Proper partitioning of the contents of a cell between two daughters requires integration of spatial and temporal cues. The anaphase array of microtubules that self-organize at the spindle midzone contributes to positioning the cell-division plane midway between the segregating chromosomes1. How this signalling occurs over length scales of micrometres, from the midzone to the cell cortex, is not known. Here we examine the anaphase dynamics of protein phosphorylation by aurora B kinase, a key mitotic regulator, using fluorescence resonance energy transfer (FRET)-based sensors in living HeLa cells and immunofluorescence of native aurora B substrates. Quantitative analysis of phosphorylation dynamics, using chromosome- and centromere-targeted sensors, reveals that changes are due primarily to position along the division axis rather than time. These dynamics result in the formation of a spatial phosphorylation gradient early in anaphase that is centred at the spindle midzone. This gradient depends on aurora B targeting to a subpopulation of microtubules that activate it. Aurora kinase activity organizes the targeted microtubules to generate a structure-based feedback loop. We propose that feedback between aurora B kinase activation and midzone microtubules generates a gradient of post-translational marks that provides spatial information for events in anaphase and cytokinesis.
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We thank: the Department of Biochemistry and Molecular Genetics, University of Virginia School of Medicine, for its support; D. Burke for many discussions; W. Lan for contributions to this manuscript; Y.-L. Wang (University of Massachusetts Medical School) for the aurora B-green fluorescent protein (aurora B-GFP) plasmid; H. Nakagawa (University of Tokyo) for the anti-MKLP-2 antibody; C. D. Allis (Rockefeller University) for the anti-phospho H3 serine 10 antibody; A. Newton (University of California San Diego) for the CKAR plasmid; A. North and the Rockefeller University Bioimaging facility. This work was supported by: the American Lung foundation (P.T.); the National Institutes of Health grants to T.M.K., P.T.S. and D.L.B.; a Francis Goulet Fellowship at Rockefeller University (M.A.L.); a National Institute of Child Health and Human Development T32 Training Grant ‘Cellular and Physiologic Mechanisms in Reproduction’ at the University of Virginia (B.G.F.); and the Pew Charitable Trust. E.A.F. is a Robert Blount Family Fellow of the Damon Runyon Cancer Research Foundation. We thank N. Kraut and Boehringer Ingelheim for hesparadin.
Author Contributions Development of the aurora B and Plk phosphorylation sensors, and FRET imaging and analysis, were done in the Kapoor laboratory by M.A.L. with E. A.F. B.G.F. performed immunofluorescence experiments. S. R.-N. and P.T. performed the kinase assays and P-LISA experiments, respectively. K.V.L. performed live imaging of aurora B-GFP. M.A.L. and B.G.F. wrote the paper.
The file contains Supplementary Figures S1-S11 and Legends; Supplementary Methods; Supplementary Tables S1-S4; additional references and Supplementary Movies 1-9 Legends. (PDF 6741 kb)
The file contains Supplementary Movie 1. YFP signal (SV1) and YFP/CFP emission ratio (SV2) from a cell expressing the centromere-targeted Aurora B sensor, with MAD2 depleted by RNAi. The video corresponds to the images in Fig. 1c. (AVI 133 kb)
The file contains Supplementary Movie 2. YFP signal (SV1) and YFP/CFP emission ratio (SV2) from a cell expressing the centromere-targeted Aurora B sensor, with MAD2 depleted by RNAi. The video corresponds to the images in Fig. 1c. (AVI 133 kb)
The file contains Supplementary Movie 3. YFP signal (SV3) and YFP/CFP emission ratio (SV4) from a cell expressing the chromatin-targeted Aurora B sensor. The video corresponds to the images in Fig. 2a. (AVI 277 kb)
The file contains Supplementary Movie 4. YFP signal (SV3) and YFP/CFP emission ratio (SV4) from a cell expressing the chromatin-targeted Aurora B sensor. The video corresponds to the images in Fig. 2a. (AVI 277 kb)
The file contains Supplementary Movie 5. DIC (SV5), YFP (SV6), and YFP/CFP emission ratio (SV7) during monopolar anaphase. The video corresponds to the images in Fig. 4a. (AVI 2141 kb)
The file contains Supplementary Movie 6. DIC (SV5), YFP (SV6), and YFP/CFP emission ratio (SV7) during monopolar anaphase. The video corresponds to the images in Fig. 4a. (AVI 385 kb)
The file contains Supplementary Movie 7. DIC (SV5), YFP (SV6), and YFP/CFP emission ratio (SV7) during monopolar anaphase. The video corresponds to the images in Fig. 4a. (AVI 371 kb)
The file contains Supplementary Movie 8. Aurora B-GFP (SV8) and DIC (SV9) during monopolar anaphase. The video corresponds to the images in Fig. S11c-d. (AVI 3258 kb)
The file contains Supplementary Movie 9. Aurora B-GFP (SV8) and DIC (SV9) during monopolar anaphase. The video corresponds to the images in Fig. S11c-d. (AVI 6341 kb)
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Fuller, B., Lampson, M., Foley, E. et al. Midzone activation of aurora B in anaphase produces an intracellular phosphorylation gradient. Nature 453, 1132–1136 (2008) doi:10.1038/nature06923
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