SUPPLEMENTARY INFORMATION

Transcription

1 SUPPLEMENTARY INFORMATION 1

2 SUPPLEMENTARY INFORMATION 2

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5 SUPPLEMENTARY INFORMATION Supplementary Movies Movie S1 Animation of the isosurface reconstruction set presented in Fig. 1a. The time lapse is 5 min and the total time elapsed is 40 min, from prometaphase to telophase/g1. Movie S2 Photoactivation experiment similar to Fig. 2a, but initiated in mid anaphase, after the last pair of sister telomeres had separated. Time interval between frames is ~1.5 min and the total time elapsed is 18 min, from mid-anaphase to telophase/g1. Representative example of n = 30. Consistent with our previous study6, chromosome segments marked by photoactivation kept their relative position and orientation throughout mitosis. Movie S3 Entire control time-lapse experiment set presented in Supplementary Information, Fig. S1a. The interval between frames is 30 s. and the total time elapsed is 15 min, from metaphase to early telophase. Movie S4 Entire taxol time-lapse experiment presented in Fig. 3c. The interval between frames is 30 s. and the total time elapsed is ~40 min, from metaphase to G1. 5

6 Supplementary Information Methods Measuring the volume occupied by Chromatin through mitosis Fluorescent chromatin was automatically tracked and focused during imaging using in-house developed macros 1. The raw fluorescence signal in each optical section in a stack was background-subtracted and filtered by two-dimensional anisotropic diffusion in each optical section (Fig. 1a). Then, the intensity sum of all voxels in each 3D stack was equalized to the first time-point to correct for possible photobleaching effects (typically below 5% during the entire experiment). A threshold to segment chromatin was set interactively in metaphase, when chromatin borders can sharply be distinguished from the cytoplasm. Within a narrow range, this threshold was then automatically adjusted for each 3D stack such that the intensity sum contained in the segmented chromatin volume was equal to the metaphase reference. Variations of the metaphase segmentation threshold by ± 10% did not significantly affect the measured kinetics of compaction (Not shown, see also 2 ). The inverse of this volume, normalized to the interphase value, was used as a relative measure of compaction. The volume automatically segmented by this method is illustrated by isosurface reconstructions in Fig. 1a and Supplementary Information, Fig. S5). The interpretation of the results of this assay must take into account its inherent limitations, such as a relatively low and anisotropic spatial resolution, and the limited signal-to-noise ratio and sampling resolution compatible with an unperturbed progression through mitosis. Furthermore, this measurement can be biased by the relative arrangement of chromosomes along the optical axis during late mitotic stages.

7 Measuring the entire length of single chromatid arms during anaphase We differentially labelled pericentromeric heterochromatin by transient expression of MeCP2-EGFP 3, with Hoechst counter-staining as general chromatin marker. Typical stacks of 256*256 pixels*7 images, (xyzt resolution 0.11*0.11*0.4 µm*1-4min) were acquired around the centre of the chromatids of interest. Measuring kinetics of microtubule-perturbing drugs during anaphase To test how quickly microtubules could be perturbed during anaphase, we applied drugs to NRK cells stably expressing EGFP-α-tubulin during time-lapse imaging, immediately after segregation of the last pair of sister chromatids. Then we measured the kinetics of action of the drugs by analyzing the amount of structured tubulin signal in 4D data sets. Mitotic spindles (megfp-α-tubulin) and chromatin (Hoechst) were recorded by dual-colour imaging from metaphase until telophase. Typical stacks of three 256*256 images (xyzt resolution: 0.11*0.11*2 µm*30 s), were acquired around the pole-pole axis of the mitotic spindle (Supplementary Information, Fig. S2). To perturb microtubules as rapidly and acutely as possible, pre-warmed solutions of nocodazole and taxol were added to the imaging medium to a final concentration of 20 µm. To inhibit Aurora kinase, hesperadin was added to a final concentration of 30 µm. For automated and unbiased quantitation of drug activity kinetics, we developed macros in ImageJ ( Maximum intensity projections of the fluorescence tubulin signal were generated and the entire cells were automatically segmented by Gaussian filtering and thresholding with a single value defined interactively. The extracellular background was subtracted from the raw intracellular signal in the segmented images and the mean fluorescence intensity and standard deviation (SD) of all pixel intensities within the cell was calculated as a measure of

8 heterogeneity of signal distribution. Cellular structures, like a mitotic spindle result in high SD, whereas diffuse signal, like soluble cytoplasmic tubulin results in low SD. The SD was then normalized to the corresponding mean fluorescence intensity in each image to account for cell movements and focus shifts. This robust and simple method can be applied to the analysis of other structures (unpublished observations). Results: in mock- and hesperadin-treated cells, spindle microtubules displayed a constant fluorescence intensity distribution during metaphase, which became more homogeneous when K-fibres started depolymerising normally in early anaphase, thus lowering the SD by 30%. Taxol strongly hyperstabilized and aggregated polymerized tubulin within 1-2 min of addition, as shown by an increase in the local brightness and a 50% increase in SD, indicating a strong perturbation of dynamic anaphase microtubules. Taxol did not strongly affect other aspects of chromosome dynamics, as all treated cells decompacted chromatin normally, and underwent cytokinesis and reattachment to the substrate, albeit with a delay (see Fig. 3c). Nocodazole was comparably efficient, as the proportion of polymerized tubulin, and thus the amount of microtubules, strongly decreased within 2 min, as shown by a lowering in the SD by a further 30% compared to mocks, consistent with our previous findings in fixed NRK cells 4. All nocodazole-treated cells decondensed chromatin in telophase; however, as expected, most did not complete cytokinesis and substrate reattachment in G1. (See Supplementary Information, Fig. S3). Testing the activity of roscovitine. To further test whether the inhibition of anaphase chromatid shortening was a specific effect of Aurora kinase inhibition, or whether it could be achieved with any mitotic kinase inhibitor, we used roscovitine, an inhibitor of Cyclin-Dependent Kinase (Fig.

9 3g-h). We confirmed that roscovitine (Calbiochem 5 ) was active by applying an acute dose (360.0 µm) to NRK cells stably-expressing megfp-γ-tubulin in prometaphase. As expected, these cells quickly arrested in prometaphase with a perturbed spindle assembly (Supplementary Information, Fig. S4).

10 Supplementary Information, References 1. Rabut, G. & Ellenberg, J. Automatic real-time three-dimensional cell tracking by fluorescence microscopy. J Microsc 216, (2004). 2. Gerlich, D., Beaudouin, J., Gebhard, M., Ellenberg, J. & Eils, R. Fourdimensional imaging and quantitative reconstruction to analyse complex spatiotemporal processes in live cells. Nat Cell Biol 3, (2001). 3. Brero, A. et al. Methyl CpG-binding proteins induce large-scale chromatin reorganization during terminal differentiation. J Cell Biol 169, (2005). 4. Beaudouin, J., Gerlich, D., Daigle, N., Eils, R. & Ellenberg, J. Nuclear envelope breakdown proceeds by microtubule-induced tearing of the lamina. Cell 108, (2002). 5. Bach, S. et al. Roscovitine targets, protein kinases and pyridoxal kinase. J Biol Chem 280, (2005). 6. Gerlich, D. et al. Global chromosome positions are transmitted through mitosis in mammalian cells. Cell 112, (2003).