A mitotic chromatin phase transition prevents perforation by microtubules – Nature

Cell lines and cell culture

All of the cell lines used in this study have been regularly tested negative for mycoplasm contamination. All cell lines in this study were derived from a HeLa ‘Kyoto’ cell line that was previously described in ref. ⁵¹. The cells were cultured in Dulbecco’s modified Eagle medium (DMEM) (IMP/IMBA/GMI Molecular Biology Service/Media Kitchen) containing 10% (v/v) fetal bovine serum (FBS; Gibco, 10270-106, 2078432), 1% (v/v) penicillin–streptomycin (Sigma-Aldrich), 1% (v/v) GlutaMAX (Gibco; 35050038) and selected antibiotics according to the respective expression constructs: blasticidin S (6 µg ml⁻¹, Thermo Fisher Scientific), puromycin (0.5 µg ml⁻¹, Calbiochem), hygromycin B (0.25 mg ml⁻¹) and G418 (0.5 mg ml⁻¹, Invitrogen). HeLa cells were cultured at 37 °C in a 5% CO₂-containing atmosphere. Chromatin was visualized by stable expression of histone 2B labelled with mCherry (Figs. 2a–g and 4 and Extended Data Figs. 2, 6e, 7a–h, 8e–h and 10) or with the aurora B-FRET sensor (CFP/YFP) (Extended Data Fig. 8a–h) or alternatively by labelling with Hoechst 33342 (1.62 µM, Invitrogen, H1399) (Figs. 1a–c, 2m and 3a,c and Extended Data Figs. 1a,d,f, 3a,c,e,g, 4a–d, 5a,c,e and 9a,c,e,g,i,k). Tubulin was visualized by stable expression of an N-terminally tagged eGFP–α-tubulin fusion (Extended Data Figs. 10a,c) or by labelling with SiR–tubulin⁵² (Spirochrome, SC002, Figs. 1a and 4a and Extended Data Figs. 4a,c, 5c, 6e and 9e,g (100 nM) and Extended Data Fig. 2a–c (50 nM))⁵². Centromeres were visualized by stable expression of an N-terminally tagged eGFP–CENP-A fusion (Fig. 4a) or an N-terminally tagged meGFP–CENP-A fusion (Extended Data Figs. 2a–c and 6e). Ki-67 was visualized in live cells either by stable expression of endogenous eGFP–Ki-67³¹ (Fig. 2e–g) or by transient expression of Ki-67-mNeonGreen (Extended Data Fig. 8m,n). To determine the apoptotic index, cells were stained with pSIVA⁵³ and prodidium iodide (PI) (Bio-Rad, APO004) (Extended Data Figs. 3e and 7a,b).

Live-cell imaging was performed in DMEM containing 10% (v/v) FBS (Gibco, 10270-106, 2078432), 1% (v/v) penicillin–streptomycin (Sigma-Aldrich) and 1% (v/v) GlutaMAX (Gibco; 35050038) but omitting phenol red and riboflavin to reduce autofluorescence (imaging medium)⁵¹. Cells were grown in 10 cm or 15 cm Cellstar (Greiner) dishes, in 75 cm² or 175 cm² Nunc EasYFlask cell culture flasks (Thermo Fisher Scientific) or in 96-well, 48-well, 12-well or 6-well Nunclon Delta Surface multiwell plates (Thermo Fisher Scientific). For live-cell imaging, cells were cultivated on Nunc LabTek II chambered cover glass (Thermo Fisher Scientific), on µ-Slide 8-well covered coverslips (Ibidi), in µ-Dish 35 mm high imaging dishes with a polymer or glass bottom (Ibidi).

Generation of stable fluorescent reporter and genetically engineered cell lines

Cell lines stably expressing fluorescently labelled marker proteins were generated by random plasmid integration (for the transfection conditions, see below) or by a lentiviral vector system pseudotyped with a mouse ecotropic envelope that is rodent-restricted (RIEP receptor system). Construction of the RIEP receptor parental cell lines and the subsequent generation of stable cell lines that express fluorescent marker proteins was performed as described previously⁵⁴.

HeLa genome editing was performed using a CRISPR–Cas9-mediated integration approach, using the chimeric Cas9–human Geminin fusion (Cas9–hGem) modification for enhanced editing efficiency⁵⁵. Single-guide RNA (sgRNA) was cloned into pX330-U6-Chimeric_BB-CBh-hSpCas9-hGem-P2A-mCherry*aBpil (gift from S. Ameres). For tagging of endogenous SMC4 genes with an auxin-inducible degron tag⁵⁶, a repair template for targeting the C terminus of the protein was designed, with homology flanks of 800 and 901 bp length for the 5′ and 3′ flanks, respectively. The repair template containing the coding sequence for a C-terminal mAID-HaloTag, including mutations of the protospacer adjacent motif, was synthesized as a gBlock (IDT) and cloned into plasmid pCR2.1 for amplification. sgRNA/Cas9 and homology repair template plasmids were co-transfected using the Neon transfection system (Thermo Fisher Scientific). Transfection was performed according to the manufacturer’s guidelines (for HeLa cells: pulse voltage, 1.005 V; pulse width, 35 ms; pulse number, 2), using the 100 µl tip cuvette, with 10 µg homology repair template and 10 µg gRNA/Cas9–hGEM for 1 × 10⁶ cells. Then, 9 days after electroporation, cell pools were stained with Oregon Green HaloTag (Promega, G2801) ligand and single clones were isolated using fluorescence-activated cell sorting (FACS) and sorted into 96-well plates. Genotyping was performed as in a previous study⁵⁴.

Integration of the OsTIR1(F74G) ligase into the adeno-associated virus integration site (AAVS1, designated safe harbour) locus for establishment of AID2 auxin-inducible degradation of SMC4 was achieved using a sgRNA and homology repair template strategy described in ref. ⁵⁷. Expression of the E3-ubiquitin ligase OsTIR1(F74G) enables degradation of degron-tagged proteins by labelling them for proteasomal degradation in the presence of a small-molecule auxin analogue. sgRNA was cloned into pX330-U6-Chimeric_BB-CBh-hSpCas9-hGem-P2A-mCherry*aBpil. For integration of the OsTIR1(F74G), a homology template containing an OsTIR1(F74G)-SnapTAG-MycA1-NLS and homology flanks of 804 and 837 bp length for the 5′ and 3′ flanks, respectively, was generated. Co-transfection of sgRNA/Cas9 and homology repair template plasmids was performed as described above. Then, 8 days after electroporation, transfected cells were selected with 6 µg ml⁻¹ blasticidin. Single clones were isolated from the stable pool by single-cell dilution into 96-well plates. To identify clones that depleted SMC4-mAID-Halo, clones were treated for 90 min with 1 µM 5-Ph-IAA24, stained with Oregon Green HaloTag (Promega, G2801) ligand and then analysed by flow cytometry using the iQue Screener Plus system.

Transfection of plasmids and small interfering RNA

For expression of fluorescently labelled markers, the respective genes were cloned into bicistronic vectors containing an IRES or a 2A ‘self-cleaving’ peptide sequence with antibiotic resistance genes that enable the protein of interest and the resistance gene to be expressed from the same transcript. For transient transfection or transfection for subsequent selection and colony picking, plasmids were transfected using X-tremeGENE 9 DNA transfection reagent (Roche, 6365787001) according to the manufacturer’s protocol (1 µg plasmid, 4.5 µl X-tremeGENE 9 in 100 µl serum-free OptiMEM) or PEI transfection reagent (1 mg ml⁻¹ stock, Polyscience 24765-2, 4 µg of transfection reagent per 1 µg of plasmid). For stable expression, plasmids were transfected using PEI, 10 µg plasmid for one ~70% confluent 15 cm dish and incubated for 48 h before antibiotic selection.

Small interfering RNAs (siRNAs) were delivered with lipofectamine RNAiMax (Invitrogen) according to the manufacturer’s instructions. hKid (Kif22) was targeted using 16 nM custom silencer select siRNA (sense strand CAAGCUCACUCGCCUAUUGTT, Thermo Fisher Scientific, including a 3′ overhang TT dinucleotide for increased efficiency). Kif4A was targeted using 16 nM custom silencer select siRNA (sense strand GCAAGAUCCUGAAAGAGAUTT, Thermo Fisher Scientific, including a 3′ overhang TT dinucleotide for increased efficiency). Custom silencer select siXWNeg (sense strand UACGACCGGUCUAUCGUAGTT, Thermo Fisher Scientific, including a 3′ overhanging TT dinucleotide for increased efficiency) was used as a non-targeting siRNA control. Cells were imaged 30 h after siRNA transfection. Co-transfection of KIF22 and KIF4A siRNAs was performed using 16 nM final concentrations each. Both hKid and KIF4A siRNAs were described in ref. ²².

Inhibitors and small molecules

To degrade Smc4-mAID-Halotag, cells were incubated in 1 µM 5-phenylindole-3-acetic acid (5-PhIAA) (Bio Academia, 30-003) for 2.5–3 h. To induce histone hyperacetylation, cells were incubated with 5 µM TSA⁵⁸ (Sigma-Aldrich, T8552). To arrest cells in prometaphase with monopolar spindle configuration, cells were incubated in 5 µM STLC⁵⁹ (Enzo Life Sciences, ALX-105-011-M500) for 2–3 h. To arrest cells in prometaphase, cells were incubated for 2–3 h in nocodazole (200 ng ml⁻¹ for live-cell imaging only) or 100 ng ml⁻¹ for microinjection and/or subsequent washout; Sigma-Aldrich, M1404). Nocodazole washout was performed by washing cells five times with prewarmed imaging medium on the microscope. To arrest cells in metaphase for microinjection, cells were incubated with 12 µM proTAME (R&D systems, I-440-01M) for 2 h. To synchronize cells to the G2–M boundary, cells were first synchronized with a double thymidine block followed by a single RO3306⁶⁰ block. One day after seeding, cells were transferred into medium containing 2 mM thymidine (Sigma-Aldrich, T1895). Then, 16 h later, cells were washed three times with prewarmed medium and released for 8 h. The thymidine block was repeated once. Then, 6 h after the second thymidine release cells were transferred into medium containing 8 µM RO3306 (Sigma-Aldrich, SML0569) for 4 h. After G2 arrest, RO3306 was removed, and the cells were washed three times with imaging medium containing 1.5 µM okadaic acid (LC laboratories, O-5857). To induce apoptosis as a positive control for apoptotic index measurements, cells were treated with 5 µM anisomycin (Sigma-Aldrich, A9789) for 3 h. To induce DNA double-stranded breaks as a positive control for DNA damage measurements, cells were treated with 50 ng ml⁻¹ neocazinostatin (Sigma-Aldrich, N9162) for 3 h.

Immunofluorescence

For all immunofluorescence experiments, cells were grown on sterilized 18 mm round Menzel cover glasses in 24-well plates, except immunofluorescence against acetylated histones, which was performed on Nunc LabTek II chambered cover glass. For co-staining spindle poles and centromeres, cells were fixed and extracted at the same time with 1× PHEM (60 mM K-PIPES (Sigma-Aldrich), pH 6.9, 25 mM K-HEPES (Sigma-Aldrich), pH 7.4, 10 mM EGTA (Merck, 324626), 4 mM MgSO₄·7H₂O (Sigma-Aldrich)) containing 0.5% Tween-20 (Sigma-Aldrich) and 4% methanol-free formaldehyde (Thermo Fisher Scientific) for 10 min. The fixation reaction was quenched with 10 mM Tris-HCl (Sigma-Aldrich), pH 7.4, in phosphate-buffered saline (PBS), washed again with PBS and blocked in 10% normal goat serum (Abcam), 0.1% Tween-20 (Sigma-Aldrich) for 1 h.

For staining of acetylated histone tails or cyclin B1, cells were fixed in PBS containing 4% methanol-free formaldehyde (Thermo Fisher Scientific, 28906) for 10 min. The fixation was quenched with 10 mM Tris-HCl (Sigma-Aldrich) pH 7.4 in PBS for 5 min, and cells were permeabilized with PBS containing 0.5% Triton X-100 for 15 min, washed again with PBS and blocked using 2% bovine serum albumin (BSA) (Sigma-Aldrich, A7030) in PBS containing 0.2% Tween-20 (Sigma-Aldrich) for 1 h. Antibody dilution buffer was composed of PBS with 2% BSA (Sigma-Aldrich, A7030), containing 0.1% Tween-20 (Sigma-Aldrich).

For staining of γH2A.X, cells were fixed in PBS containing 4% methanol-free formaldehyde (Thermo Fisher Scientific, 28906) for 10 min. The fixation was quenched with 100 mM glycin (Sigma-Aldrich) in PBS for 10 min, and the cells were permeabilized with PBS containing 0.5% Triton X-100 for 15 min, washed again with PBS and blocked using 10% normal goat serum (Abcam), 0.1% Tween-20 (Sigma-Aldrich) in PBS for 1 h. After incubations with primary and secondary antibodies, the samples were washed three times with PBS containing 0.1% Tween-20 (Sigma-Aldrich) for 10 min each time. Cyclin B1 was detected with a monoclonal rabbit antibody (Cell Signaling, 12231S, 7, 1:800) and visualized using a donkey anti-rabbit Alexa Fluor 488 secondary antibody (Molecular Probes, A21206, 1:1,000) (Extended Data Fig. 1d). CENP-A was detected with a monoclonal mouse antibody (Enzo Life Sciences, ADI-KAM-CC006-E, 10161910, 1:1,000) and visualized using a goat anti-mouse Alexa Fluor 488 secondary antibody (Molecular Probes, A11001, 1:1,000) (Extended Data Fig. 1f). Pericentrin was detected with a recombinant rabbit antibody (Abcam, ab220784, GR3284309-1, 1:2,000) and visualized using a goat anti-rabbit Alexa Fluor 633 secondary antibody (Molecular Probes, A21071, 1:1,000) (Extended Data Fig. 1f). Acetylated histone2B was detected using a polyclonal rabbit antibody (Millipore, 07-373, 3092508, 1:500) and visualized using a donkey anti-rabbit Alexa Fluor 488 secondary antibody (Molecular Probes, A21206, 1:1,000) (Extended Data Figs. 3a–d and 5a). Acetylated histone 3 was detected using a polyclonal rabbit antibody (Merck, 06-599, 3260200, 1:500) and visualized using a donkey anti-rabbit Alexa Fluor 488 secondary antibody (Molecular Probes, A21206, 1:1,000) (Extended Data Fig. 3a–d). Acetylated histone 4K16 was detected using a recombinant rabbit antibody (Abcam, ab109463, GR284778-8, 1:400) and visualized using a donkey anti-rabbit Alexa Fluor 488 secondary antibody (Molecular Probes, A21206, 1:1,000) (Extended Data Figs. 3a–d). γH2A.X was detected with a monoclonal mouse antibody (BioLegend, 613402, B283251, 1:1,000) and visualized using a goat anti-mouse Alexa Fluor 488 secondary antibody (Molecular Probes, A11001, 1:1,000) (Extended Data Fig. 3g). Immunofluorescence samples prepared in Nunc LabTek II chambered cover glass wells were stored in PBS containing 1.62 µM Hoechst 33342 (Invitrogen). Immunofluorescence samples prepared on 18 mm round Menzel cover glasses were embedded in Vectashield with or without DAPI (Vectorlabs, H-1000 or H-1200).

Preparation of 384-well microscopy plates and coverslips for in vitro assays

µCLEAR microscopy plates (384-well; Greiner Bio-One, 781906) were washed with 5% Hellmanex III (Lactan, 105513203) in ≥18 MΩ MonoQ H₂O at 65 °C in a tabletop Incu-Line oven (VWR) for 4 h and then rinsed 10 times with ≥18 MΩ MonoQ H₂O. Silica was etched with 1 M KOH (Sigma-Aldrich) for 1 h at room temperature rinsed again 10 times with ≥18 MΩ MonoQ H₂O. The etched multiwell plate was treated with 5k-mPEG-silane (Creative PEGworks, PLS-2011) suspended in 95% ethanol (VWR) for 18 h at room temperature. The plate was washed once with 95% ethanol, then 10 times with ≥18 MΩ MonoQ H₂O and dried in a clean chemical hood overnight. Until individual wells were used, the plate was sealed with adhesive PCR plate foil (Thermo Fisher Scientific) and kept in a dry and dark space. Before an experiment, the foil above individual wells was cut and 50 µl of 100 mg ml⁻¹ BSA (Sigma-Aldrich, A7030) was added.

For microtubule/nucleosome array droplet experiments (Fig. 3e), thin layer cover glass sandwiches were constructed from passivated 24 × 60 mm Menzel coverglass (VWR). To clean coverslips, they were vertically stacked into a Coplin jar (Canfortlab, LG084). Coplin jars were then sonicated in acetone (Sigma-Aldrich) for 15 min, in 100% ethanol (VWR) for 15 min and then washed 10 times with ≥18 MΩ MonoQ H₂O. All of the sonication steps were performed using an ultrasonic cleaning bath (Branson, 2800 Series Ultrasonic Cleaner, M2800). In a separate Erlenmeyer flask, 31.5 ml 30% aqueous hydrogen peroxide solution (Sigma-Aldrich, 31642) was added to 58.5 ml concentrated sulfuric acid (Sigma-Aldrich, 258105) (piranha acid). The flask was gently swirled until bubbling and heating occurred, then the whole contents of the flask was added to the coverslips in the Coplin jars, ensuring that all coverslip surfaces were covered. The jar was transferred into a 95 °C water bath and heated for 1 h. Afterwards, the piranha solution was discarded, and the cover glasses were washed once with ≥18 MΩ MonoQ H₂O and etched with 0.1 M KOH for 5 min. The cover glasses were transferred to a fresh, dry Coplin jar and dried to completion in a 65 °C benchtop oven, and afterwards left to cool to room temperature. In a separate Erlenmeyer flask, 4 ml dichlorodimethylsilane (DCDMS) (Sigma-Aldrich, 440272, anhydrous) was injected into 80 ml heptane (Sigma-Aldrich, 246654, anhydrous). The contents of the flask were immediately transferred to the jar containing the cover glasses, and the jar was incubated for 1 h at room temperature. Next, silane was decanted, and the cover glasses were sonicated in chloroform (Sigma-Aldrich) for 5 min, washed in chloroform once, sonicated in chloroform again for 5 min and sonicated twice in ≥18 MΩ MonoQ H₂O. Finally, the cover glasses were washed once more in chloroform, air dried and stored in a sealed container in a dry and dark, dust-free space (for up to 6 months).

To passivate cover glasses, on the day of an experiment, a cover glass was transferred to a drop of 5% Pluronic F-127 (Thermo Fisher Scientific, P6866) dissolved in BRB80 buffer (80 mM K-PIPES, pH 6.9 (Sigma-Aldrich, P6757), 1 mM MgCl₂ (Sigma-Aldrich), 1 mM EGTA (Merck, 324626)) for >2 h at room temperature. Directly before assembly of the imaging chamber, the cover glass was rinsed once with ≥18 MΩ MonoQ H₂O and once with BRB80 (80 mM K-PIPES, pH 6.9 (Sigma-Aldrich, P6757), 1 mM MgCl₂ (Sigma-Aldrich) and 1 mM EGTA (Merck, 324626)).

Expression and purification of GFP proteins

pET-based constructs for the expression and purification of meGFP(−7e) and scGFP(+7e) were a gift from D. Liu⁴³. An overnight culture of Rosetta 2 (pLysS) Escherichia coli (Novagen) transformed with pET_−7GFP or pET_+7GFP plasmids encoding GFP with a theoretical peptide charge of −7e or +7e, respectively, were grown on an agar plate by replating a single transformant on LB supplemented with 100 ng µl⁻¹ of ampicillin at 37 °C. The bacterial lawn was suspended in LB supplemented with the 100 ng µl⁻¹ of ampicillin and grown at 37 °C to an optical density at 600 nm of 0.4, cooled over 1 h to 18 °C, and recombinant protein expression was induced by addition of IPTG to 0.5 mM for 18 h at 18 °C. The cells were collected by centrifugation, resuspended in NiNTA lysis buffer (50 mM HEPES·NaOH, pH 7, 150 mM NaCl, 10% (w/v) glycerol, 15 mM imidazole, 5 mM β-mercaptoethanol, 1 mM benzamidine, 100 µM leupeptin, 100 µM antipain, 1 µM pepstatin), the cellular suspension was flash frozen in liquid N₂ and stored at −80 °C.

Bacterial cultures with expressed GFP proteins in NiNTA lysis buffer were thawed in a water bath and lysed by multiple passages through an Avestin Emulsiflex-C5 high-pressure homogenizer at around 10,000 psi. An equal volume of NiNTA dilution buffer (50 mM HEPES·NaOH, pH 7, 1.85 M NaCl, 10% (w/v) glycerol, 15 mM imidazole, 5 mM β-mercaptoethanol, 1 mM benzamidine, 100 µM leupeptin, 100 µM antipain, 1 µM pepstatin) was added to the lysate to increase NaCl concentration to 1 M. Soluble bacterial lysate was isolated by centrifugation of cellular debris in a Beckman Avanti J-26 XPI centrifuge in a JA25.5 rotor at 20,000 rpm. Soluble lysate was incubated with NiNTA resin (Qiagen) equilibrated in NiNTA wash buffer A (50 mM HEPES·NaOH, pH 7, 1 M NaCl, 10% (w/v) glycerol, 15 mM imidazole, 5 mM β-mercaptoethanol, 1 mM benzamidine, 100 µM leupeptin, 100 µM antipain, 1 µM pepstatin) for 2 h in batch with end-over-end mixing. NiNTA resin was poured into a Bio-Rad EconoColumn and the resin was washed with 20 column volumes of NiNTA wash buffer A and 20 column volumes of NiNTA wash buffer B (50 mM HEPES·NaOH, pH 7, 150 mM NaCl, 10% (w/v) glycerol, 15 mM imidazole, 5 mM β-mercaptoethanol, 1 mM benzamidine, 100 µM leupeptin, 100 µM antipain, 1 µM pepstatin) before elution in NiNTA elution buffer (50 mM HEPES·NaOH, pH 7, 150 mM NaCl, 10% (w/v) glycerol, 350 mM imidazole, 5 mM β-mercaptoethanol, 1 mM benzamidine, 100 µM leupeptin, 100 µM antipain, 1 µM pepstatin).

GFP proteins were diluted with 9 volumes of buffer SA (20 mM HEPES·NaOH, pH 7, 10% (w/v) glycerol, 1 mM DTT) and applied to Source 15S (meGFP) or Source15Q (scGFP) resin (GE Healthcare) equilibrated in 98.5% buffer SA and 1.5% buffer SB (20 mM HEPES·NaOH, pH 7, 1 M NaCl, 10% (w/v) glycerol, 1 mM DTT) and eluted with a linear gradient to 100% buffer SB. Fractions containing GFP proteins were concentrated in a centrifugal concentrator with a 3,000 Da MWCO and purified further by size-exclusion chromatography using a Superdex 200 10/300 GL gel filtration column equilibrated with gel filtration buffer (20 mM Tris·HCl, pH 8, 150 mM NaCl, 10% (w/v) glycerol, 1 mM DTT). Peak fractions of purified meGFP(−7e) and scGFP(+7e) proteins were concentrated in a centrifugal concentrator, as described above, and quantified by measuring protein absorbance at 280 nm using their calculated molar extinction coefficient (https://web.expasy.org/protparam/) of 23,380 M⁻¹ cm⁻¹ for meGFP and 18,910 M⁻¹ cm⁻¹ for scGFP. Purified proteins were flash-frozen in liquid N₂ and stored at −80 °C in single-use aliquots.

Nucleosome array in vitro experiments

Generation, fluorophore labelling, assembly and quality control of nucleosome arrays has been described previously¹³. Here 12X601 nucleosome arrays carrying no label or an Alexa Fluor 488 or Alexa Fluor 594 label were used. The covalently fluorescently labelled nucleosome arrays used for microinjection were labelled at an 8% fluorophore density (8 in 100 histone2B proteins labelled with a fluorophore) and dialysed against TE (10 mM Tris-HCl (Sigma-Aldrich), pH 7.4 (Sigma-Aldrich), 1 mM EDTA (Sigma-Aldrich), pH 8.0, 1 mM DTT (Roche, 10708984001)) to remove glycerol.

To induce phase separation of nucleosome arrays (Extended Data Figs. 4e and 5e,g), arrays were first equilibrated in chromatin dilution buffer (25 mM Tris·OAc, pH 7.5 (Sigma-Aldrich), 5 mM DTT (Roche, 10708984001), 0.1 mM EDTA (Sigma-Aldrich), 0.1 mg ml⁻¹ BSA (Sigma-Aldrich, A7030), 5% (w/v) glycerol (Applichem, A0970)) in the presence of 2.03 µM Hoechst 33342 (Invitrogen, H1399) when using unlabelled arrays for 10 min at room temperature. Phase separation was induced by addition of 1 volume of phase separation buffer (25 mM Tris·OAc, pH 7.5 (Sigma-Aldrich), 5 mM DTT (Roche, 10708984001), 0.1 mM EDTA (Sigma-Aldrich), 0.1 mg ml⁻¹ BSA (Sigma-Aldrich, A7030), 5% (w/v) glycerol (Applichem, A0970), 300 mM potassium acetate (Sigma-Aldrich), 1 mM Mg[OAc]₂, 2 µg ml⁻¹ glucose oxidase (Sigma-Aldrich, G2133), 350 ng ml⁻¹ catalase (Sigma-Aldrich, C1345) and 4 mM glucose (AMRESCO, 0188)). For in vitro assays containing tubulin, EGTA was substituted for EDTA in the dilution and phase separation buffers. After addition of phase-separation buffer, the final concentration of nucleosome arrays per reaction was 500 nM, and the reactions were incubated at room temperature for 10 min before transferring the suspension to the imaging chambers.

To visualize soluble tubulin partitioning relative to chromatin droplets in vitro, 20 µl of chromatin droplet suspension was transferred to a passivated well of a 384-well microscopy plate. Chromatin droplets were allowed to sediment for 15 min. Next, 5 µl of soluble TRITC-labelled tubulin (Cytoskeleton, TL590M) in phase-separation buffer containing 500 ng ml⁻¹ nocodazole was added for a final tubulin concentration of 5 µM. Soluble tubulin was equilibrated for 10 min before images were recorded.

To visualize GFP surface charge variant partitioning relative to chromatin droplets in vitro, 20 µl of chromatin droplet suspension was transferred to a passivated well of a 384-well microscopy plate. Chromatin droplets were allowed to sediment for 15 min, after which 5 µl of GFP solution in phase-separation buffer was added to a final concentration of 5 µM. GFP proteins were equilibrated for 10 min before images were recorded.

To visualize chemically modified dextran partitioning into chromatin droplets in vitro, 20 µl of chromatin droplet suspension was transferred to a passivated well of a 384-well microscopy plate. Chromatin droplets were allowed to sediment for 15 min, after which 5 µl dextran solution in phase-separation buffer was added to a final concentration of 500 µg ml⁻¹. The dextran used was a fluorescein (FITC)-labelled 4.4 kDa dextran fraction (negative overall charge conferred to dextran by fluorophore charge) (Sigma-Aldrich, FD4) or a FITC-labelled 4.4 kDa dextran fraction modified with diethylaminoethyl (DEAE) groups conferring an overall positive charge (TdB, DD4).

All of the images of macromolecule partitioning relative to chromatin droplets in vitro (Fig. 3e and Extended data Fig. 9m,o) were recorded on the incubated stage of a customized Zeiss LSM980 microscope using a ×40 1.4 NA oil-immersion DIC plan-apochromat objectives (Zeiss).

Nucleosome array acetylation in vitro

To generate acetylated nucleosome arrays for microinjection, recombinant p300 histone acetyl transferase domain was generated (VBCF Protein technologies Facility) using plasmid pETduet+p300_HAT according to a purification strategy described previously¹³. To induce histone acetylation of nucleosome arrays, arrays with 8% Alexa Fluor 488 or Alexa Fluor 594 label density at a concentration of 3.85 µM were incubated with 6.12 µM p300-HAT (enzyme stock 61.2 µM in gel-filtration buffer (20 mM Tris·HCl, pH 8.0, 150 mM NaCl, 10% (w/v) glycerol, 1 mM DTT)) in the presence of 750 µM Acetyl-CoA (Sigma-Aldrich, A2056) in gel-filtration buffer at room temperature for 2 h with occasional agitation. The acetylation was next stopped by the addition of A-485 (Tocris, 6387) to a final concentration of 9 µM and the reaction mixture was stored in the dark. To 10 µl of quenched acetylation reaction, 10 µl dilution buffer containing 5 µM A-485 (Tocris, 6387) was added and the reaction was allowed to equilibrate at room temperature for 10 min. Next, 1 volume of phase-separation buffer containing 5 µM A-485 (Tocris, 6387) was added and the mixture was incubated for 10 min at room temperature before transferring the suspension to a well of a passivated 384-well microscopy plate.

Microtubule in vitro polymerization

To generate stabilized microtubule seeds for nucleation of microtubule networks in vitro, 22 µl of 5 mg ml⁻¹ tubulin protein (Cytoskeleton, T240) was mixed with 2 µl of 5 mg ml⁻¹ TRITC-labelled tubulin protein (Cytoskeleton, TL590M) and 1 µl of 5 mg ml⁻¹ Biotin-XX-labelled tubulin (Pursolutions, 033305). All tubulin storage solutions were prepared in BRB80 (80 mM K-PIPES, pH 6.9 (Sigma-Aldrich, P6757), 1 mM MgCl₂ (Sigma-Aldrich), 1 mM EGTA (Merck, 324626)). The tubulin mixture was centrifuged at 4 °C for 15 min in a tabletop centrifuge (Eppendorf, 5424R) at 21,000g. To 22.5 µl of the resulting supernatant, 2.5 µl 10 mM guanylyl-(alpha,beta)-methylene-diphosphonate (Jena Biosciences, NU-405) was added to a final concentration of 1 mM and the resulting solution was incubated at 37 °C in a water bath in the dark for 30 min. The resulting seeds were sheared using a 22-gauge needle Hamilton syringe (Sigma-Aldrich, 20788). The resulting suspension was stored in the dark. Seeds were prepared freshly each day and used for subsequent experiments.

Soluble tubulin polymerization mix containing 50 µM tubulin protein (Cytoskeleton, T240), 1 µM TRITC-labelled tubulin protein (Cytoskeleton, TL590M) and 1 mM GTP (Sigma-Aldrich, G8877) in BRB80 (80 mM K-PIPES, pH 6.9 (Sigma-Aldrich, P6757), 1 mM MgCl₂ (Sigma-Aldrich), 1 mM EGTA (Merck, 324626)) was centrifuged at 4 °C for 15 min in a tabletop centrifuge (Eppendorf, 5424R) at 21,000g. The resulting supernatant was used to assemble a microtubule polymerization mix containing 20 µM total soluble tubulin (19.61 µM unlabelled tubulin dimers, 0.39 µM labelled tubulin), 50 mM DTT (Roche, 10708984001), 120 µM glucose (AMRESCO, 0188), 1 mM GTP (Sigma-Aldrich, G8877), 20 µg ml⁻¹ glucose oxidase (Sigma-Aldrich, G2133), 175 ng ml⁻¹ (Sigma-Aldrich, C1345) in BRB80 (80 mM K-PIPES, pH 6.9 (Sigma-Aldrich, P6757), 1 mM MgCl₂ (Sigma-Aldrich), 1 mM EGTA (Merck, 324626)). Composition of soluble tubulin and tubulin polymerization mix was adapted from a procedure for visualization of microtubule plus-end tracking (+TIP) proteins⁶¹.

To visualize chromatin droplets along with polymerized microtubules, imaging chambers were constructed as described previously⁶². Before Pluronic F-127 passivation, each silanized 24 × 60 mm cover glass was cut into a 24 × 25 mm (top piece) and 24 × 35 mm (bottom piece) using a diamond-tipped steel scribe (Miller, DS-60-C). The bottom coverslip was mounted into the support slide and fixed in place with preheated VALAP (1 part Vaseline (Sigma-Aldrich, 16415), 1 part lanolin (Sigma-Aldrich, L7387), 1 part paraffin wax (Sigma-Aldrich, 327204); all parts by weight). In the central region of the bottom cover glass, a two-well silicon culture-insert (Ibidi, 80209) was attached and 40 µl of nucleosome array droplet suspension was added to one of the wells. The suspension was sedimented for 15 min in a humidified chamber at room temperature. Next, 32 µl of buffer was removed from the well, the culture-insert was removed from the coverslip and 20 µl soluble tubulin mix was added to the remaining chromatin droplet suspension. The top coverslip was added on top with the passivated side facing the reaction mixture, and the droplet was allowed to spread fully (~10 s). The sample was next sealed with VALAP to prevent evaporation and thermal streams within the liquid film. This procedure yielded a liquid film of ~30 µm thickness. The assembled imaging cell was transferred to the incubated stage of a customized Zeiss LSM980 microscope combined with the Airyscan2 detector, using a ×63 1.4 NA oil-immersion DIC plan-apochromat objective (Zeiss). The sample was incubated at 37 °C for >30 min before images were recorded.

CLEM and electron tomography

HeLa cells stably expressing H2B–mCherry were cultured on carbon-coated Sapphire discs (0.05 mm thick, 3 mm diameter; Wohlwend). To enrich prometaphase cells, cells were synchronized to G2–M using a double thymidine (Sigma-Aldrich) block for 16 h with 2 mM thymidine each and a subsequent RO3306 (Sigma-Aldrich) block for 6 h with 8 µM RO3306. During the RO-3306 block, the cells were treated with 5 µM TSA for 3 h before RO3306 washout. RO3306 was washed out by rinsing the cells three times with prewarmed imaging medium. The cells were observed on a customized Zeiss LSM780 microscope, using a ×20 0.5 NA EC PlnN DICII air objective (Zeiss). Then, 25 min after the RO3306 washout, most of the cells reached prometaphase (based on DIC and chromatin morphology), and the cells were subsequently processed for electron microscopy analysis. Immediately before freezing, the cells were immersed in cryoprotectant solution (imaging medium containing 20% Ficoll PM400; Sigma-Aldrich), and instantly frozen using a high-pressure freezing machine (HPF Compact 01, Wohlwend).

Frozen cells were freeze-substituted into Lowicryl HM20 resin (Polysciences, 15924-1) using freeze-substitution device (Leica EM AFS2, Leica Microsystems) as follows: cells were incubated with 0.1% uranyl acetate (UA) (Serva Electrophoresis, 77870) in acetone at −90 °C for 24 h. The temperature was increased to −45 °C at a rate of 5 °C h⁻¹ and the cells were incubated for 5 h at −45 °C. Cells were washed three times in acetone at −45 °C and then incubated in increasing concentrations of resin Lowicryl, HM20 in acetone (10, 25, 50 and 75% for 2 h each) while the temperature was further increased to −25 °C at a rate of 2.5 °C h⁻¹. Cells were then incubated in 100% resin Lowicryl, HM20 at −25 °C for a total of 16 h, changing the pure resin solution after 12 h, 14 h and 16 h. The resin was polymerized under ultraviolet light at −25 °C for 48 h. The temperature was increased to 20 °C (5 °C h⁻¹) and ultraviolet polymerization was continued for another 48 h.

After resin polymerization, sections of 250 nm thickness were cut with an ultramicrotome (Ultracut UCT; Leica) and collected on copper–palladium slot grids (Science Services) coated with 1% formvar (Plano). The sections were first observed on the Zeiss LSM710 microscope, using a ×40 1.4 plan-apochromat oil-immersion objective (Zeiss). H2B fluorescence was used to choose cells and subcellular ROIs for tomography. The sections were then post-stained with 2% UA in 70% methanol at room temperature for 7 min and lead citrate at room temperature for 5 min. The sections were observed by Tecnai F20 transmission EM (200 kV; FEI). For tomography, a series of tilt images was acquired over a −60° to +60° tilt range with an angular increment of 1° using Serial EM software⁶³ at a final xy pixel size of 1.14 nm. Tomograms were reconstructed using the R-weighted back projection method implemented in the IMOD software package⁶⁴.

Western blotting

Samples were separated by Novex NuPAGE SDS–PAGE system (Thermo Fisher Scientific) using 4–12% Bis-Tris in MES running buffer according to the manufacturer’s guidelines, and transferred to a 0.45 µm nitrocellulose membrane (Bio-Rad) by tank-blot wet transfer (Bio-Rad) at room temperature for 1 h. Blocking, primary antibody incubations (4 °C, 16 h) and secondary antibody incubations (room temperature, 1.5 h) were performed in 5% (w/v) milk (Maresi, Fixmilch instant milk powder) in PBS, containing 0.05% Tween-20 (Sigma-Aldrich). SMC4 was probed using a rabbit polyclonal antibody (Abcam, ab229213, GR3228108-5, 1:1,000). GAPDH was probed with a rabbit polyclonal antibody (Abcam, ab9485, GR3212164-2, 1:2,500). hKid was probed with a monoclonal rabbit antibody (Abcam, ab75783, GR129278-4, 1:1,000). KIF4A was probed with a recombinant rabbit antibody (Abcam, ab124903, GR96215-7, 1:1000). Horseradish peroxidase (HRP)-conjugated anti-mouse or anti-rabbit secondary antibodies (Bio-Rad, 1:10,000) were visualized using ECL Plus Western Blotting Substrate (Thermo Fisher Scientific) on a Bio-Rad ChemiDoc Imager operated by analysed using Bio-Red Image Lab v.6.0.1, which was also used for analysis.

Microscopy

Images of G2-to-mitosis inductions, DsRed transfections, HaloTag stainings of wild-type and Smc4-mAID-HaloTag cells, histone acetylation, p300 overexpression and fluorescence recovery after photobleaching (FRAP) experiments were recorded on a customized Zeiss LSM780 microscope, using ×40 or ×63 1.4 NA oil-immersion DIC plan-apochromat objectives (Zeiss), operated by ZEN Black 2011 software. Images of chromatin density, live-microtubule stains, AluI-digestion time-lapse videos, nucleosome array microinjections, tubulin microinjections, CENP-A/pericentrin immunofluorescence experiments and cyclin B1 staining were recorded on a customized Zeiss LSM980 microscope combined with the Airyscan2 detector, using ×40 or ×63 1.4 NA oil-immersion DIC plan-apochromat objectives (Zeiss), operated by ZEN3.3 Blue 2020 software. For all of the confocal microscopes, an incubator chamber (EMBL) provided a humidified atmosphere and a constant 37 °C temperature with 5% CO₂.

Images of fields of cells to determine the apoptotic index were recorded on an inverted Axio Observer Z1 with a sCMOS camera equipped with a ×20/0.5 plan-neofluar air objective, controlled by ZEN Blue. For detection of Hoechst 33342 fluorescence, a Ex 377/350 nm, Em 447/60 nm filter was used; for detection of pSIVA fluorescence, a Ex 470/40 nm, Em 525/50 nm filter was used; and for detection of PI fluorescence, a Ex 530/75 nm, Em 560/40 nm was used.

For FRAP experiments, selected image regions were bleached using a laser intensity 200-fold higher than the laser intensity used for image acquisition, and each bleached pixel was illuminated 20 times with the pixel dwell time used for acquisition (1.79 µs). Images were acquired every 25 ms for the course of the experiment.

Images of nucleosome array droplets and in vitro polymerized microtubules were recorded on a customized Zeiss LSM980 microscope combined with the Airyscan2 detector, using the ×40 or ×63 1.4 NA oil-immersion DIC plan-apochromat objectives (Zeiss), operated by ZEN3.3 Blue 2020 software. For mounting 384-well microscopy plates, the Pecon Universal Mounting Frame KM adapter was used. For mounting microtubule imaging slides, a custom aluminium mounting block for 24 mm × 24–60 mm coverslips was used (IMP/IMBA workshop and BioOptics).

Microinjection experiments

Live-cell microinjection experiments were performed using a FemtoJet 4i microinjector in conjunction with an InjectMan 4 micromanipulation device (Eppendorf). All microinjections were performed using pre-pulled Femtotips injection capillaries (Eppendorf). The microinjection device was directly mounted onto a customized confocal Zeiss LSM780 (Fig. 2c and Extended Data Fig. 8a–h) or a customized Zeiss LSM980 microscope (Fig. 2a,e and Extended Data Figs. 3a, 4a,  7a,c,e,g, 8k,l,o, 9a,c,l,k and 10a,c) with live-cell incubation (37 °C, 5% CO₂). For all microinjections, cells were cultured in µ-Dish 35 mm high-wall imaging dishes with a polymer or glass bottom (Ibidi) to reach near-confluency on the day of the injection.

For injection of AluI (Fast Digest, Thermo Fisher Scientific, FD0014), 1.5 µl AluI stock was added to 3 µl of 5 mg ml⁻¹ FITC-labelled 500 kDa dextran fraction (Sigma-Aldrich, FD500S) dissolved in injection buffer (50 mM K-HEPES, pH 7.4 (Sigma-Aldrich, H3375), 5% glycerol (Applichem, A0970), 1 mM Mg(OAc)₂ (Sigma-Aldrich, M5661)). Using an Eppendorf Microcapillary Microloader (Eppendorf, 5242956003), 3 µl diluted enzyme was loaded into a Femtotip microinjection capillary. Microinjection of mitotic cells (Figs. 2a,c,e,g and 4a and Extended Data Figs. 7a,c,e,g, 8g and 10a,c) was performed using injection settings of 130–150 hPa injection pressure, 0.15 to 0.25 s injection time and 30 hPa compensation pressure. Microinjection of G2 interphase cells (Extended Data Fig. 8a–h) was performed using injection settings of 120 hPa injection pressure, 0.4 to 0.5 s injection time and 20 hPa compensation pressure.

For injection of nucleosome arrays (Extended Data Fig. 8k), 1.5 µl injection buffer (50 mM K-HEPES, pH 7.4, 25% glycerol buffer) was added to 1.5 µl unmodified and 1.5 µl acetylated nucleosome array solution (1.3 µM final concentration for each nucleosome in the injection solution). Microinjection of mitotic cells was performed using injection settings of 180–190 hPa injection pressure, 0.35 s injection time and 85 hPa compensation pressure.

For injection of tubulin protein (Fig. 3a), TRITC-labelled tubulin (Cytoskeleton, TL590M) was dissolved in 5% GPEM (80 mM K-PIPES, pH 6.9 (Sigma-Aldrich, P6757), 1 mM MgCl₂ (Sigma-Aldrich, 63065), 1 mM EGTA (Merck, 324626)) supplemented with 1 mM GTP (Sigma-Aldrich, G8877) to a concentration of 0.5 mg ml⁻¹. Protein was clarified by centrifugation at 4 °C for 15 min in a tabletop centrifuge at 21,000g. The supernatant was microinjected into mitotic cells using injection settings of 175 hPa injection pressure, 0.25 s and 85 hPa compensation pressure. For microinjection of cells arrested in prometaphase with nocodazole, the G-PEM was additionally supplemented with 100 ng ml⁻¹ nocodazole (Sigma-Aldrich, M1404).

For microinjection of GFP surface charge variants (Extended Data Fig. 9a), recombinant meGFP(−7e) or scGFP(+7e) were dissolved in injection buffer (50 mM K-HEPES, pH 7.4 (Sigma-Aldrich, H3375), 25% glycerol (Applichem, A0970)) to a concentration of 15 µM. Microinjection of mitotic cells was performed using injection settings of 125 hPa injection pressure, 0.2 s injection time and 40 hPa compensation pressure.

For microinjection of charge-modified dextran fractions (Extended Data Fig. 9c), a FITC-labelled 4.4 kDa dextran fraction (negative overall charge conferred to dextran by fluorophore charge) (Sigma-Aldrich, FD4) or a FITC-labelled 4.4 kDa dextran fraction modified with DEAE groups conferring an overall positive charge (TdB, DD4) were dissolved in injection buffer (50 mM K-HEPES, pH 7.4 (Sigma-Aldrich, H3375), 25% glycerol (Applichem, A0970)) to a concentration of 5 mg ml⁻¹. Microinjection of mitotic cells was performed using injection settings of 125 hPa injection pressure, 0.1–15 s injection time and 20 hPa compensation pressure.

Image analysis

DNA congression analysis

To quantify DNA congression to the spindle equator in live cells, the DNA distribution along a line profile parallel to the pole-to-pole axis was measured (for Fig. 1a,b, 7.06 µm width, 22.5 µm length; for Extended Data Fig. 5c,d, 12.04 µm width, 22.5 µm length). Along each line profile, the accumulated DNA density was measured in the central 5 µm interval around the centre position between the poles (determined by highest SiR–tubulin peak intensities) and divided by the total DNA density along the entire profile, after subtraction of the extracellular background fluorescence. Each line profile represents an average intensity projection of z-slices around the pole-to-pole axis (for Fig. 1a,b, 2.4 µm range; for Extended Data Fig. 5c,d, 0.75 µm range).

Chromatin/DNA compaction analysis

To quantify DNA density, in a central z-section of a mitotic cell (determined by visual inspection on the basis of the highest SiR–tubulin staining intensity at the poles (Fig. 1a,c and Extended Data Figs. 4a–d and 5c,e) the DNA channel was denoised using a Gaussian blur filter (σ = 2) and thresholded using the Otsu dark method in Fiji. The resulting binary mask was converted into a selection and the DNA mean fluorescence within this ROI was measured. All data points were normalized to the mean of unperturbed control cells.

In STLC-treated cells used for AluI microinjection experiment (Fig. 2a,b,e,f and Extended Data Figs. 7e–h and 8g,h), the DNA density was measured in line profiles. In a single z-slice, a line profile (3 pixels wide, 2 µm long) through a chromosome parallel to the imaging plane (before AluI injection), a chromatin droplet (20 min after AluI injection) or the diffuse chromatin mass in TSA treated cells (20 min after AluI injection) was measured. The mean histone 2B fluorescence intensity in a 200 nm interval around the peak value was measured. Values were normalized to the mean of the non-injected control (Fig. 2b,f and Extended Data Figs. 7f and 8h) or the non-injected condensin-degraded control (Extended Data Fig. 7h).

In cells subjected to G2–mitosis induction (Extended Data Fig. 8a–h), the YFP channel was denoised using a Gaussian blur filter (σ = 2) and thresholded using the Otsu dark method in Fiji. The resulting mask was converted into a ROI and the histone 2B–YFP mean fluorescence recorded within this ROI. The values were normalized to the mean of the G2 measurements.

DNA displacement analysis after mitotic entry with monopolar spindle configuration

To quantify DNA displacement away from the spindle pole after mitotic entry in the presence of STLC to induce a monopolar spindle geometry, the DNA/chromatin distribution was measured along a radial line profile having the centre of mass of SiR–tubulin fluorescence as a seeding point. From videos of mitotic entry, the time point corresponding to 20 min after prophase entry was used for measurement (Extended Data Fig. 2a–c). The measurement was performed in a maximum intensity z-projection of 2 confocal slices around the centre of mass of SiR–tubulin fluorescence, indicating the centre of the monopolar spindle (z-offset of 2.5 µm). A radial line profile was drawn, using the ImageJ Plugin Radial Profile Angle (v.1-14-2014 by P. Carl and K. Miura, based on Radial Profile from P. Baggethun), with an integration angle Θ = 180°. Along the integrated histone 2B fluorescence around the monopole, the fluorescence signal within the inner 30% distance of the pole to the outer edge of chromatin distance (~99% of cumulative histone 2B fluorescence) was divided by the outer 70%. The resulting value represents the relative amount of chromatin detectable in pole-proximal regions.

Kinetochore displacement analysis

In z-projections of Airyscan or confocal slices (7 Airyscan z-sections with a z-offset of 0.15 µm for Extended Data Fig. 1f, 2 confocal z-sections with a 2.5 µm z-offset for Extended Data Fig. 2a–c), the histone 2B channel was denoised using a Gaussian blur filter (σ = 2) and thresholded using the Otsu dark method in Fiji and converted into a mask. Kinetochores were detected after denoising the CENP-A channel using a Gaussian blur filter (σ = 1) using the Find Maxima function in Fiji (strict, excluding edge maxima, prominence = 100 (Extended Data Fig. 1g), prominence = 2,000 (Extended Data Fig. 2e)). Of the total number of detected kinetochores, the fraction of kinetochores outside chromatin (>0.5 µm distance to the nearest chromatin mask surface) was quantified manually.

Electron tomography analysis

Analysis of electron tomograms were performed in IMOD/3dmod, v.4.11. Annotation of all structures was performed in the Slicer Window view, using running z average intensity projections to increase contrast (microtubule annotation: projection of 10–20 z-slices, chromatin annotation: 35–50 z-slices). To account for the loss in image sharpness towards the top and bottom of the recorded tomograms, for all of the tomograms, only the centre section of the recorded volume was analysed (tomograms with 120 to 150 slices, no annotation in the top and bottom 20 slices). For microtubule annotation, a zoom factor of 0.8 to 1.0 was used. For chromatin annotation, a zoom factor of 0.35–0.45 was used. Assignment of microtubules was based on ultrastructural morphology, and assignment of chromatin boundaries was performed on the basis of local grain size, considering the transition of a large-grained, coarsely interspersed particle-containing area (cytoplasm with ribosomes; typical coefficient of variation (CV) > 0.4) to a fine-grained, finely interspersed particle containing area (chromatin with nucleosomes; typical CV < 0.3 for control, CV < 0.4 for TSA) as the chromosome surface. The electron density as a determining factor of a chromatin surface was only used as a secondary direction as TSA treatment perturbs the chromatin density/morphology and therefore decreases the annotation accuracy. On average, an annotation landmark was set every 5–10 nm for both microtubules and the chromatin surface. After manual annotation of microtubules and a chromatin surface, a model of a meshed chromatin surface was generated by averaging five consecutive annotated slices to increase surface smoothness. Microtubule segment length was measured within the cytoplasm and chromatin-internal regions and normalized to the total volume of the respective domain.

Mitotic duration measurements in the absence or presence of TSA

Fields of asynchronous cells stably expressing histone 2B–mRFP were imaged for up to 4 h. The timing of nuclear envelope breakdown and anaphase onset was determined by visual inspection of chromatin morphology.

FRAP analysis of chromatin mobility

To measure FRAP, raw data measurement, background subtraction, data correction and normalization were performed according to ref. ⁶⁵. The measurement of FRAP ROI and background fluorescence signal was performed directly in ZEN software. Total chromatin fluorescence per time-lapse movie was measured using the Time Series Analyzer v.3 (J. Balaji).

FRET analysis of aurora B–FRET biosensor

To determine the mitotic state of control and AluI-injected cells at the G2 to M transition (Extended data Fig. 8a–h), CFP, FRET and YFP signals of the histone 2B fused Aurora B-FRET biosensor⁶⁶ were recorded at the same time. To quantify FRET efficiency, a custom Fiji script was used. In average-intensity projections of 9 z-slices (1 µm each), a nuclear mask was generated using the YFP fluorescence, after denoising using a Gaussian blur filter (σ = 5), by thresholding using the Otsu dark method. For each time point, the background for the FRET and CFP channel was measured in an extracellular ROI (square of around 1 µm × 1 µm). After background subtraction, FRET and CFP signals within the nuclear ROI were denoised using a Gaussian blur filter (σ = 5) and a FRET/CFP ratio was calculated. The resulting ratiometric image was depicted using the Fire lookup table in Fiji, at an interval of 0 to 1.4 for FRET/CFP.

Measurement of Ki-67 surface confinement

To determine the surface confinement of Ki-67 to the surface of mitotic chromatin under various conditions, the distribution of Ki-67 was measured along line profiles across the chromatin–cytoplasm boundary.

In mitotic cells arrested in taxol, chromosomes were identified, which were perpendicular to the imaging section in 3 consecutive slices (z-offset between Airyscan slices of 0.15 µm). The centre slice was used for analysis. A line profile of 10 px width was manually drawn through one chromatid in a single Airyscan section, orthogonal to the surface of the chromatid and near-orthogonal to the longer axis of the chromosome. The Ki-67 fluorescence was measured along the line and aligned to the first peak in fluorescence intensity (Fig. 2e,f). For measuring Ki-67 distribution across the chromatin–cytoplasm boundary in cells after AluI-digestion of mitotic chromatin, the line profile was drawn perpendicular to the chromatin droplet surface, otherwise following the same measurement parameters as described above. To quantify the surface confinement of Ki-67, the fluorescence value along the line profile for the highest value of the control (surface) was divided by the value in the centre of the chromatid for control (inside). The same positions along the line profiles were used to calculate the ratios for TSA-treated and AluI-digested cells. (Fig. 2f,g).

To measure the Ki-67 distribution on mitotic chromatin after degradation of SMC4 or degradation of SMC4 and TSA treatment, a line profile of 10 px width was manually drawn across the chromatin surface in a central (based on visual inspection) Airyscan z-section (z-offset, 0.15 µm). The Ki-67-fluorescence was measured along these profiles and aligned around the 50% maximum value of the histone 2B fluorescence, indicative of the edge of the chromatin mass (Extended Data Fig. 8e,f).

Macromolecule partitioning relative to chromatin

In images of microinjected nucleosome arrays into metaphase cells (Extended Data Fig. 8k), the DNA channel was denoised using a Gaussian blur filter (σ = 2) and thresholded using the Otsu dark method in Fiji. The chromatin mask was converted into a ROI by using the Analyze Particles function in Fiji. For the two injected nucleosome arrays of different colours, the mean fluorescence intensity was measured in the DNA ROI and divided by the fluorescence intensity in the cytoplasm measured in a circular ROI of ~5 µm diameter, with at least 1 µm distance to the chromosomes.

The TRITC–tubulin partition coefficient (Fig. 3a,b) was measured >10 min after microinjection in nocodazole-arrested cells. At the surface of a chromosome at the edge of the chromosome cluster, mean fluorescence intensity of TRITC was measured along a line profile (5 px width, 1.3 µm length) orthogonal to the chromosome surface. Values 1 µm apart within and outside of chromatin were divided, resulting in the measured equilibrium partition coefficient.

The distribution of differently expressed DsRed fusion proteins (Fig. 3c,d) was measured using a custom Fiji script. A chromatin mask was generated using thresholding (default method, Fiji), after Gaussian blur filtering (σ = 2) the chromatin channel. The mask was then shrunk by 0.2 µm and the mean fluorescence was measured within the mask. Cytoplasmic fluorescence was measured within a ring (width, 0.5 µm), generated by extending the mask by 1.5 µm. Background mean fluorescence was measured within a small circular region outside the cell and subtracted from chromatin and cytoplasmic values. Partitioning coefficients were obtained by dividing chromatin by cytoplasmic fluorescence values. Measurements were performed on the central z-slice.

The distribution of microinjected differently charged GFPs (Extended Data Fig. 9a,b) and differently modified FITC-labelled dextran fractions (Extended Data Fig. 9c,d) was measured analogously along line profiles orthogonal to the metaphase plate at the orthogonal surface of the chromatin mass (5 px width, 2.5 µm length). Values 1 µm apart within and outside of chromatin were divided, resulting in the measured equilibrium partition coefficient.

The distribution of soluble TRITC–tubulin dimers (Fig. 3e,f), differently charged GFPs (Extended Data Fig. 9m,n) and differently modified FITC-labelled dextran fractions (Extended Data Fig. 9o,p) within nucleosome array droplets in vitro was measured by determining the mean fluorescence intensity in the droplet in a central section of the droplet, in a small circular region covering ~25% of the droplet area in the respective section. In a large, rectangular buffer ROI without droplets, the mean fluorescence in the buffer was measured and the partition coefficient was calculated for each field of droplets.

Chromatin droplet distribution analysis in cells

Chromatin distribution in pole-peripheral regions was measured at 36 min after nocodazole washout. The spindle-pole position was determined as the z-slice with highest tubulin signal (SiR–tubulin (Fig. 4a) or eGFP–α-tubulin (Extended Data Fig. 10a,c)) and the total histone 2B (Fig. 4a and Extended Data Fig. 10a,c) and eGFP–CENPA fluorescence intensity in circular ROIs (r = 5 µm) in maximum intensity projections of 5 z-slices (1 µm offset between slices) around the central section was subtracted from the total cellular fluorescence in maximum intensity projections (the cell outline was determined by DIC channel). Each dataset was normalized to the mean of the total fluorescence intensities of all cells.

Microtubule density distribution in vitro

After deconvolution in ZEN, Airyscan images of fields of microtubules were denoised using a Gaussian blur filter (σ = 2). The background was then removed using the subtract background function in Fiji, using a rolling ball radius of 740 px. The image was converted into 8-bit, and thresholded using the Auto Local Threshold (v.1.10.1) plugin in Fiji, with the Phansalkar method and a radius of 100. The resulting binary image was skeletonized using the Skeletonize function in Fiji. The resulting skeleton length was measured. The chromatin channel was denoised using a Gaussian blur filter (σ = 2) and thresholded using the Otsu method in Fiji. The resulting binary image was transformed into a ROI using the Analyze particles function (size range, 5–infinity). Microtubule skeleton lengths were measured within chromatin ROIs and the surrounding buffer, and the ratio of total segment length in chromatin/buffer was calculated per image.

Validation of auxin-inducible degradation of SMC4-mAID-HaloTAG in live cells

In images of fields of live cells stained with OregonGreen488 HaloTAG ligand, the total fluorescence intensity in average intensity projection was recorded in Fiji. To normalize the values to the cell count, the ratios of HaloTAG fluorescence over SiR–DNA intensity were calculated, and the values were normalized to the mean of the Hela WT cells.

Acetylated histone fluorescence intensity measurements

After indirect immunofluorescence analysis of the respective histone-acetyl marks, a mask of the DNA channel was generated by thresholding using the Otsu method in Fiji. Next, the obtained binary image was converted to ROIs using the Analyze particles function (size range, 5–infinity). Mitotic and interphase cells were differentiated manually on the basis of chromatin morphology. Within the obtained ROI per cell, the mean fluorescence intensity of the acetyl mark was recorded, the extracellular background measured in a rectangular ROI was subtracted and a ratio of the histone mark over DNA mean intensity was generated to account for changes in DNA compaction between the different conditions. For comparison of untreated interphase and mitotic cells, obtained ratios were normalized to the mean of untreated interphase cells (Extended Data Fig. 3a,b). For comparison of untreated and TSA-treated mitotic cells, ratios were normalized to the mean of untreated mitotic cells (Extended Data Fig. 3c,d). For comparison of p300-expressing mitotic cells, ratios were normalized to the mean of the mock-plasmid transfected mitotic cells (Extended Data Fig. 5a,b).

Measurement of cyclin B1 stain

In interphase mitotic cells, determined by the absence or presence of cell rounding, in the central section of the recorded z-stack (7 z-slices, 1 µm each, central section manually determined), the cell outline in DIC images was used to determine a ROI per cell. The total fluorescence intensity of the cyclin B1 fluorescence within this ROI was recorded in Fiji. The values were normalized to the mean of the cyclin B1 stain in interphase cells.

Quantification of the apoptotic index

In fields of cells, the DNA channel was denoised using a Gaussian blur filter (σ = 2) and thresholded using the Otsu dark method in Fiji, and one ROI per cell was derived using the Analyze Particle function in Fiji. Within each ROI, the mean fluorescence signal of pSIVA and PI was quantified and any cell with a mean fluorescence signal of greater than 1.2× the median of all untreated control cells was considered to be positive for the respective marker and therefore dead/apoptotic. The fraction of all cells scoring positive for pSIVA and PI stain reflects the apoptotic index (Extended Data Fig. 3e,f).

Quantification of DNA damage foci by γH2A.X staining

A confocal stack of 21 slices with a z-offset of 0.5 µm was converted into a maximum intensity projection. The z-projection was denoised using a Gaussian blur filter (σ = 2), and γH2A.X foci were detected using the Find Maxima tool in Fiji (strict detection, 3,000 prominence) within a segmented chromatin mask obtained by thresholding the DNA channel using the Otsu dark method in Fiji (Extended Data Fig. 3g,h).

Determination of coefficient of variation of chromatin droplets in vitro

In entire fields of chromatin droplets or nucleosome array solution (Extended Data Fig. 8c) (both recorded ~3 µm above the cover glass using laser powers adjusted to the unmodified condition), the mean fluorescence intensity and s.d. was measured, and the CV (CV = α µ⁻¹) was calculated.

Airyscan processing and deconvolution

Raw images recorded using the Airyscan2 detector using AS-SR or multiplex airyscan modes were processed using ZEN3.3 Blue 2020 software.

Fiji version details

The Fiji-integrated distribution/version used for analyses in this study was ImageJ v.1.53c, using Java v.1.8.0_66 (64 bit), with in part custom ImageJ plugins as indicated.

Statistical analyses and data reporting

No statistical methods were used to predetermine sample size. To test significance, the robust, nonparametric Mann–Whitney U-test was used. The statistical test used are indicated and the exact P values are been provided wherever possible. In some cases, the P values calculated were below the precision limit of the data type and algorithm used. In those cases, this has been noted in the legend and an upper bound was given. NS, P > 0.05; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.

Statistics and reproducibility

Figure 1a,b: representative examples and quantification of chromatin fraction at spindle centre: control (n = 51 cells), ΔCondensin (n = 65 cells), ΔCondensin + TSA (n = 34 cells), TSA (n = 61). Figure 1c: quantification of DNA density: control (n = 31 cells), ΔCondensin (n = 89 cells), ΔCondensin + TSA (n = 99 cells), TSA (n = 74). Figure 1d–f: representative examples and quantification of microtubule densities in chromatin (Fig. 1e) and cytoplasm (Fig. 1f): control (n = 10 tomograms from 7 different cells), TSA (n = 10 tomograms from 8 different cells). Figure 2a,b: representative example and quantification of 11 cells, 3 ROIs per cell. Figure 2c,d: representative examples and quantification of 8 cells (undigested) and 10 cells (AluI-digested). Figure 2e,f: representative example and quantification of 11 cells, 3 ROIs per cell. Figure 2g–j: line scans and ratios measured for control (n = 19 cells), TSA (n = 24 cells) and AluI (n = 22 cells). Figure 3a: representative example of an untreated metaphase cell (n = 27). Figure 3a,b: representative examples and quantification of nocodazole treated cells: control (n = 46), TSA (n = 31). Figure 3c,d: representative examples and quantification of DsRed (n = 26), DsRed(−7e) (n = 26), DsRed(+9e) (n = 26). Figure 3e,f: representative examples and quantification of: nocodazole (n = 94 droplets from 45 fields), polymerized microtubules (n = 13 fields of droplets). Figure 4a,b: representative example and quantification of n = 15 cells.

Extended Data Figure 1a,b: representative examples and quantification of HeLa WT (n = 25), HeLa Smc4-mAID-Halo (n = 25), HeLa Smc4-mAID-Halo + 5-PhIAA (n = 25). Extended Data Figure 1d,e: representative examples and quantification of interphase (n = 60), mitotic (n = 89), ΔCondensin (n = 59), ΔCondensin + TSA (n = 83) and TSA (n = 67). Extended Data Figure 1f,g: representative examples and quantification of control (n = 40), ΔCondensin (n = 59), ΔCondensin + TSA (n = 35) and TSA (n = 40). Extended Data Figure 2a–e: representative examples and quantification of control (n = 42), ΔCondensin (n = 38) and ΔCondensin + TSA (n = 60) cells (Extended Data Fig. 2d) and control (n = 42), ΔCondensin (n = 42) and ΔCondensin + TSA (n = 62) cells (Extended Data Fig. 2e). Extended Data Figure 3a,b: representative examples and quantification of interphase (H2B-Ac (n = 20), H3-Ac (n = 20), H4K16-Ac (n = 20)) and mitotic (H2B-Ac (n = 20), H3-Ac (n = 20), H4K16-Ac (n = 20)) cells. Extended Data Figure 3c,d: representative examples and quantification of control (Extended Data Fig. 2b) and TSA-treated (H2B-Ac (n = 20), H3-Ac (n = 20), H4K16-Ac (n = 20)). Extended Data Figure 3e,f: representative fields of cells and quantification of untreated (13 fields with n = 4,574 cells), 500 nM TSA (13 fields with n = 4,926 cells), 5 µM TSA (13 fields with n = 4,653) and anisomycin (13 fields with n = 4,188 cells). Extended Data Figure 3g,h: representative examples and quantification of control (n = 48), TSA (n = 48) and NCS (n = 32) cells. Extended Data Figure 4a,b: representative examples and quantification of control (n = 39), 500 nM TSA/washout (n = 35), 5 nM TSA (n = 40) and 500 nM TSA (n = 40) cells. Extended Data Figure 4c,d: representative examples and quantification of ΔCondensin control (n = 45), 500 nM TSA/washout (n = 47), 5 nM TSA (n = 40) and 500 nM TSA (n = 40) cells. Extended Data Figure 5a,b: representative examples and quantification of mock (n = 20), p300_HAT (n = 26), p300(D1399Y) (n = 20) transfected cells. Extended Data Figure 5c–e: representative examples and quantification of chromatin fraction at spindle centre (Extended Data Fig. 5d) and chromatin density (Extended Data Fig. 5e) of mock (n = 20), ΔCondensin + p300_HAT (n = 20), ΔCondensin + p300(D1399Y) (n = 24) transfected cells. Extended Data Figure 6a–c: more examples of electron tomograms in the absence or presence of TSA; control prometaphase (n = 3), control metaphase (n = 7), TSA prometaphase (n = 5) and TSA metaphase (n = 5) examples drawn from 10 tomograms per condition from 7 different cells each. Extended Data Figure  6e: quantification of mitotic duration in control (n = 44) and TSA-treated (n = 36) cells. Extended Data Figure 6f: 3 representative examples of control anaphase cells (top row) and 3 representative examples of TSA treated anaphase cells (bottom row). Extended Data Figure 6g,h: quantification of control (n = 64) and TSA-treated (n = 110) cells from 3 biological replicates. Extended Data Figure 7a,b: representative examples and quantification of control (n = 77), AluI-injected (n = 41) and anisomycin-treated (n = 83) cells. Extended Data Figure 7c,d: representative examples and quantification of control (n = 75) and AluI-injected (n =  60) cells. Extended Data Figure 7e,f: representative example of a p300 expressing cell during AluI digestion and quantification of n = 12 cells, 3 ROIs for each cell. Extended Data Figure 7g,h: representative example of a condensin-depleted cell during AluI digestion and quantification of n = 7 cells, 3 ROIs each. Extended Data Figure 8a,b: representative example and quantification of 13 cells. Extended Data Figure 8c,d: representative example and quantification of 8 cells. Extended Data Figure 8e,f: representative example and quantification of n = 11 cells. Extended Data Figure 8g,h: representative example and quantification of 10 cells. Extended Data Figure 8i,j: representation examples and quantification of fields of unmodified (AF488, n = 26; AF594, n = 25) and acetylated (AF488, n = 25; AF594, n = 30) nucleosome arrays. Extended Data Figure 8k,l: representative example and quantification of 28 cells. Extended Data Figure 8m,n: representative examples and quantification of control (n = 5), ΔCondensin (n = 7) and ΔCondensin + TSA (n = 8) cells, 2–3 ROIs per cell. Extended Data Figure 8o,p: representative example of a MKI67-KO cell during AluI digestion and quantification of n = 10 cells, 3 ROIs each. Extended Data Figure 9a,b: representative examples and quantification of microinjected meGFP(−7) (n = 17) and scGFP(+7) (n = 20). Extended Data Figure 9c,d: representative examples and quantification of microinjected FITC–dextran (−) (n = 21) and FITC–DEAE-dextran (+) (n = 10). Extended Data Figure 9e,f: representative examples of metaphase cells (n = 4) and quantification of nocodazole-treated cells for control (n = 13) and TSA (n = 8). Extended Data Figure 9g,h: representative examples of metaphase cells (n = 4) and quantification of nocodazole-treated cells for control (n = 33) and TSA (n = 31). Extended Data Figure 9i,j: representative examples of 20 kDa dextran partitioning in control (n = 33) and TSA treated (n = 33) cells. Extended Data Figure 9k,l: representative examples of 70 kDa dextran partitioning in control (n = 22) and TSA-treated (n = 33) cells. Extended Data Figure 9m,n: representative examples and quantification of nucleosome array droplets with meGFP(−7) (n = 69) and scGFP(+7) (n = 73). Extended Data Figure 9o,p: representative examples and quantification of nucleosome array droplets with FITC–dextran (−) (n = 69) and FITC–DEAE-dextran (+) (n = 57). Extended Data Figure 10a,b: representative example and quantification of n = 13 cells. Extended Data Figure 10c,d: representative example and quantification of n = 16 cells. All of the experiments in this study were performed in at least two biological replicates.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this paper.