fig. 6.
