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. 2022 Sep;609(7925):183-190.
doi: 10.1038/s41586-022-05027-y. Epub 2022 Aug 3.

A mitotic chromatin phase transition prevents perforation by microtubules

Maximilian W G Schneider  1   2 , Bryan A Gibson #  3 , Shotaro Otsuka #  4 , Maximilian F D Spicer  5   6 , Mina Petrovic  5   6 , Claudia Blaukopf  5 , Christoph C H Langer  5 , Paul Batty  5   6 , Thejaswi Nagaraju  5 , Lynda K Doolittle  3 , Michael K Rosen  3 , Daniel W Gerlich  7
Affiliations

A mitotic chromatin phase transition prevents perforation by microtubules

Maximilian W G Schneider et al. Nature. 2022 Sep.

Abstract

Dividing eukaryotic cells package extremely long chromosomal DNA molecules into discrete bodies to enable microtubule-mediated transport of one genome copy to each of the newly forming daughter cells1-3. Assembly of mitotic chromosomes involves DNA looping by condensin4-8 and chromatin compaction by global histone deacetylation9-13. Although condensin confers mechanical resistance to spindle pulling forces14-16, it is not known how histone deacetylation affects material properties and, as a consequence, segregation mechanics of mitotic chromosomes. Here we show how global histone deacetylation at the onset of mitosis induces a chromatin-intrinsic phase transition that endows chromosomes with the physical characteristics necessary for their precise movement during cell division. Deacetylation-mediated compaction of chromatin forms a structure dense in negative charge and allows mitotic chromosomes to resist perforation by microtubules as they are pushed to the metaphase plate. By contrast, hyperacetylated mitotic chromosomes lack a defined surface boundary, are frequently perforated by microtubules and are prone to missegregation. Our study highlights the different contributions of DNA loop formation and chromatin phase separation to genome segregation in dividing cells.

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Conflict of interest statement

M.K.R. is a co-founder of Faze Medicines. The other authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Acetylation-regulated chromatin compaction prevents microtubule perforation in mitosis.
a, The contribution of condensin and histone deacetylases to mitotic chromosome compaction and congression to the spindle centre. HeLa cells with homozygously mAID-tagged SMC4 were treated with 5-PhIAA to deplete condensin (ΔCondensin) or with TSA to suppress mitotic histone deacetylation as indicated. Live-cell images with microtubules stained by SiR–tubulin; DNA was stained with Hoechst 33342. Projection of 5 z-sections. b, Quantification of chromosome congression by the fraction of chromatin localizing to the central spindle region. n = 51 (control), n = 65 (ΔCondensin), n = 34 (ΔCondensin + TSA), n = 61 (TSA) cells. The bars indicate the mean. Significance was tested using two-tailed Mann–Whitney U-tests (P < 10−15 (ΔCondensin + TSA); P < 10−15 (TSA); precision limit of floating-point arithmetic). c, Quantification of chromatin density in cells treated as described in a. n = 31 (control), n = 89 (ΔCondensin), n = 99 (ΔCondensin + TSA) and n = 74 (TSA) cells. The bars indicate the mean. Significance was tested using two-tailed Mann–Whitney U-tests (P < 10−15 (ΔCondensin + TSA); P < 10−15 (TSA); precision limit of floating-point arithmetic). AU, arbitrary units. d, Electron tomography analysis of wild-type prometaphase HeLa cells in the absence or presence of TSA. Magenta, chromatin surfaces; green, microtubules in cytoplasm; cyan, microtubules in chromatin. The red circles show the perforation sites. e,f, Quantification of microtubule density in chromatin (e) and cytoplasmic (f) regions as shown in d. n = 10 tomograms from 7 cells for each condition. The bars indicate the mean. Significance was tested using two-tailed Mann–Whitney U-tests (P = 1.083 × 10−5 (e); P = 0.247 (f)). Biological replicates: n = 2 (a–f). Scale bars, 5 µm (a), 2 µm (d, 250 nm section); 200 nm (tomogram slices and 3D model). Source Data
Fig. 2
Fig. 2. Acetylation regulates chromatin solubility in mitotic cytoplasm.
a, Chromosome fragmentation in live mitotic HeLa cells by AluI injection (t = 0 min). Chromatin was visualized with H2B–mCherry. Projection of 3 z-sections. Time is shown as min:s. b, Quantification of chromatin density for cells as in a. n = 11 cells, 3 regions of interest (ROIs) each. The bars indicate the mean. Significance was tested using a two-tailed Mann–Whitney U-test (P = 0.332). c, Chromatin mobility in undigested metaphase chromosomes and after AluI injection, measured by fluorescence recovery after photobleaching in live metaphase cells expressing H2B–mCherry. The circles indicate the photobleaching region at t = 0 s. Time is shown as s. d, Quantification of fluorescence in n = 8 (undigested) or n = 10 (AluI-digested) cells as described in c. Data are mean ± s.d. e, AluI injection as described in a for a TSA-treated mitotic cell. Time is shown as min:s. f, Quantification of chromatin density, normalized to the mean of untreated pre-injection cells shown in b. n = 11 cells, 3 ROIs each. The bars indicate the mean. Significance was tested using a two-tailed Mann–Whitney U-test (P < 10−15; precision limit of floating-point arithmetic). g–i, Ki-67 localization in mitotic cells. g, HeLa cells expressing eGFP–Ki-67 and H2B–mCherry were treated with taxol for mitotic arrest (control); cells were treated with TSA or microinjected with AluI as indicated. Ki-67 localization was analysed in chromosomes oriented perpendicularly to the optical plane (insets). h, Line profiles across the chromatin–cytoplasm boundary as indicated by the yellow lines in g were aligned to the first peak in eGFP–Ki-67 fluorescence and normalized to the mean of Ki-67 fluorescence at the first peak of control. n = 19 (control), n = 24 (TSA) and n = 22 (AluI) cells. Data are mean ± s.d. i, Quantification of Ki-67 surface confinement by the ratio of Ki-67 fluorescence on the surface (S) over inside (I). n = 19 (control), n = 24 (TSA) and n = 22 (AluI) cells. The bars indicate the mean. Significance was tested using two-tailed Mann–Whitney U-tests (P = 9.305 × 10−10 (TSA); P = 0.476 (AluI)). Biological replicates: n = 3 (a,b,g–i); n = 2 (c–f). Scale bars, 5 µm (a, e and g, main images), 1 µm (a, e and g, insets) and 3 µm (c). Source Data
Fig. 3
Fig. 3. Chromatin condensates limit access of tubulin and other negatively charged macromolecules.
a, The localization of tubulin (tub.) relative to mitotic chromosomes. Rhodamine-labelled tubulin was injected into live mitotic cells that were untreated, treated with nocodazole alone (control) or in combination with TSA. b, Quantification of the tubulin concentration for the data shown in a. n = 27 cells. The bars indicate the mean. Significance was tested using a two-tailed Mann–Whitney U-test (P < 1 × 10−15; precision limit of floating-point arithmetic). c, Live-cell images of a HeLa cell expressing DsRed or DsRed fused at its N terminus to electrically charged polypeptides. DNA was stained with Hoechst 33342. The numbers in parentheses indicate the predicted elementary charge of the tetramers formed by DsRed fusion constructs. d, Quantification of DsRed concentration for the data shown in c. n = 26 (DsRed), n = 26 (DsRed(−7e)), n = 26 (DsRed(+9e)) cells. The bars indicate the mean. Significance was tested using two-tailed Mann–Whitney U-tests (P = 0.4 × 10−14 (DsRed(−7e)); P = 0.4 × 10−14 (DsRed(+9e)). e, The localization of tubulin relative to reconstituted nucleosome (nuc.) array droplets. Nucleosome array droplets were formed by incubation in phase separation buffer and fluorescently labelled tubulin was then added in the presence of nocodazole, or in the absence of nocodazole with subsequent temperature increase to 20 °C to induce microtubule polymerization. f, Quantification of the tubulin concentration or microtubule density in nucleosome array condensates relative to buffer for the data shown in e. n = 94 droplets, n = 13 fields of polymerized microtubules. The bars indicate the mean. Biological replicates: n = 2 (a–d); n = 3 (e,f). Technical replicates: n = 3 (a,b); n = 2 (c,d); n = 3 (e,f). For a, c and e, scale bars, 5 µm (main images) and 1 µm (insets). Source Data
Fig. 4
Fig. 4. Microtubules push liquified chromatin away from the spindle pole.
a, Time-lapse microscopy analysis of liquified chromatin during monopolar spindle assembly. AluI was injected into live mitotic HeLa cells expressing H2B–mCherry and meGFP–CENP-A, stained with SiR–tubulin, in the presence of nocodazole (noco) and STLC. Nocodazole was then removed at t = 0 min during time-lapse imaging to induce monopolar spindle assembly. Projection of 5 z-sections. Time is shown as min:s. b, Quantification of bulk chromatin (H2B–mCherry) and centromeric chromatin (meGFP–CENP-A) localizing at the cell periphery relative to the region around the spindle monopole at t = 36 min. n = 15 cells. The bars indicate the mean. Significance was tested by a two-tailed Mann–Whitney U-test (P = 1.289 × 10−8). c, Model of chromatin compaction and condensin-mediated DNA looping in mitotic chromosome and spindle assembly. The illustration shows a top-down view of a chromosome cross-section. Biological replicates: n = 3 (a,b). Scale bars, 5 µm. Source Data
Extended Data Fig. 1
Extended Data Fig. 1. Characterization of HeLa Smc4-mAID-HaloTAG cells and analysis of mitotic phenotypes.
a-c, Smc4 expression analysis. a, HeLa cells with homozygous Smc4-mAID-Halo alleles and stably expressing OsTIR(F74G) were analysed with or without treatment of 5-Phenylindole-3-eacetic acid (5-PhIAA) for 3 h and stained with OregonGreen-488 HaloTAG ligand; HeLa wild-type (WT) cells serve as control to measure fluorescence background. DNA was stained with Hoechst 33342. Representative examples of HeLa WT (n = 25), HeLa Smc4-mAID-Halo (n = 25), HeLa Smc4-mAID-Halo + 5-PhIAA (n = 25). b, Quantification of HaloTAG fluorescence in live mitotic cells as shown in a. n = 25 for WT, n = 25 for Smc4-mAID-Halo without 5-PhIAA, n = 25 for Smc4-mAID-Halo with 5-PhIAA. Bars indicate mean; significance was tested by a two-tailed Mann-Whitney test (Smc4-mAID-Halo with 5-PhIAA, P = 1.4−14). c, Immunoblot analysis of Smc4-mAID-Halo cells with or without 3 h 5-PhIAA treatment. Representative examples of n = 2 experiments. For gel source data, see Supplementary Figure 1a. d, e, Analysis of cell cycle state by immunofluorescence staining against Cyclin B1. HeLa cells with homozygously mAID-tagged SMC4 were treated with 5-PhIAA to deplete condensin (ΔCondensin) or with TSA to suppress mitotic histone deacetylation as indicated. DNA was stained with Hoechst 33342. Classification of interphase and mitotic cell is based on overall cell shape and spindle morphology. e, Quantification of Cyclin B1 fluorescence in cells as in d. Data normalized to the mean of untreated interphase cells. n = 60 (interphase), n = 89 (mitotic), n = 59 (ΔCondensin), n = 83 (ΔCondensin+TSA), n = 67 (TSA) cells. Bars indicate mean; significance was tested by a two-tailed Mann-Whitney test (CTRL, P<10−15; ΔCondensin, P<10−15; ΔCondensin+TSA, P<10−15; TSA, P<10−15, precision limit of floating-point arithmetic). f, g, Immunofluorescence analysis of kinetochores and spindle poles in mitotic Smc4-mAID-Halo cells after 3 h degradation of Smc4 (ΔCondensin) and/or 3 h treatment with TSA as indicated. Projection of 7 Z-sections with Z-offset of 0.15 µm. g, Quantification of fraction of kinetochores outside chromatin regions (>0.5 µm distance from chromatin surface) of cells as in f. n = 40 cells for control n = 59 for ΔCondensin, n = 35 for ΔCondensin+TSA, n = 40 for TSA. Bars indicate mean, significance was tested by a two-tailed Mann-Whitney test (ΔCondensin, P<10−15, precision limit of floating-point arithmetic; ΔCondensin+TSA, P = 0.852; TSA, P = 0.911). Biological replicates: n = 2 (a–e); n = 3 (f,g). Scale bars, 5 µm. Source Data
Extended Data Fig. 2
Extended Data Fig. 2. Chromosome and spindle organization in condensin-depleted and TSA-treated cells entering mitosis.
a–c, 3D confocal time-lapse microscopy of live HeLa cells entering mitosis in the presence of STLC to induce monopolar spindle geometry. Cells have homozygous Smc4-mAID-Halo alleles and stably express OsTIR(F74G), H2B-mCherry, and CENP-A-meGFP and are stained with SiR-tubulin. Images show projection of 2 Z-sections with Z-offset of 2.5 µm, centred around the spindle pole. a, Mitotic entry of a control cell. b, Cell entering mitosis after treatment with 5-PhIAA to deplete Smc4 (ΔCondensin). c, Cell entering mitosis after treatment with 5-PhIAA to deplete Smc4 and TSA to suppress mitotic histone deacetylation (ΔCondensin+TSA). d, Quantification of chromatin distribution relative to spindle monopole of cells in a-c 20 min after prophase onset. Total H2B-mCherry fluorescence in pole-proximal region divided by total H2B-mCherry-fluorescence of pole-distal regions. n = 42 cells for control, n = 38 for ΔCondensin, n = 60 for ΔCondensin+TSA. Bars indicate mean, significance was tested by a two-tailed Mann-Whitney test (ΔCondensin, P = 0.988; ΔCondensin+TSA, P<10−15, precision limit of floating-point arithmetic). e, Quantification of fraction of kinetochores outside chromatin (>0.5 µm distance from chromatin surface) of cells as in a–c 20 min after prophase onset. n = 42 cells for control, n = 42 for ΔCondensin, n = 62 for ΔCondensin+TSA. Bars indicate mean, significance was tested by a two-tailed Mann-Whitney test (ΔCondensin, P<10−15, precision limit of floating-point arithmetic; ΔCondensin+TSA, P = 0.061). Biological replicates: n = 2 (a,c–e); n = 3 (b). Scale bars, 10 µm. Source Data
Extended Data Fig. 3
Extended Data Fig. 3. Histone acetylation during cell cycle progression and effect of TSA.
a,b, Immunofluorescence analysis of histone acetylation in interphase and mitosis. a, HeLa cells were fixed and stained with antibodies against different acetylated histones as indicated. b, Quantification of histone acetylation by immunofluorescence as in a, by the ratio of antibody fluorescence to DNA reference staining by Hoechst 33342. For each acetylated histone, all data points were normalized to the mean of interphase cells. n = 20 for H2B-Ac, Interphase, n = 20 for H3-Ac, Interphase, n = 20 for H4K16-Ac, Interphase, n = 20 for H2B-Ac, Mitosis, n = 20 for H3-Ac, Mitosis, n = 20 for H4K6-Ac, Mitosis. Bars indicate mean; significance was tested by a two-tailed Mann-Whitney test (H2B-Ac, P = 2.117x10−7; H3-Ac, Mitosis, P = 4.72x10−4; H4K16-Ac, P = 1.451x10−11). c,d, Histone acetylation in mitotic cells after TSA treatment. c, HeLa cells were treated with TSA for 3 h, fixed, and histone acetylation analysed by immunofluorescence as in a. d, Quantification of histone acetylation as in b for mitotic cells 3 h after TSA treatment. For each antibody, all data points were normalized to the mean of control metaphase cells. n = 20 for H2B-Ac, control, n = 20 for H3-Ac, control, n = 20 for H4K16-Ac, control, n = 20 for H2B-Ac, TSA, n = 20 for H3-Ac, TSA, n = 20 for H4K16-Ac, TSA. Bars indicate mean; significance was tested by a two-tailed Mann-Whitney test (H2B-Ac, P = 1.451x10-11; H3-Ac, P = 1.451x10−11; H4K16-Ac, TSA, P = 1.451x10−11). e,f, Analysis of apoptosis after TSA treatment. e, Fields of cells stained with Hoechst 33342, pSIVA, and PI to detect apoptotic cells were untreated (control) or treated for 3 h with 500 nM or 5 µM TSA, or 5 µM anisomycin as positive control. f, Quantification of apoptotic index after drug treatment of asynchronous cells, each dot representing a field of cells shown in e, with n = 4574 for untreated, n = 4926 for 500 nM TSA, n = 4653 for 5 µM TSA, and n = 4188 anisomycin cells in total. Bars indicate mean, significance was tested by a two-tailed Mann-Whitney test (500 nM TSA, P = 2.971x10-4; 5 µM TSA, P = 0.078; anisomycin, P = 1.923x10−7). g,h, Immunofluorescence analysis of yH2A.X DNA damage foci in mitosis after treatment with 5 µM TSA or the DNA-damaging agent neocarzinostatin (NCS) as positive control. g, Mitotic cells were stained for yH2A.X foci and DNA by Hoechst 33342 after indicated treatments. Z-projection of 11 Z-sections with Z-offset of 0.5 µm. h, Quantification of DNA-damage foci in cells as shown in g. n = 48 cells for control, n = 48 for TSA, n = 32 for NCS. Bars indicate mean, significance was tested by a two-tailed Mann-Whitney test (TSA, P = 0.103; NCS, P<10−15, precision limit of floating-point arithmetic). Biological replicates: n = 2 (a–d,g,h); n = 3 (e,f). Scale bars, a,c,e, 10 µm, g, 5 µm. Source Data
Extended Data Fig. 4
Extended Data Fig. 4. Effect of various TSA treatments on mitotic chromosome organization.
a,b, Mitotic chromosome morphology and DNA density after various TSA treatment conditions. a, Live HeLa cells stained with Hoechst 33342 and SiR-tubulin were analysed without perturbations (control), after 3 h treatment with 500 nM TSA followed by 8 h removal of TSA (500 nM TSA/washout), or after 3 h treatment with 5 nM or 500 nM TSA, as indicated. Mitotic cells were identified based on their rounded morphology and the presence of a bipolar spindle. Z-projection of 4 Z-sections with Z-offset of 0.25 µm. b, Quantification of DNA density in mitotic chromatin for cells as shown in a. Data normalized to mean of control mitotic cells. n = 39 cells for control, n = 35 for TSA washout, n = 40 for 5 nM TSA and n = 40 for 500 nM TSA. Bars indicate mean, significance was tested by a two-tailed Mann-Whitney test (P<10−15, precision limit of floating-point arithmetic). c,d, Mitotic chromosome morphology and DNA density in condensin-depleted cells, in various TSA treatment conditions. HeLa cells with homozygous Smc4-mAID-Halo alleles and stably expressing OsTIR(F74G) were treated with 5-PhIAA for 3 h to degrade Smc4 (ΔCondensin). c, Live cells stained with Hoechst 33342 and SiR-tubulin were analysed without additional perturbations (control), after 3 h treatment with 500 nM TSA followed by 8 h removal of TSA (500 nM TSA/washout), or after 3 h treatment with 5 nM or 500 nM TSA, as indicated. Mitotic cells were identified based on their rounded morphology and the presence of a bipolar spindle. Z-projection of 4 confocal slices with Z-offset of 0.25 µm. d, Quantification of DNA density in mitotic chromatin for cells as shown in c. Data normalized to mean of control mitotic cells. n = 45 cells for control, n = 47 for TSA washout, n = 40 for 5 nM TSA and n = 40 for 500 nM TSA. Bars indicate mean, significance was tested by a two-tailed Mann-Whitney test (P<10−15, precision limit of floating-point arithmetic). Biological replicates: n = 2 (a–d). Scale bars, 5 µm. Source Data
Extended Data Fig. 5
Extended Data Fig. 5. Histone acetylation and chromosome organization in cells overexpressing eGFP-p300.
a, b, Immunofluorescence analysis of histone acetylation in mitotic cells overexpressing p300 histone acetyltransferase or catalytically dead p300(D1399Y). a, Cells were transfected with a plasmid coding for eGFP-p300 or eGFP-p300(D1399Y) as indicated and fixed after 48 h. Histone 2B acetylation was analysed by immunofluorescence. DNA was stained with Hoechst 33342. b, Quantification of histone acetylation in metaphase cells as in a. Data points were normalized to the mean of mock-transfected mitotic cells. n = 20 for mock-transfected, n=26 for p300, n = 20 for p300(D1399Y). Bars indicate mean; significance was tested by a two-tailed Mann-Whitney test (P = 3.57x10−13). c–e, Analysis of chromatin density and chromosome congression to the spindle centre in cells after SMC4-AID-Halo degradation (ΔCondensin) and overexpression of p300 or catalytically dead p300(D1399Y). c, Cells were transfected with a plasmid coding for eGFP-p300 or eGFP-p300(D1399Y) as indicated and analysed by live-cell imaging after 48 h. DNA was stained with Hoechst 33342 and microtubules stained by SiR-Tubulin to identify mitotic cells with bipolar spindles. Projection of 5 Z-sections. d, Quantification of chromosome congression by the fraction of chromatin localizing to the central spindle region. n = 20 for mock-transfected, n = 24 for ΔCondensin+p300, n=20 for ΔCondensin+p300(D1399Y). Bars indicate mean; significance was tested by a two-tailed Mann-Whitney test (P = 1.451x10−11). e, Quantification of chromatin density in cells treated as in c. n = 20 for mock-transfected, n = 24 for ΔCondensin+p300, n = 20 for ΔCondensin+p300(D1399Y). Bars indicate mean; significance was tested by a two-tailed Mann-Whitney test (P = 7.86x10−13). Biological replicates: n = 2 (a,b); n = 3 (c–e). Scale bars, 5 µm. Source Data
Extended Data Fig. 6
Extended Data Fig. 6. Correlative fluorescence and electron microscopy of mitotic cells and analysis of chromosome segregation by live imaging after TSA treatment.
a, b, Electron tomograms of prometaphase WT Hela cells, untreated (a) or treated with TSA (b). Magenta: chromatin surfaces; green: microtubules in cytoplasm; cyan: microtubules in chromatin; red circles: microtubule perforation sites at chromosome surface. Representative example regions for control prometaphase (n = 3), control metaphase (n = 7), TSA prometaphase (n = 5) and TSA metaphase (n = 5); example regions are from 10 tomograms per condition from 7 different cells each. c, Quantification of microtubule density in chromatin regions of prometaphase or metaphase cells in the absence or presence of TSA. Data shown in Fig.1e, f separated by mitotic stage, n = 10 tomograms from 7 different cells for each condition. Bars indicate mean. d, Correlative transmission electron microscopy and fluorescence microscopy of chromatin/H2B-mCherry in prometaphase WT Hela cells (related to a, control cell #1 and b, TSA cell #1). e, Mitotic progression analysis by time-lapse microscopy of HeLa cells expressing H2B-mRFP, in untreated control and TSA-treated cells. n = 44 for control from 5 biological replicates, n = 36 for TSA from 4 biological replicates. Time is relative to nuclear envelope disassembly (NEBD). f, Chromosome missegregation analysis by Airyscan imaging of live anaphase HeLa cells expressing H2B-mCherry and meGFP-CENP-A and stained with SiR-tubulin. Representative images of n=64 control cells and n = 110 TSA-treated cells. Single Z-sections. g, Quantification of chromosome missegregation of cells as illustrated in f. Dots indicate biological replicates, bars indicate mean. n=64 cells for control, n = 110 for TSA. h, Quantification of number of lagging chromosomes in cells as illustrated in f. Fraction of cells with 1, 2, or 3 lagging chromosomes. n = 64 cells for control, n = 110 for TSA. Biological replicates: n = 2 (a–h). Scale bars, a,b, 250 nm section, 2 µm; tomogram slices and 3D model, 200 nm; d, 2 µm; f, 5 µm. Source Data
Extended Data Fig. 7
Extended Data Fig. 7. Analysis of AluI-fragmented chromosomes.
a,b, Measurement of early apoptosis marker pSIVA after AluI-injection. a, Hela cells expressing H2B-mCherry were treated with STLC and microinjected with AluI to induce chromatin fragmentation as in Fig. 2a, or treated with anisomycin to induce apoptosis as positive control. AluI-injected cells were imaged 1 h after injection. b, Quantification of pSIVA fluorescence on cell surface of untreated, AluI-injected, and anisomycin treated cells. n = 77 cells for control, n = 41 for AluI-injected, n = 83. Bars indicate mean, significance was tested by a two-tailed Mann-Whitney test (AluI-injected, P = 0.446; anisomycin, P<10−15, precision limit of floating-point arithmetic). c,d, Analysis of Smc4 localization after AluI-injection. c, Hela Smc4-HaloTAG cells expressing H2B-mCherry were stained with OregonGreen-488 HaloTAG ligand and mitotic cells were injected with AluI to fragment chromosomes. d, Quantification of Smc4-Halo fluorescence on chromatin relative to the cytoplasm of cells as in c. n = 75 uninjected cells, n =  60 AluI-injected cells. Bars indicate mean, significance was tested by a two-tailed Mann-Whitney test (P<10−15, precision limit of floating-point arithmetic). e,f, Chromatin fragmentation in cells overexpressing p300-HAT. e, AluI injection (t = 0 min) during time-lapse microscopy of cells expressing H2B-mCherry and expressing p300-HAT. f, Quantification of chromatin density in cells as in e, normalized to the mean of mock-transfected, non-injected cells. n = 12 cells, 3 ROIs per cell. Bar indicates mean, significance was tested by a two-tailed Mann-Whitney test (P = 2.075x10−10). g,h, AluI-fragmentation after condensin depletion. g. Smc4-AID HeLa cells expressing H2B-mCherry were treated 3 h with 5-PhIAA to deplete condensin and mitotic cells were then injected with AluI (t = 0 min) during time-lapse microscopy. h, Quantification of chromatin density before and after injection of AluI, normalized to the mean of untreated pre-injection cells. n = 7 cells, 3 ROIs each. Bars indicate mean, significance was tested by a two-tailed Mann-Whitney test (P = 0,887). Biological replicates: n = 3 (a,b,e,f); n = 2 (c,d); n = 5 (g,h). Scale bars, a,c,e,g 5 µm; insert c,e,g 1 µm. Source Data
Extended Data Fig. 8
Extended Data Fig. 8. Analysis of chromatin phase transitions and role of Ki-67.
a, Chemical induction of G2-to-mitosis transition. HeLa cell expressing Aurora B-FRET biosensor was synchronized to G2 by RO3306 and then induced to enter mitosis by removing RO3306 and adding okadaic acid (OA). Mitotic entry was detected by chromosome compaction and FRET signal. Projection of 9 Z-sections. b, Quantification of chromatin density in G2 and mitosis as in a for n = 13 cells. Bars indicate mean; significance was tested by a two-tailed Mann-Whitney test (P = 1.923x10−7). c, Chromatin was fragmented in G2 cells by injection of AluI and mitosis subsequently induced as in a. Projection of 9 Z-sections. d, Quantification of chromatin density in G2 and mitosis as in c for n = 8 cells. Bars indicate mean; significance was tested by a two-tailed Mann-Whitney test (P = 1.554x10−4). e,f, Chromatin fragment localization in G2-arrested cells. e, HeLa cells expressing Aurora B-FRET biosensor were synchronized to G2 by RO3306 and microinjected in the nucleus with AluI. G2 state was retained in presence of RO3306 as indicated by FRET signal. t = 0 minutes refers to the first time point of the recorded time-lapse. f, Quantification of chromatin density in cells as in e, normalized to the mean of t = 0 min. n=11 cells. Bars indicate mean; significance tested by a two-tailed Mann-Whitney test (P = 0,438). g, Chromatin was fragmented in TSA-treated G2 cells by injection of AluI and mitosis was subsequently induced as in a. Projection of 9 Z-sections. h. Quantification of chromatin density in G2 and mitosis as in g for n=10 cells. Bars indicate mean; significance was tested by a two-tailed Mann-Whitney test (P = 0.481). i, j, In vitro liquid-liquid phase separation behaviour of unmodified or acetylated nucleosome arrays. i, 12X601 Nucleosome arrays labelled with fluorophores as indicated were treated with recombinant p300 histone acetyltransferase or no enzyme and then subjected to identical phase separation buffers for 30 min. j, Quantification of nucleosome array self-association into condensates by coefficient of variation (CV =  σ/µ) in images as in i. n = 26 for AlexaFlour488 array (AF488), n = 25 for acetylated AlexaFluor488 array (AF488-p300), n = 25 for AlexaFluor594 array (AF594), n = 30 for acetylated AlexaFluor594 array (AF594-p300). Bars indicate mean; significance tested by a two-tailed Mann-Whitney test (AF488-Ac, P = 0.8x10−14; AF594-Ac, P<10−15, precision limit of floating-point arithmetic). k, Microinjection of synthetic nucleosome arrays that were either untreated or pre-incubated with p300 acetyltransferase into live mitotic cells, for n = 28 cells. Unmodified and acetylated nucleosome arrays were labelled by distinct fluorescent dyes. DNA was counterstained with DAPI. l, Quantification of unmodified and acetylated nucleosome array partitioning into mitotic chromatin. Bars indicate mean; significance was tested by a two-tailed Mann-Whitney test (P = 1.645x10−9). m,n, Ki-67 localization in mitotic cells after Smc4-degradation in the absence and presence of TSA. m, Cells expressing H2B-mCherry were transfected with a construct for expression of mNeonGreen-tagged Ki-67 and imaged without further perturbations (control) or treated with 5-PhIAA for 3 h to degrade Smc4 (ΔCondensin) or 5-PhIAA and TSA to additionally suppress mitotic histone deacetylation (ΔCondensin+TSA). Single Airyscan Z-section. n, Distribution of Ki-67 across the surface of mitotic chromatin in cells as in e. Line profiles were drawn perpendicularly across the chromatin/cytoplasm boundary in a single Airyscan Z-section. n = 5 cells for control, n = 7 for ΔCondensin, n = 8 for ΔCondensin+TSA. 2-3 line profiles per cell. Curves indicate mean +/− SD. o,p AluI chromatin fragmentation in Ki-67 knockout cells. o, Mitotic Ki-67 knockout HeLa cell expressing H2B-mCherry was injected with AluI (t = 0 min) during time-lapse microscopy. p, Quantification of chromatin density before and after injection of AluI, normalized to the mean of untreated pre-injection cells. n = 10 cells, 3 ROIs each. Bars indicate mean, significance was tested by a two-tailed Mann-Whitney test (P = 0,201). Biological replicates: n = 2 (a–l); n = 3 (m,n); n = 2 (o,p). Scale bars, 5 µm, inserts 1 µm. Source Data
Extended Data Fig. 9
Extended Data Fig. 9. Partitioning of proteins and dextrans relative to mitotic chromatin and synthetic nucleosome array condensates.
a, b, Partitioning of GFP surface charge variants relative to chromatin in metaphase cells. a, Live metaphase cells after injection of recombinant mEGFP(−7e) or scGFP(+7e). DNA was stained with Hoechst 33342. b, Quantification of GFP fluorescence in chromatin relative to cytoplasm. n = 17 for mEGFP(−7e), n=20 for scGFP(+7e). Bars indicate mean; significance tested by a two-tailed Mann-Whitney test (P = 1.257x10−10). c, d, Partitioning of charge-modified fluorescent dextrans relative to chromatin in metaphase cells. c, Live metaphase cells after injection of negatively or positively charged 4.4 kDa FITC-dextrans. DNA was stained with Hoechst 33342. d, Quantification of dextran fluorescence in chromatin relative to cytoplasm. n = 21 for 4.4 kDa FITC-dextran(−), n = 10 for 4.4 kDa FITC-dextran(+). Bars indicate mean; significance tested by a two-tailed Mann-Whitney test (P = 4.509x10−8). e–h Partitioning of microtubule-associated proteins (MAPs) with mitotic chromatin in the absence and presence of TSA. DNA was stained with Hoechst 33342 and microtubules with SiR-Tubulin. e, HeLa cells expressing p80-Katanin-mNeongreen were treated with nocodazole and TSA for 3 h as indicated and imaged live. Single confocal Z-sections. f, Quantification of p80-Katanin-mNeongreen fluorescence in mitotic chromatin relative to cytoplasm. n = 13 cells for control, n = 8 for TSA. Bars indicate mean, significance was tested by a two-tailed Mann-Whitney test (P =  P = 3.931x10−5). g, HeLa cells expressing EB3-mNeongreen were treated with nocodazole and TSA for 3 h as indicated and imaged live. Single confocal Z-sections. h, Quantification of EB3-mNeongreen fluorescence in mitotic chromatin relative to cytoplasm. n = 33 cells for control, n = 31 for TSA. Bars indicate mean, significance was tested by a two-tailed Mann-Whitney test (P<10−15, precision limit of floating-point arithmetic). i–l, Partitioning of negatively charged higher molecular weight dextrans relative to mitotic chromatin in the absence and presence of TSA. DNA was stained with Hoechst 33342. i, Live metaphase cells after injection of 20 kDa FITC-dextran. j, Quantification of dextran fluorescence in mitotic chromatin relative to cytoplasm. n = 33 for control, n = 33 for TSA. Bars indicate mean; significance tested by a two-tailed Mann-Whitney test (P<10−15, precision limit of floating-point arithmetic). k, Live metaphase cells after injection of 70 kDa FITC-dextran. l¸ Quantification of dextran fluorescence in mitotic chromatin relative to cytoplasm. n = 22 for control, n = 33 for TSA. Bars indicate mean; significance tested by a two-tailed Mann-Whitney test (P = 4x10−14). m, n, Partitioning of GFP surface charge variants relative to nucleosome array condensates in vitro. m, Chromatin condensates were formed in vitro by exposing 12X601 nucleosome arrays to phase separation buffer. GFP charge variants were added for 10 minutes and then imaged. DNA was stained with Hoechst 33342. n, Quantification of GFP fluorescence in chromatin relative to buffer. n = 69 for mEGFP(−7e), n = 73 for scGFP(+7e). Bars indicate mean; significance tested by a two-tailed Mann-Whitney test (P<10−15, precision limit of floating-point arithmetic). o, p, Partitioning of charge modified dextrans relative to liquid nucleosome array condensates in vitro. o, Liquid chromatin droplets were formed were formed as in m. Charge modified 4.4 kDa dextrans were added for 10 minutes and then imaged. DNA was stained with Hoechst 33342. p, Quantification of dextran fluorescence in chromatin relative to buffer. n = 69 for 4.4 kDa dextran(−), n = 57 for 4.4 kDa dextran(+). Bars indicate mean; significance tested by a two-tailed Mann-Whitney test (P<10−15, precision limit of floating-point arithmetic). Biological replicates: n = 2 (a–h,m–p); n = 3 (i–l). Scale bars, 5 µm, inserts 1 µm. Source Data
Extended Data Fig. 10
Extended Data Fig. 10. Microtubules push liquified chromatin away from the spindle pole independently of hKid and Kif4A.
a, Time-lapse microscopy of liquified chromatin during chemically-induced assembly of monopolar spindles. Live mitotic HeLa cells expressing H2B-mCherry and eGFP-a-tubulin were treated with nocodazole and STLC and then injected with AluI. Nocodazole was removed at t = 0 min during time-lapse imaging. Projection of 4 z-sections. Representative example of n = 13 cells. b, Quantification of chromatin localization at the cell periphery relative to the region around the spindle monopole at = 36 min for cells as shown in a. c, Time-lapse microscopy of liquified chromatin during spindle assembly as in a for cells depleted of Kid and Kif4a by RNAi. d, Quantification as in b for hKid/Kif4a-RNAi cells. n = 16 cells. Bar indicates mean; significance tested by a two-tailed Mann-Whitney test (P = 0.215) e, Validation of RNAi efficiency by Western Blotting. Samples were collected 30 h after transfection of siRNAs targeting hKid and Kif4a and probed by antibodies as indicated; n = 2 experiments. For gel source data, see Supplementary Figure 1b. Biological replicates: n = 4 (a,b); n = 3 (c,d); n = 2 (e). Scale bars, 5 µm. Source Data
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