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References
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Acknowledgements
We thank N.Daigle and S.Alexander for their technical support; F.Uhlmann and S.Gruber for comments on the manuscript; S.Spencer (Stanford University, USA) for the DHB–mVenus construct; S.Mori and O.Gotoh for statistical analyses; and all members of the T.H. and J.E. laboratories for discussions. K.N. acknowledges a fellowship from the Japanese Society for the Promotion of the Science (JSPS). M.J.R. was supported by a Humboldt Foundation postdoctoral fellowship. The EMBL Advanced Light Microscopy Facility is acknowledged for support in image acquisition and analysis. This work was supported by research grants from JSPS and the Ministry of Education, Culture, Sports and Technology of Japan (MEXT) to T.H., from EU-FP7-Systems Microscopy NoE (Grant Agreement 258068) and EU-FP7-MitoSys (Grant Agreement 241548) to J.E., and by the European Molecular Biology Laboratory (M.J.H., M.J.R., J.E.).
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Division of Experimental Pathology, Cancer Institute of the Japanese Foundation for Cancer Research, 3-8-31 Ariake, Koto-ku, Tokyo135-8550, Japan
Kota Nagasaka&Toru Hirota
Department of Biological Sciences, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama, Kanagawa226-8501, Japan
Kota Nagasaka
European Molecular Biology Laboratory, Cell Biology and Biophysics Unit, Meyerhofstraße 1, 69117Heidelberg, Germany
M. Julius Hossain,M. Julia Roberti&Jan Ellenberg
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- Kota Nagasaka
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- M. Julius Hossain
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- M. Julia Roberti
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- Jan Ellenberg
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- Toru Hirota
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Contributions
K.N. designed and performed the experiments. M.J.H. generated the fully automated pipeline for resolution analysis. M.J.R. provided technical advice for imaging and generating the pipeline. J.E. designed and supervised quantitative imaging analyses. T.H. directed and coordinated the study.
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Correspondence to Jan Ellenberg or Toru Hirota.
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Integrated supplementary information
Supplementary Figure 1 Chromosome segmentation with constraints.
(a) An overview of image processing pipeline for volumetric analyses of sister chromatids. Single labeled chromosomes that were stained for BrdU, F-ara-EdU and DAPI are shown in slices from raw image stacks (first row) and corresponding Gaussian filtered stacks (second row). Based on these filtered images, segmented chromosome masses were acquired through a computational pipeline, and superimposed on the filtered image (third row). Note that these segmented chromosome masses are largely comparable with the chromosomes in filtered image in the second row, with minor exceptions (for example, arrowhead indicates a small chromatid mass undetected by the computational pipeline). Chromosome segmentation with the constraints (+ cons.) using DAPI-positive volume as a reference (see text) allowed to detect theses undetected masses, and a superimposed image shows that segmented chromosome masses now principally overlap with the filtered images of chromatids. (fourth row). Isosurface rendering images of 3D reconstructed chromosomes based on segmented masses with the constraint are shown (fifth row). (b–g) Data sets in Fig.2 were validated for the use of constraints in chromosome segmentation. (b,c) Comparison of chromosome volumes quantified from segmented chromosome or DAPI-positive masses, in the absence (b) or presence (c) of constraints (mean ± s.d.). Note that constrains were applied in a way that compensate the slight differences of chromosome volumes, as indicated by 1:1 ratio. (c,d) Comparison of EdU- and BrdU-positive chromosome masses, in the absence (d) or presence (e) of constraints (mean ± s.d.). Note that constrains were applied in a way that compensate the slight differences between the two, as indicated by 1:1 ratio. (f,g) Analysis of total chromosome volume (f) and non-overlapping rate (g) through mitosis as in Fig.2c, d, but based on segmented chromosome value without constraints. Note that similar if not identical results were obtained from data sets without constraints, validating the image processing pipeline developed to analyze differentially labeled chromosomes.
Supplementary Figure 2 Chromosome volumetric analysis is little affected by the fixation.
(a) Representative microscopic images for RPE1 cells that stably express EGFP-H2B at indicated mitotic phases before (Living) and after (Fixed) the fixation are shown. Gird size, 2 μm. (b) Quantification of chromosome volumes shown in (a) were normalized and summarized in the histogram (8 cells in prophase, 7 in prometphase, and 8 in metaphase were analyzed; mean ± s.d.).
Supplementary Figure 3 Inactivation of Wapl and TopoIIα do not detectably affect chromosome compaction.
(a) HT-1080 and HeLa cells transfected with a control or a Wapl targeting siRNA were analyzed by immunoblotting. HT1080 cells were used in the sister labeling experiment because Wapl can be efficiently depleted more than other cell lines tested and differential labeling of sister chromatids can be effectively processed. We used HeLa cells to biochemically estimate the amount of cohesin on chromosomes, because they are advantageous to obtain highly synchronized cell populations. The asterisk indicates nonspecific signals. (b) Wapl depleted HT-1080 cells were processed for immunofluorescence microscopy, and representative segmented images of single labeled chromosomes in G2 phase, prophase and prometaphase cells are shown. (c) Total volume of chromosomes analyzed in Fig.3c are summarized in the histogram (mean ± s.d.). (d) Control or Topo IIα depleted RPE1 cells were analyzed by immunoblotting after 36 h later. (e) Control or TopoIIα depleted cells were processed for immunofluorescence microscopy, and representative segmented images of single labeled chromosomes in G2 phase, prophase and prometaphase cells are shown. (f,g) The total volume of chromosomes (μm3) analyzed in Fig.4b, c and were summarized in (f) and (g) respectively. (mean ± s.d.). (h) Analysis of chromosome compaction during prophase in control-, CAP-D3 depleted- and ICRF-193 treated-HeLa Kyoto cell lines that stably express EGFP-H2B and DHB-mKO2 by live cell imaging. The image sequences before the NEBD, which was defined by complete loss of nuclear periphery were aligned. The scale bar represents 5 μm. (i) Chromatin volumes during prophase were quantified by using a slightly modified pipeline previously reported26, normalized to the G2 chromatin volume and plotted over time (n = 8 from control-, 11 from CAP-D3 depleted- and 12 from ICRF-193 treated-cells). Note that live cell imaging analysis also revealed no delay in the reduction rate of total chromosomal volume in TopoIIα inactivated cells, unlike in CAP-D3 depleted cells, indicating that ICRF193-induced chromosome condensation defect is not reflected as a change in total chromosomal mass.
Supplementary Figure 4 Condensin I does not essentially contribute to the increase of resolution after the NEBD occurs in CondensinII depleted cells.
(a) RPE1 cells were depleted of condensinI orII by transfecting siRNA targeting CAP-H or CAP-D3, respectively. After 36 h later, knockdown efficiency was analyzed by immunoblotting. (b) Immunofluorescence microscopy reveals that chromosome compaction before the NEBD is delayed in condensinII depleted cells. Cells in G2 phase, early- and late- prophase were indicated by discrete pH3S10 staining, clear pH3S10 staining without nuclear cyclin B1 and nuclear influx of cyclin B1, respectively. Scale bar, 5 μm. (c) The total volume of chromosomes analyzed in Fig.5b were summarized in the histogram (mean ± s.d.). (d) RPE1 cells transfected with CAP-H and CAP-D3 targeting siRNAs simultaneously were analyzed by immunoblotting after 36 h. (e,f) Following RNAi-mediated knockdown cells were processed for immunofluorescence microscopy. The ratio of Non-OLs (%) (e) and total volume of chromosomes (μm3) (f) in control, CAP-D3, and both of CAP-H and CAP-D3 depleted cells in G2, pro- and prometa- phases were analyzed and summarized in the histogram (±s.d.; n indicates the total number of cells pooled across three independent experiments). Cells arrested in metaphase by MG132 treatment for 30 min were also analyzed.
Supplementary Figure 5 Cdk1 mediated phosphorylation of CAP-D3 T1415 is required for CondensinII mediated morphological change of chromosomes during prophase.
(a) Parental RPE1 cells or cells stably expressing EGFP-tagged CAP-D3WT or T1415A were transfected with mock or siRNA specifically targeting endogenous CAP-D3. After 36 h, the knockdown efficiency was analyzed by immunoblotting. (b) Immunofluorescence microscopy of RPE1 cells where endogenous CAP-D3 were depleted or replaced with EGFP-tagged CAP-D3, either wild type (WT) or T1415A mutant (TA). Note that chromosome morphology in prophase is impaired in both CAP-D3 depleted and T1415A-replaced cells, as described earlier24. Representative late prophase cells, defined as in Supplementary Fig.4b, are shown. Scale bar, 5 μm. (c) Total chromosome volume (μm3) analyzed in Fig.5c were measured and summarized in box plots as in Fig.5c.
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Nagasaka, K., Hossain, M., Roberti, M. et al. Sister chromatid resolution is an intrinsic part of chromosome organization in prophase. Nat Cell Biol 18, 692–699 (2016). https://doi.org/10.1038/ncb3353
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DOI: https://doi.org/10.1038/ncb3353
