Sensing chromosome bi-orientation by spatial separation of aurora B kinase from kinetochore substrates

Abstract

Successful cell division requires that chromosomes attach to opposite poles of the mitotic spindle (bi-orientation). Aurora B kinase regulates chromosome-spindle attachments by phosphorylating kinetochore substrates that bind microtubules. Centromere tension stabilizes bi-oriented attachments, but how physical forces are translated into signaling at individual centromeres is unknown. Using fluorescence resonance energy transfer-based biosensors to measure localized phosphorylation dynamics in living cells, we found that phosphorylation of an Aurora B substrate at the kinetochore depended on its distance from the kinase at the inner centromere. Furthermore, repositioning Aurora B closer to the kinetochore prevented stabilization of bi-oriented attachments and activated the spindle checkpoint. Thus, centromere tension can be sensed by increased spatial separation of Aurora B from kinetochore substrates, which reduces phosphorylation and stabilizes kinetochore microtubules.

Figure 1

Phosphorylation of a kinetochore Aurora B substrate depends on the microtubule attachment state. (A-B) Hela cells with both incorrect (green boxes, A) and bi-oriented (red boxes, A) attachments were fixed and stained for kinetochores (CREST) and phospho-CENP-A. Insets show stronger phospho-CENP-A staining on unaligned (1) vs. aligned (2) kinetochores. The phospho-CENP-A/CREST ratio was calculated at individual aligned (N=146) and unaligned (N=89) kinetochores from multiple cells (B). (C-F) Hela cells expressing either the CENP-B-targeted (C,E) or Mis12-targeted (D,F) sensor were fixed and stained for either Aurora B (C,D) or Hec1 (E,F) as markers for the inner centromere and outer kinetochore, respectively. YFP emission (green) shows sensor localization relative to Aurora B or Hec1 (red). Insets show individual centromere pairs used for linescans. Scale bars 5 μm.

Figure 2

Phosphorylation of Aurora B substrates depends on spatial separation of kinase from substrate. Hela cells expressing either the kinetochore (KT)-targeted or centromere (Cen)-targeted sensors were treated as indicated and imaged live. (A) Cells were treated with nocodazole, monastrol, or MG132, and the YFP/TFP emission ratio was calculated. An increase in emission ratio indicates dephosphorylation. Each bar represents an average over multiple cells, ≥30 kinetochores analyzed per cell. Note that y-axis is scaled to include the range of the data. Schematic shows sensor localization relative to Aurora B, as in Fig. S1A. (B-C) Cells were treated as in Fig. 1A, then imaged live before and after ZM447439 washout. The emission ratio was calculated at aligned and unaligned centromeres/kinetochores at the indicated timepoints (B). Each point represents ≥14 kinetochores/centromeres analyzed over multiple cells. Images (C) show sensor localization (YFP) and a color-coded representation of the emission ratio 10 min after ZM447439 washout. Boxes indicate aligned (red) and unaligned (green) centromeres/kinetochores. Scale bar 5 μm.

Figure 3

Positioning Aurora B closer to the kinetochore leads to increased phosphorylation of kinetochore substrates. (A) U2OS cells expressing the indicated vsv-tagged INCENP fusion proteins were fixed and stained for Aurora B (green) and Hec1 (red). Scale bars 5 μm. (B) Hela cells were transfected with a phosphorylation sensor together with mCherry-tagged wt-INCENP or CB-INCENP as indicated, imaged live, and the YFP/TFP emission ratio was calculated. Cells were grouped based on levels of the INCENP fusion proteins at centromeres, calculated based on mCherry fluorescence intensity (wt-INCENP was not present at as high levels as CB-INCENP). Each bar represents an average over multiple cells, ≥30 kinetochores analyzed per cell.

Figure. 4

Positioning Aurora B closer to the kinetochore activates the spindle checkpoint and destabilizes kinetochore-microtubule attachments. (A) U2OS cells expressing the indicated proteins were released from a G1/S block with or without ZM447439 for 14 hrs. The mitotic index was determined by propidium iodide/MPM2 mAb labeling and FACS analysis. One representative experiment out of 3 is shown. (B) Box-and-whisker plots (median, interquartile range, full range) showing time from nuclear envelope breakdown to metaphase (NEB-M) and from metaphase to anaphase (M-A) of U2OS cells expressing the indicated INCENP fusion proteins. (C-D) Hela cells expressing vsv-tagged CB-INCENP or Mis12-INCENP were treated with or without ZM447439, then analyzed for cold-stable microtubules (green) and vsv immunofluorescence (red). Brightness of vsv-Mis12-INCENP staining is enhanced relative to vsv-CB-INCENP so that it is visible. The percent of kinetochores with cold-stable microtubules was determined and plotted vs. expression level of the INCENP fusion proteins (D). Each data point represents >80 kinetochores from one cell.