Figure 1. Inner centromere localization of the CPC is required for stable chromosome bi-orientation.

(a) Scheme of human CPC (bor, borealin; surv, survivin; AurB, Aurora B) and of INCENPΔCEN (deletion of aa 1–48), survivin-INCENPΔCEN (surv-INCENP) and CENP-B-INCENPΔCEN (CB-INCENP). In all experiments HeLa Flp-In T-REx cells expressing the indicated mCherry-tagged INCENP variants were used.+ ind., expression induced by doxycycline, − ind., no induction of expression. (b) IF of Aurora B, RFP (to detect mCherry), and CENP-C on chromosome spreads of nocodazole treated cells. 1D line graphs of Aurora B (green) and CENP-C (red) are shown on the right. Scale bar, 2 μm. Of note, although surv-INCENP and Aurora B accumulate at the inner centromere, in surv-INCENP-expressing prometaphase cells we also observed some localization of surv-INCENP and Aurora B over the chromosomal arms. Since survivin directly interacts with pH3T3 (refs ⁴⁷, ⁴⁸) and we frequently detected pH3T3 along the chromosomal arms, this most likely explains the additional arm localization. (c) Scheme of the bi-orientation assay (that is, release from a monastrol-induced mitotic arrest into medium containing MG132) and examples of the alignment categories. Scale bar, 5 μm. (d) Cells were transfected with siRNAs for Luciferase (siLUC) or INCENP (siINC) and subjected to the bi-orientation assay and chromosome alignment was assessed (n=2 exp., ≥150 cells per condition, error bars are s.e.m.). Representative images of two conditions (scale bar, 5 μm), and enlargements of selected image regions are shown on the right (scale bar, 2 μm). DNA is visualized using DAPI.