Aurora B dynamics at centromeres create a diffusion-based phosphorylation gradient

Abstract

Aurora B kinase is essential for successful cell division and regulates spindle assembly and kinetochore-microtubule interactions. The kinase localizes to the inner centromere until anaphase, but many of its substrates have distinct localizations, for example on chromosome arms and at kinetochores. Furthermore, substrate phosphorylation depends on distance from the kinase. How the kinase reaches substrates at a distance and how spatial phosphorylation patterns are determined are unknown. In this paper, we show that a phosphorylation gradient is produced by Aurora B concentration and activation at centromeres and release and diffusion to reach substrates at a distance. Kinase concentration, either at centromeres or at another chromosomal site, is necessary for activity globally. By experimentally manipulating dynamic exchange at centromeres, we demonstrate that the kinase reaches its substrates by diffusion. We also directly observe, using a fluorescence resonance energy transfer-based biosensor, phosphorylation spreading from centromeres after kinase activation. We propose that Aurora B dynamics and diffusion from the inner centromere create spatial information to regulate cell division.

Figure 1.

Local concentration of Aurora B is required for kinase activity. (A) HeLa cells were transfected with either INCENP-mCherry or CB^DBD–INCENP-mCherry with or without Borealin siRNA as indicated and fixed and stained for DNA, Borealin, and Aurora B. (B) A schematic showing targeting of phosphorylation sensors by fusion to Hec1 (kinetochores), CB (centromeres), or histone H2B (bulk chromatin). (C) Cells were transfected as described in A, together with an Aurora B phosphorylation sensor targeted to chromatin, and imaged live. The YFP/CFP emission ratio was analyzed to measure phosphorylation changes and averaged over multiple cells (n = 12 cells for each bar). 2 µM ZM was used to dephosphorylate the sensor, which is indicated by an increased emission ratio. The letters to the left of the vertical axis indicate how normalized phosphorylation was calculated: (c − b)/(a − b) for wt-INCENP or (d − b)/(a − b) for CB^DBD–INCENP. (D) The experiment described in C was repeated with sensors targeted either to centromeres or to chromatin. Normalized values were calculated for each experiment as in C and averaged over three independent experiments. (E) U2OS-LacO cells were transfected with mCherry-LacI or with LacI-INCENP–mCherry with or without Borealin siRNA as indicated and then were fixed and stained for DNA, Borealin, and Aurora B. (F) Cells were transfected as described in E, together with the chromatin-targeted Aurora B phosphorylation sensor, and imaged live. The normalized YFP/CFP emission ratio was averaged over three independent experiments. Bars, 5 µm.

Figure 2.

Aurora B activity at a distance depends on phosphorylation of INCENP. (A) HeLa cells were transfected with either CB^DBD-INCENP–mCherry or CB^DBD-INCENP^TSS/AAA–mCherry, together with an Aurora B phosphorylation sensor targeted either to centromeres, chromatin, or kinetochores. Cells were also treated with or without Borealin siRNA and imaged live. The normalized YFP/CFP emission ratio or YFP/TFP for the kinetochore-targeted sensor was calculated as described in Fig. 1 C and averaged over three independent experiments. (B and C) Cells were transfected with the indicated constructs with or without INCENP siRNA as indicated and then were fixed and stained for phospho-Dsn1 (pDsn1) Ser100 or phospho-H3 (pHH3) Ser10. Representative images are shown (B), and normalized phospho-Dsn1 or phospho-H3 staining was quantified and averaged over three independent experiments (C). (D and E) Cells transfected as described in B and C were fixed and analyzed for cold-stable microtubules. (D) Images are maximum intensity projections of confocal stacks; the insets are optical sections showing individual kinetochores on the right. (E) The percentage of kinetochores with cold-stable microtubules from multiple cells was averaged over three independent experiments. Bars: (B and D) 5 µm; (D, insets) 2.5 µm.

Figure 3.

Aurora B activity at a distance depends on INCENP dynamics at centromeres. (A and B) Cells were transfected with either CB^DBD-INCENP–GFP or CB^FL-INCENP–GFP and treated with monastrol to induce monopolar spindles, which facilitates tracking of individual centromeres (indicated by white boxes). Images (A) were acquired before and after bleaching a single pair of centromeres (at time = 0) using a 405-nm laser, and fluorescence intensity was measured at each time point and averaged over multiple cells (each point represents n ≥ 8 cells; B). (C) Cells were transfected with either CB^DBD-INCENP–mCherry or CB^FL-INCENP–mCherry with or without INCENP siRNA as indicated and fixed and stained for Aurora B. (D) Cells were transfected as described in C, together with an Aurora B phosphorylation sensor targeted either to centromeres or to chromatin, and imaged live. The normalized YFP/CFP emission ratio was calculated as described in Fig. 1 C and averaged over three independent experiments. (E and F) Cells were transfected with either CB^DBD-INCENP–mCherry or CB^FL-INCENP–mCherry, together with INCENP siRNA and the chromatin-targeted Aurora B phosphorylation sensor. Cells were treated with monastrol to induce monopolar spindles with centromeres oriented toward the middle and imaged live. (E) The left panels show centromeres (mCherry) and chromosomes (YFP emission), and the right panels show the YFP/CFP emission ratio, color coded as indicated by the color scale. Spatial phosphorylation patterns were analyzed along lines drawn manually extending outward from mCherry-labeled centromeres (white lines). (F) The emission ratio was averaged over multiple line scans (each line represents n ≥ 5 cells, five centromeres per cell).

Figure 4.

Partial Aurora B inhibition reveals a spatial phosphorylation gradient. (A–C) HeLa cells were transfected with the chromatin-targeted Aurora B phosphorylation sensor together with CB-mCherry to label centromeres and then were treated for 1 h with monastrol to orient centromeres toward the middle, MG132 to prevent mitotic exit, and ZM at the indicated concentrations. (A) Cells were imaged live, and the YFP/CFP emission ratio was averaged over multiple cells (n ≥ 6 for each concentration) to determine concentrations in which phosphorylation is most sensitive to local kinase activity. (B) Images show centromeres and chromosomes and the color-coded YFP/CFP emission ratio. (C) Spatial phosphorylation patterns were analyzed along lines extending out from centromeres (B, white lines). Each curve represents n ≥ 6 cells, at least four centromeres per cell. (D and E) U2OS-LacO cells were transfected with the chromatin-targeted Aurora B phosphorylation sensor, INCENP siRNA vector, and siRNA-resistant LacI-INCENP–mCherry. Cells were treated for 1 h with nocodazole and ZM at the indicated concentrations and imaged live. (D) Images show LacI-INCENP and chromosomes and the color-coded YFP/CFP emission ratio. (E) Phosphorylation was analyzed along lines extending from the LacI spot (D, white lines). Each curve represents n ≥ 8 cells, three line scans per cell. Bars, 5 µm.

Figure 5.

Real-time observation of phosphorylation spreading from centromeres. (A–C) Cells were transfected with the chromatin-targeted Aurora B phosphorylation sensor together with CB-mCherry to label centromeres and then were treated with monastrol, MG132, and ZM. Cells were imaged live during activation of Aurora B by ZM washout. (A) The YFP/CFP emission ratio was averaged over multiple cells (n = 11) to determine the kinetics of phosphorylation during ZM washout. The arrow indicates the time point analyzed in C. (B, top) Centromeres (CB-mCherry) and chromosomes (YFP emission) for a single time point. (bottom) Color-coded YFP/CFP emission ratio at different time points. The timestamp (minutes and seconds) is relative to ZM washout at t = 0. Bar, 5 µm. (C) The spatial phosphorylation gradient was analyzed by averaging the emission ratio over lines extending outward from mCherry-labeled centromeres (B, white lines) at t = 8 min (n = 11 cells, four centromeres per cell).