Ki-67 acts as a biological surfactant to disperse mitotic chromosomes.
Journal: 2016/August - Nature
ISSN: 1476-4687
Eukaryotic genomes are partitioned into chromosomes that form compact and spatially well-separated mechanical bodies during mitosis. This enables chromosomes to move independently of each other for segregation of precisely one copy of the genome to each of the nascent daughter cells. Despite insights into the spatial organization of mitotic chromosomes and the discovery of proteins at the chromosome surface, the molecular and biophysical bases of mitotic chromosome structural individuality have remained unclear. Here we report that the proliferation marker protein Ki-67 (encoded by the MKI67 gene), a component of the mitotic chromosome periphery, prevents chromosomes from collapsing into a single chromatin mass after nuclear envelope disassembly, thus enabling independent chromosome motility and efficient interactions with the mitotic spindle. The chromosome separation function of human Ki-67 is not confined within a specific protein domain, but correlates with size and net charge of truncation mutants that apparently lack secondary structure. This suggests that Ki-67 forms a steric and electrostatic charge barrier, similar to surface-active agents (surfactants) that disperse particles or phase-separated liquid droplets in solvents. Fluorescence correlation spectroscopy showed a high surface density of Ki-67 and dual-colour labelling of both protein termini revealed an extended molecular conformation, indicating brush-like arrangements that are characteristic of polymeric surfactants. Our study thus elucidates a biomechanical role of the mitotic chromosome periphery in mammalian cells and suggests that natural proteins can function as surfactants in intracellular compartmentalization.
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Discussion board
Nature. Jul/13/2016; 535(7611): 308-312

Ki-67 acts as a biological surfactant to disperse mitoticchromosomes


Eukaryotic genomes are partitioned into chromosomes, which during mitosisform compact and spatially well-separated mechanical bodies13.Thisenables chromosomes to move independently of each other for segregation ofprecisely one copy of the genome to each of the nascent daughter cells. Despiteinsights into the spatial organization of mitotic chromosomes4 and the discovery of proteins at thechromosome surface3,5,6, the molecular andbiophysical basis of mitotic chromosome individuality have remained unclear. Wereport that Ki-67, a component of the mitotic chromosome periphery, preventschromosomes from collapsing into a single chromatin mass after nuclear envelopedisassembly, thus enabling independent chromosome motility and efficientinteractions with the mitotic spindle. The chromosome separation function ofKi-67 is not confined within a specific protein domain but correlates with sizeand net charge of truncation mutants that apparently lack secondary structure.This suggests that Ki-67 forms a steric and electrical barrier, similar tosurface-active agents (surfactants) that disperse particles or phase-separatedliquid droplets in solvents. Fluorescence correlation spectroscopy showed a highsurface density of Ki-67 and dual-color labeling of both protein terminirevealed an extended molecular conformation, indicating brush-like arrangementsthat are characteristic for polymeric surfactants. Our study thus elucidates abiomechanical role of the mitotic chromosome periphery and suggests that naturalproteins can function as surfactants in intracellular compartmentalization.

To identify molecular factors that contribute to spatial separation of mitoticchromosomes we used an automated live-cell imaging pipeline. We visualized chromosomemorphologies in HeLa cells stably expressing histone 2B (H2B) fused to a fluorescenceresonance energy transfer (FRET) biosensor, which probes phosphorylation by the kinaseAurora B and thereby discriminates mitotic from interphase cells7. Addition of nocodazole excluded the effect of mitotic spindleperturbations. In this assay, we expected that depletion of any protein essential forchromosome separation would induce clusters of mitotic chromosomes, in contrast to thescattered chromosome distribution observed in control cells (Fig. 1a). We hence quantified the area of segmented chromosomes oflive mitotic cells to detect clustering phenotypes (Fig.1b).

Screening a small interfering RNA (siRNA) library targeting 1295 candidate genes,including a comprehensive list of factors required for mitosis8,9 and components of thechromosome periphery6 (Supplementary Table 1), revealeda single hit with three different siRNAs causing a chromosome clustering phenotype:Ki-67 (Fig. 1c and Extended Data Fig. 1a, b). We validated on-target specificity of the RNAiphenotype by Cas9 nickase-mediated synonymous mutations of the target region of onesiRNA in all endogenous alleles of Ki-67 (Fig. 1d,e and Extended Data Fig. 1c-g).

Ki-67 is widely used as a proliferation marker in basic research and cancerprognosis10,11, yet its molecular mechanism is unknown. Clues might come from thelocalization of Ki-67 to the chromosome surface from prophase until telophase12,13, itsrequirement to target several other components of the mitotic chromosome peripherydomain14,15, and its interaction with the kinesin Hklp216. While Ki-67 does not seem to contribute to the internalstructure of mitotic chromosomes14, its depletioncauses nucleolar reassembly defects during mitotic exit14 and misorganized interphase heterochromatin15. Given that we had screened for chromosome separationregulators, we re-examined a potential role of Ki-67 in mitotic chromosomeindividualization.

We imaged mitosis in HeLa, non-cancer hTERT-RPE1 cells, and mouse embryonic stemcells depleted of Ki-67. While metaphase plates in non-depleted control cells containedspatially separate chromosomes, they appeared as a single contiguous mass of chromatinin cells depleted of Ki-67 (Fig. 2a, Extended Data Fig. 2a-e). Time-lapse microscopyrevealed that during prophase, when most chromosomes attach to the nuclear envelope17, chromosomes condensed into separate bodies inKi-67-depleted cells, however soon after nuclear envelope breakdown (NEBD) chromosomesmerged into a single coherent mass of chromatin (Fig2b and Extended Data Fig. 2f, g).Furthermore, in wildtype cells Ki-67 relocalized from the nucleolus to chromosome armsonly during very late prophase stages, after chromosome condensation had initiated(Extended Data Fig. 2h, i), and the internalstructure of mitotic chromosomes appeared unaffected by Ki-67 depletion (Extended Data Fig. 3)14. Hence, Ki-67 is not required for the initial chromosomeindividualization and condensation during prophase, but for the maintenance of spatialseparation after nuclear envelope breakdown.

The unstructured morphology of metaphase plates in cells depleted of Ki-67 mightresult from increased adhesion between neighboring chromosomes. To test this, we trackedkinetochores in Ki-67-depleted cells stably expressing fluorescently labeled Centromereprotein A (CENP-A) after mitotic entry in the presence of nocodazole (Fig. 2c, d). In control cells, chromosomes movedextensively by free diffusion. Mitotic chromosomes of Ki-67-depleted cells were, incontrast, almost as immobile as interphase chromosomes in control cells (Fig. 2e and ExtendedData Fig. 4a-c, and Supplementary Video 1). Cell and nuclear size appeared normal and thenuclear envelope disassembled properly in Ki-67-depleted cells (Extended Data Fig. 4d-h and Supplementary Video 2). This suggests that mitotic chromosomes inKi-67-depleted cells immobilize by increased adhesion rather than by spatialconfinement.

A loss of spatial separation between mitotic chromosomes might impair spindleassembly and chromosome congression to the metaphase plate. Ki-67 depletion indeedsubstantially delayed progression from nuclear envelope disassembly until anaphase onset(Extended Data Fig. 5a). Under theseconditions, however, chromosomes were still separated at the onset of spindle assemblythrough their preceding tether to the nuclear envelope (Fig. 2b, 0 min)17, which might enablemicrotubule access to kinetochores before chromosomes coalesce. To investigate morespecifically how chromosome clustering affects chromosome congression, we depolymerizedthe spindle by nocodazole during mitotic entry and then imaged spindle assembly afternocodazole washout. Most control cells rapidly formed metaphase plates and enteredanaphase, whereas Ki-67-depleted cells consistently failed to assemble metaphase platesand almost never entered anaphase (Fig. 2f, g).Imaging cells stably expressing the microtubule plus-tip marker EB3-EGFP showed rapidgrowth of microtubules into regions between neighboring chromosomes after nocodazolewashout in control cells but much less in Ki-67-depleted cells (Extended Data Fig. 5b, c). Hence, Ki-67-dependent chromosomeseparation is important for efficient access of spindle microtubules and progression toanaphase.

To address the mechanism by which Ki-67 establishes a non-adhesive chromosomesurface, we aimed to map this function to specific domains. To test the function ofKi-67 truncation mutants (Fig. 3a), we generated aKi-67 knockout cell line, which clustered mitotic chromosomes similar to the Ki-67 RNAiphenotype (Fig. 3b, Extended Data Fig. 6a-g and Supplementary Video 3). That Ki-67 knockout cells survive despitethe drastic chromosome coalescence phenotype might be explained by the initialseparation of prophase chromosomes through tethers to the nuclear envelope17, which enables microtubule access tokinetochores prior to coalescence. However, the Ki-67 knockout cell line was sensitizedto various stress conditions (Extended Data Fig.6h), consistent with previous Ki-67 knockout studies15,18.

The N-terminus of Ki-67 contains a phosphopeptide-binding Forkhead-associated(FHA) domain19 and a Protein Phosphatase 1(PP1)-binding site14. The central region consistsof 16 tandem repeats20 and the C-terminal part isenriched in leucine and arginine (LR) residue pairs21. As expected, expression of full-length Ki-67 restored mitotic chromosomeindividualization in Ki-67 knockout cells (#1, Fig.3b). Ki-67 lacking the C-terminal LR domain does not bind to chromosomes12 and it did not restore mitotic chromosomeseparation (#2, Fig. 3b) even if expressed at veryhigh levels. A construct lacking the entire N-terminus, in contrast, restored mitoticchromosome separation, indicating that the binding sites for the known Ki-67 interactorsKlp216, NIFK22, and PP114 are not relevant forthis function (#4, Fig. 3b). A small Ki-67 fragmentcontaining only half of the repeat domain and the chromosome-targeting LR domain stillrestored chromosome individualization, but the LR domain alone did not (#5 and #6,repectively, Fig. 3b). Unexpectedly, acomplementary construct lacking the entire repeat domain also restored chromosomeindividualization (#7, Fig. 3b). Thus, any piece ofthe Ki-67 protein appears to support spatial separation as long as it is targeted tochromosomes by the LR-domain. To further test this, we designed a construct withrandomly shuffled N-terminal fragments (#8, Fig.3a). Remarkably, this construct also restored spatial separation of mitoticchromosomes in Ki-67 knockout cells (#8, Fig. 3b).Thus, Ki-67's chromosome-separating activity is not confined within a specificprotein region.

We wondered whether general physico-chemical features of the Ki-67 protein mightprovide clues about its mechanism. Ki-67 is very large (325 and 360 kDa isoforms), has avery high net electrical charge (Extended Data Table1), and is predicted to be mostly unfolded (Extended Data Fig. 7a). Furthermore, Ki-67 has an amphiphilic structure, asthe short C-terminal LR domain of Ki-67 has high attraction to chromatin, while its longN-terminal domain has high attraction to the cytoplasm and is excluded from chromatin(Fig. 3b, # 6 and #2, respectively). Anamphiphilic molecular structure combined with high electrical charge or long extensions(e. g. long hydrocarbon chains) are typical features of surface-active agents(surfactants), which are chemical reagents that disperse particles or phase-separatedliquid droplets23.Through their localization atphase boundaries, surfactants stabilize dispersions and emulsions via steric hindranceand/or electrostatic repulsion23. The strikingsimilarity of Ki-67 to surfactants prompted us to further explore this function.

If Ki-67 acted by steric or electrostatic repulsion similar to surfactants,reducing its size or charge by truncating the peptide chain should decrease theefficiency of knockout phenotype rescue.We indeed observed a more than 14-folddifference in the amount of protein required to restore spatial separation of mitoticchromosomes between the minimal truncation version bearing only half of the repeats andthe LR domain, and full-length Ki-67 protein (Fig.3c). The phenotype rescue efficiency correlated with the protein size andpredicted net charge of the constructs (Fig. 3d),suggesting that the size and overall electric charge might be important forKi-67's ability to space mitotic chromosomes apart.

If electrical charge were a key mechanism of mitotic chromosome separation, thenother highly positively charged DNA-binding proteins might be able to substitute forKi-67. Strikingly, individually overexpressed core histones separated mitoticchromosomes in Ki-67 knockout cells – yet only at very high overexpression levelsand not as far apart as Ki-67 (Fig. 3e, and Extended Data Fig. 7b-h). This suggests that anypositively charged chromosome-binding protein might separate mitotic chromosomes, yetKi-67 apparently has additional properties relevant for spacing chromosomes furtherapart, as for example its large size and its enrichment at the mitotic chromosomesurface.

To form an effective repulsive barrier, Ki-67 should cover a substantialfraction of the mitotic chromosome surface. To investigate this, we tagged allendogenous alleles of Ki-67 with EGFP and measured the cytoplasmic concentration byfluorescence correlation spectroscopy (FCS) (ExtendedData Fig. 8a-c). Using image segmentation of a reference DNA dye, we inferredthat about 270,000 Ki-67 molecules bind to mitotic chromosomes, which corresponds to asurface density of about 210 Ki-67 molecules per square micrometer (Extended Data Fig. 8d-h) and an average spacing of 69 nm betweenKi-67 molecules. Thus, Ki-67 is highly concentrated at the mitotic chromosomesurface.

Polymeric surfactants adsorb at interfaces in a specific molecularorientation23 and they form extendedbrush-like structures at high concentrations24,25. Given the very C-terminalposition of the chromatin-binding domain within Ki-67 and the exclusion of the remainingprotein parts from chromatin regions (Fig. 3a, b),we probed the molecular extension of Ki-67 by attaching different fluorophores at eachof its polypeptide ends (Extended Data Fig. 9a).Reference images of multispectral fluorescent beads showed that Gaussian fitting tofluorescence line profiles determines the relative localization of red and greenfluorophores with an accuracy of 2.9 ± 2.2 nm (Extended Data Fig. 9b-d). We imaged live mitotic cells expressingmCherry-Ki-67-EGFP using confocal microscopy and determined the mean radial position ofeach fluorophore in perpendicularly sectioned chromosome arms (Fig. 4a, b). The N-terminal mCherry tag of Ki-67 localized 87.2± 45.7 nm outwards relative to the C-terminal EGFP tag, whereby a construct withinverted fluorophore positions and a control construct with both fluorophores attachedto the N-terminus of Ki-67 yielded consistent results (Fig. 4c and Extended Data Fig. 9e-h).Thus, Ki-67 has a very elongated conformation that orients perpendicular to the surfaceof mitotic chromosomes, consistent with a brush-like arrangement.

High grafting densities of Ki-67 molecules at the chromosome surface mightincrease the height of brush-like structures, as described for non-biologicalsurface-attached polymers24 (Extended Data Fig. 9i). Consistent with this, high overexpressionlevels of Ki-67 spaced mitotic chromosomes further apart than observed in wildtype cells(Fig. 4d, e). The range of Ki-67-mediatedchromosome repulsion thus depends on molecular density.

Our study shows that Ki-67 is required to maintain individual mitoticchromosomes dispersed in the cytoplasm after their release from the mechanically rigidnuclear envelope. Ki-67 might provide this function through a surfactant mechanism atthe phase boundary between mitotic chromatin and the cytoplasm (Fig. 4f).As phase separation has emerged as an important principleunderlying the formation of many other membrane-less cell organelles like nucleoli orcentrosomes2527, it will be interesting to investigate whether and how naturalprotein surfactants might regulate other cellular phase boundaries.


Cell lines and cell culture

All cell lines used in this study have been regularly tested negativelyfor mycoplasm contamination. Their sources and authentication is summarized inSupplementary Table2. HeLa cell lines stably expressing fluorescent reporter proteinswere generated from a HeLa Kyoto cell line as previously described28. HeLa and hTERT-RPE1 cells were culturedin Dulbecco’s modified Eagle medium (DMEM; Gibco) supplemented with 10%(v/v) fetal bovine serum (FBS; Gibco), 1% (v/v) penicillin-streptomycin(Sigma-Aldrich), 500 µg ml-1 G418 (Gibco) and 0,5 µgml-1 puromycin (Calbiochem). CCE mouse embryonic stem cells weregrown in gelatin-coated dishes in DMEM supplemented with 10% (v/v) FBS, 0.1 mMnon-essential amino acids, 1 mM sodium pyruvate, 2 mM L-Glutamine, 0.05 mMβ-mercaptoethanol, 1% penicillin-streptomycin, and 106 U/mlleukemia inhibitory factor. To visualize chromatin cell lines either stably expressed histone H2Bfused to mCherry or mRFP (Fig. 2c-e, Extended Data Fig. 2h, i, 3 c-g, 4a-c, e,f, h), fused to the Aurora B FRET biosensor (Fig. 1b, c, 2b, f, g, ExtendedData Fig. 2f, g, 5a) or werelabeled with Hoechst/SiR-Hoechst as indicated. For mutation of the endogenousKi-67 locus in HeLa wildtype cells CRISPR/Cas9 nickase strategy was applied asdescribed in Extended Data Fig. 1c, d.SgRNAs were cloned into pSpCas9n(BB)-2A-GFP (pX461, Zhang lab29). A circular plasmid (pCR2.1, LifeTechnologies) carrying the mutation and 700 bp homology flanks on each site wasused as a repair template for homologous recombination. The plasmid mix of guideRNA plasmids and the repair template was transfected into HeLa cells usingX-tremeGENE 9 DNA transfection reagent (Roche). 2 days after transfection cellswere sorted for the presence of Cas9 (GFP positive) and another 4 days later forthe absence of Cas9 (GFP negative) using Fluorescence-activated cell sorting(FACS), either in batch or into 96 well plates, respectively. For endogenousEGFP tagging of Ki-67 in HeLa wildtype cells sgRNAs were cloned intopSpCas9n(BB) (pX335, Zhang lab29) and1000 bp homology flanks were used. GFP positive cells were FACS sorted 6 daysafter transfection into 96 well plates. For live-cell imaging HeLa and RPE1 celllines were grown either in 96-well plastic-bottom plates (µ clear;Greiner Bio-One), or on LabTek II chambered coverglass (Thermo Scientific).Live-cell imaging was performed in DMEM containing 10% (v/v) FBS and 1% (v/v)penicillin-streptomycin, but without phenol red and riboflavin to reduceautofluorescence28. CCE mouseembryonic stem cells were grown in LabTek II chambered coverglass(ThermoScientific) coated with 5 µg/ml Laminin-511 in PBS(BioLamina) for 2-3 h at 37°C. The fluorescence correlation spectroscopymeasurements were performed in a CO2 independent imaging medium(Gibco) supplemented with 20% FBS (v/v), 1 mM L-glutamine, 1 mM sodium pyruvateand 100 nM SiR-Hoechst.

Electron microscopy

For electron microscopy HeLa cells were grown on sapphire discs andcryo-immobilized using an EMPACT2+RTS high-pressure freezer (Leica Microsystems,Vienna, Austria). Specimens were frozen in 100 µm-deep membrane carriers(Leica) filled with growth medium containing 20% BSA (Sigma). Samples werefreeze-substituted in acetone containing 1% osmium tetroxide and 0.1% uranylacetate and thin-layer embedded in Epon-Araldite as described in 30. Serial thin sections were collected onFormvar-coated copper slot grids and imaged in a TECNAI 12 electron microscope(FEI, The Netherlands) operated at 100 kV.

Western blotting

Cells were transfected with siRNAs in 6-well plates (Thermo Scientific)and 2-3 Mio. cells/ml were lysed in 1x SDS loading buffer at indicated timepoints after transfection. Protein samples were separated on NuPAGE Novex 3-8%Tris-Acetate protein gels (Life technologies) and transferred to anitrocellulose membrane (Protran BA83, Sigma) by semidry blotting. Ki-67 wasprobed by monoclonal anti-Ki-67 SP6 antibody (abcam, ab16667) and actin bymonoclonal anti-actin clone C4 (Milipore, MAB1501). Either fluorescently labeledantibodies (IRDye 800CW, IRDye 680RD; Odyssey) were used and membranes scannedon an Odyssey IR imager (LI-COR) or horseradish peroxidase-conjugated secondaryantibodies (Biorad) were visualized using ECL Plus Western Blotting Substrate(Thermo Scientific) and films.

Plasmid and siRNA transfection

For transient or stable expression of fluorescently tagged markerproteins, the genes were subcloned into IRESpuro2 vectors that allow expressionof resistance genes and tagged proteins from a single transcript (Supplementary Table 3).Plasmids were transfected into HeLa cells using X-tremeGENE9 DNA transfectionreagent (Roche) or FuGene 6 transfection reagent (Promega) according to themanufacturer’s instructions and imaged 48 h post transfection. siRNAs(Supplementary Table4) were delivered with Lipofectamine RNAiMax (Invitrogen) at a finalconcentration of 10 nM according to the manufacturer’s instructions.XWneg9 and Scrambled were used as non-targeting siRNA controls and 4 differentsiRNAs against Ki-67 were used (see below). Ki-67 #1 corresponds to Ki-5published in Booth et al., 2014. Ki-67 siRNA #1 was used in Extended Data Fig.2b, c, e and 3c-h. Ki-67 siRNA #2 was used in Fig.2b, Extended Data Fig. 2f, g,3i and 4h, Ki-67 siRNA #4 was used in Fig.2c-e and Extended Data Fig. 3a,b and 5b, c. All 4 Ki-67 siRNAscaused a similar chromosome coalescence phenotype as in Fig. 1d and 2a. Anuclear shape phenotype as described by Booth et al., 2014 (ref 14) was only observed with Ki-67 siRNA #1,but not with the other siRNAs that depleted Ki-67 protein with similarefficiency (see Extended Data Fig. 4d-g and6f, g). All siRNAs were obtained fromLife Technolgies as Silencer Select reagents. Imaging was performed 48 –72 h post siRNA transfection as Western blotting (Extended Data Fig. 2b and 4d)indicated protein depletion to background levels.

Inhibitors and stains

Nocodazole (Sigma) was used at a final concentration of 100 ngml-1 for 1 – 3 h to arrest cells in prometaphase. Forwashout experiments (Fig. 2f, g and Extended Data Fig. 5b, c) Labtek wells werewashed 4x with 500 µl imaging medium without nocodazole before they werereleased into imaging medium. For acute addition during time-lapse imaging 400ng ml-1 was used (Extended Data Fig.3g). Hoechst 33342 (Sigma) was used at a final concentration of 0.2µg ml-1, SiR-Hoechst31at concentrations of 100 – 200nM.

Ki-67 truncations

The long isoform of Ki-67 was cloned from HeLa cDNA into an IRESpuro2vector under a CMV promoter and fused to mNeonGreen at the C-terminus.Truncation mutants were generated by PCR amplification, enzymatic digest andligation from the full length construct (3256 amino acids) and cloned into thesame plasmid. For truncation construct #2 amino acids 2929 - 3256 were removed,for construct #3 amino acids 1 - 134, for construct #4 amino acids 1 - 1002, forconstruct #5 amino acids 1 - 1970, for construct #6 amino acids 1 - 2930 and forconstruct #7 amino acids 995 - 2945. For construct #8, the N-terminal regionfrom aa 1 - 994 was divided into 4 pieces and the order of the fragments wasrearranged. The final construct contained the Ki-67 fragments in the followingorder: aa 507 - 726, aa 52 - 506, aa 727 - 994, aa 1 - 51 from N- toC-terminus.

RNAi screen

1295 genes were targeted by either two or three siRNAs. This target genelist included the MitoCheck genome-wide RNAi screen validation dataset with 1128genes8, 100 predicted chromosomecondensation factors 9 and 67 known andpredicted chromosome periphery proteins (43 based on literature search, andadditionally 24 based on Gene Ontology term and domain analysis in the datasetpresented in 6). All siRNAs had beenmapped against the 2013 human genome (ENSEMBL V70) to ensure unique targetspecificity. siRNAs were delivered using solid-phase reverse transfection32 in 384 well imaging plates (Falcon).Cells were seeded with 1 µM TO-PRO-3 Iodide (Life Technologies) on thescreening plates using a Multidrop Reagent Dispenser (Thermo Scientific). 43 hafter seeding nocodazole was added with the same device to a final concentrationof 100 ng ml-1 and 6 hours later plates were imaged on a MolecularDevices ImageXpressMicro XL screening microscope (see below) using a x20, 0.75NA S Fluor dry objective (Nikon) and acquiring 4 positions with 520 µm x520 µm in each well. To compensate for inhomogeneous illumination, allimages were flatfield corrected with the Metamorph software (Molecular Devices)using background images acquired in empty wells. Automated image analysis wasperformed using the in-house-developed CellCognition software33 and nuclei and sets of mitoticchromosomes were segmented by local adaptive thresholding. As a quality controlfor RNAi efficiency and specificity, supervised classification of cellmorphologies was applied to the screening data and expected phenotypes8 were observed for all positive controls(siRNAs targeting INCENP, KIF11, PLK1, CDC20) and negative controls (empty,Scrambled or XWneg9 siRNA). For chromosome area measurements only live mitoticcells were taken into account using a 2 step gating strategy. First, mitoticcells were identified using the Aurora B FRET biosensor as depicted in Fig. 1b. Aurora B phosphorylation of thebiosensor at the onset of mitosis causes a conformational change in the sensorthat reduces FRET between a CFP for energy transfer (CyPet) donor and a YFP forenergy transfer (YPet) acceptor7. Todetermine the range of FRET/YPet ratio of mitotic cells nocodazole arrestedcells were identified by supervised classification using images from twountransfected wells of each plate. The 2.5th and 97.25th quantile of thebackground-subtracted FRET/YPet intensity of all prometaphase cells was used aslower and upper classification border, respectively (0.6 < FRET/YPet> 0.82). To identify dead cells and exclude them from further analysis, aTO-PRO-3 intensity threshold was applied (TO-PRO-3 > 15). From all livemitotic cells of each well the median size of the segmented chromosome area wascalculated and plotted either for each siRNA individually (Extended Data Fig. 1a) or as the mean of 2-3 siRNAs (Fig. 1c). Wells with > 20% apoptoticcells or < 20 live mitotic cells were excluded from the final analysis,as the chromosome area could not be reliably quantified under theseconditions.

Live-cell microscopy

Automated wide-field fluorescence microscopy (Fig. 1) was performed on a Molecular DevicesImageXpressMicro XL screening microscope equipped with reflection-based laserautofocus and a x20, 0.75 NA S Fluor dry objective (Nikon), controlled byin-house-developed Metamorph macros33.Cells were maintained in a microscope stage incubator at 37°C in ahumidified atmosphere of 5% CO2. To image the Aurora B FRET cell linethe following two filter sets were used: a FRET filter cube with an excitationfilter 426 – 450 nm, emission filter 528.5 – 555.5 nm and adichromatic mirror of 458 nm and a YFP filter cube with an excitation filter 488- 512 nm, emission filter 528.5 – 555.5 nm, and a dichromatic mirror of520 nm.

Confocal microscopy was performed on a customized Zeiss LSM780microscope using a x40, 1.4 NA. Oil DIC Plan-Apochromat objective (Zeiss),controlled by ZEN 2011 software and an autofocus macro (AutofocusScreen, provided by J.Ellenberg. The microscope was equipped with an incubation chamber (EuropeanMolecular Biology Laboratory (EMBL), Heidelberg, Germany), providing ahumidified atmosphere at 37°C with 5% CO2.

Fast time-lapse imaging with the EB3-GFP cell line was performed on aspinning-disk confocal microscope (UltraView VoX, PelkinElmer) with a x100, 1.45NA objective controlled by Volocity software and equipped with an incubationchamber (EMBL) for imaging at 37°C with 5% CO2.

For fluorescence recovery after photobleaching (FRAP) experiments,selected image regions were bleached using a laser intensity 600-fold higherthan the laser intensity used for image acquisition, and the pixel dwell timewas increased 20-fold above that used for image acquisition.

Immunostaining of mouse embryonic stem cells

Mouse embryonic stem cells were fixed in 3.7% formaldehyde in PBS for 10min, washed twice with PBS containing 0.05% Tween20 and permeabilized with 0.5%Triton X-100 in PBS. Cells were blocked for 10 min with 10% FBS in PBS with0.05% Tween20 and stained for 2 hours with a rabbit polyclonal anti-Ki-67antibody (abcam, ab66155, 1:300). After several washes cells were stained withanti-rabbit IgG Alexa Fluor 488 (Molecular Probes, 1:600). DNA was stained with0.8 µg ml-1 Hoechst 33342 (Sigma).

Mitotic chromosome spreads

HeLa cells treated with siRNA for 48 h were trypsinized and resuspendedin 75 mM KCl for 16 min at 37°C. Cells were then fixed by 3:1 ice-coldmethanol:acetic acid for 15 min at 4°C. After 2 washes with 3:1 ice-coldmethanol:acetic acid cells were dropped on cleaned and pre-chilled glass slidesfrom a height of 30 cm. Cells were dried on the slide and mounted in Vectashieldmounting medium with 1.5 µg/ml DAPI (Vector Laboratories).

Micrococcal nuclease hypersensitivity assay

HeLa cells treated with siRNA for 48 h were trypsinized and washed twicewith PBS. 10 Million cells per sample were resuspended in lysis buffer (25 mMTris pH 7.5, 100 mM NaCl, 5 mM MgCl2, 0.2% NP-40, 1x CompleteEDTA-free protease inhibitor (Roche)) and lysed on ice by passing 20 timesthrough a needle (0.4 mm). Chromatin fraction was pelleted by centrifugation,washed once with micrococcal nuclease (MNase) digestion buffer (10 mM Tris pH7.4, 15 mM NaCl, 60 mM KCl, 1 mM CaCl2), resuspended in MNasedigestion buffer and aliquoted. Aliquots were prewarmed to 37°C for 2 minand subsequently incubated with threefold serial dilutions of micrococcalnuclease from 0.06 U/ml to 45 U/ml (Thermo Scientific) for 20 min at 37°Cin MNase digestion buffer. The reaction was stopped by addition of 25 mM EDTA.1% SDS was added, the sample was diluted 1:4 with deionized water and 500 mMNaCl was added. DNA was phenol-chloroform extracted and run on a 1.5% agarosegel.

Colony formation assay

200 HeLa wildtype or Ki-67 knockout cells were seeded into 6-wellplates. After two days indicated drugs were added at increasing concentrationsand plates were incubated for 10 – 17 days. Colonies were then fixed with4% formaldehyde for 20 minutes, washed with water, stained for 20 – 30min with Crystal Violet, washed with water several times and dried.

Secondary structure and charge predictions

Folded and charged regions within Ki-67 were visualized with FoldIndex( and the EMBOSS chargeprediction tool ( Aproteome-wide net charge analysis was performed on the human proteome assemblyGRCh38 using a customized R script based on the net charge calculation functionof the ‘seqinr’ R package and pK values from EMBOSS (Extended Data Table 1).

Statistical analysis and sample numbers

All experiments were repeated several times and indicated experimentnumbers always refer to biological replicates. Data were tested for normalityand equal variances with Shapiro-Wilk and Levene’s tests (α =0.05), respectively. The appropriate statistical test was chosen as follows:Unpaired normal distributed data were tested with a two-tailed t-test (in caseof similar variances) or with a two-tailed t-test with Welch’s correction(in case of different variances). Unpaired not normal distributed data weretested with two-tailed Mann-Whitney test (in case of similar variances) or witha two-tailed Kolmogorov-Smirnov test (in case of different variances). Pairednot normal distributed data were tested with a Wilcoxon matched-pairs signedrank test.

Sample numbers of Fig. 1e: 3experiments with the following sample numbers in wildtype cells: No siRNA (n =435, 242, 327), siControl (n = 856, 472, 506), siKi-67 #1 (n = 422, 241, 201),siKi-67 #2 (n = 480, 318, 331), siKi-67 #3 (n = 961, 444, 466), siKi-67 #4 (n =859, 560, 492). 3 experiments with the following sample numbers in siKi-67 #2resistant cells: No siRNA (n = 331, 325, 235), siControl (n = 932, 291, 367),siKi-67 #1 (n = 427, 227, 71), siKi-67 #2 (n = 498, 223, 185), siKi-67 #3 (n =331, 385, 298), siKi-67 #4 (n = 847, 223, 245).

Extended Data

Extended Data Figure 1

Generation of a Ki-67 siRNA #2-resistant HeLa cell line by homozygousmutation of endogenous Ki-67 genomic loci.

a, Quantification of chromosome area as in Fig. 1b, but displayed for eachindividual siRNA, shows that the three siRNAs causing strongest chromosomeclustering all target Ki-67. Each data point corresponds to the medianchromosome area of all live mitotic cells in a specific siRNA condition.Median, quartiles and 1.5 * interquartile range of controls (siControls anduntransfected) and the siRNA library are indicated. The top four siRNAscausing an increased chromosome area all target proteins involved incytokinesis. Hence the chromosome area increase is likely to be aconsequence of polyploidization by cytokinesis failure in precedingdivisions. b, Chromosome areas of all individual live mitoticcells from the original RNAi screening data. Each data point corresponds tothe chromosome area in a single cell. This reveals that the clusteringphenotype is very penetrant within the cell population. c,CRISPR/Cas9 nickase strategy to mutate the siKi-67 #2 target site withoutchanging the amino acid coding sequence. Red triangles indicate DNA strandnicking sites. PAM: Protospacer Adjacent Motif. sgRNA: single guide RNA.d, Schematic of genotyping strategy. A newly generatedHindIII (or BglI) restriction site generated by CRISPR/Cas9 nickase asdepicted in c was used to detect correctly mutated alleles. e,HindIII and BglI restriction fragments were detected by gel electrophoresisfollowing the assay depicted in d, showing successful recombination of allthree Ki-67 alleles present in HeLa cells. f, DNA sequencingchromatogram of the siKi-67 #2 target site of a wildtype and theCRISPR/Cas9-mutated cell line, respectively. Asterisks indicate mutatednucleotides. g, Western blot performed on whole cell lysates ofwildtype or siKi-67 #2 resistant cell lines 48 h after indicated siRNAtransfections demonstrates that all siRNAs used in this study (siKi-67 #1-4)efficiently depleted Ki-67 in wildtype cells and that the CRISPR/Cas9mutated cell line was fully resistant against siKi-67 #2 but still sensitiveto siKi-67 #1. The two bands labeled by anti Ki-67 antibody correspond tothe two Ki-67 isoforms with predicted molecular masses of 320 and 359kDa.

Extended Data Figure 2

Ki-67 is not required for initial chromosome individualization andcondensation but for maintenance of chromosome separation.

a, Quantification of phenotype penetrance in Fig. 2a. Live metaphase cells recorded byan automated imaging pipeline were classified as coherent or normal bysupervised machine learning (3 independent experiments with total samplenumbers of n = 111 (no siRNA), n = 88 (siControl), n = 112 (siKi-67 #1), n =131 (siKi-67 #2)). b, Western blot analysis of HeLa wildtypecells, performed at indicated time points after siKi-67 #1 transfection,showed efficient depletion of Ki-67 at 48 – 72 h post siRNAtransfection. This time window was hence used for all further experiments.For gel source data, see Supplementary Fig. 1. c, Live hTERT-RPE1 cellsstained with Hoechst were imaged 48 h after siRNA transfection. Controlcells (n = 21) had spatially separate chromosomes, whereas Ki-67siRNA-transfected cells (n = 17) had metaphase plates that appeared as acontiguous mass of chromatin. d, Mouse embryonic stem cellswere fixed 48 h after siRNA transfection and stained using anti-Ki-67antibody and Hoechst. Metaphase plates that lacked Ki-67 signal hadchromosomes merged into an unstructured mass of chromatin (lower panel),whereas all cells with residual levels of Ki-67, owing to incompleteRNAi-mediated protein depletion, had normal metaphase plate morphologies.Representative examples for n = 20 cells. e, Electronmicrographs of HeLa cells transfected with siRNAs as indicated demonstratethat chromosomes appeared as a single contiguous mass. Representative imagesfor n = 10/10 control and 9/11 for Ki-67 RNAi cells. Closed arrowheads markdark chromatin areas; open arrowheads mark lighter grainy layer ofchromosome periphery. f, Quantification of prophase chromosomecondensation using pixel intensity standard deviation of the H2B-mCherryfluorescence as a measure for homogeneity of chromatin. Curves indicate meanand SD of 16 cells per condition. g, Representative examplecells of the chromosome condensation assay in f. h, Ki-67localization on chromosomes in live prophase HeLa cells expressingH2B-mCherry/Ki-67-EGFP (NEBD, t = 0 min) i, was quantified(excluding nucleoli) during mitotic entry. A significant change (p <0.01 by Student’s t-test) in chromosome condensation (marked by redcircle; compared to the initial 4 frames) occurs 11 min before a significantincrease in Ki-67 localization (green circle). Curves indicate mean and SDof 13 cells. Bars, 5 µm in e, all others 10 µm.

Extended Data Figure 3

Internal chromosome organization is not affected by Ki-67depletion.

a, Hoechst-stained chromosomes of control orKi-67-depleted HeLa cells were segmented by thresholding, subsequently aconvex hull was fitted around segmented chromosomes (red line) andb, the segmented chromatin area and area of a convex hullfit were quantified. The calculated chromatin area of Ki-67 depleted cellswas similar to control depleted cells, which demonstrates that Ki-67depletion has no detectable effect on chromosome-internal compaction. Ki-67depleted cells lack inter-chromosomal space as the convex hull area isalmost identical with the chromatin area (n = 16 for each siRNA, barsindicate mean ± SD, **** indicates p < 0.0001 by pairedWilcoxon matched-pairs signed rank test). c, Live HeLa cellexpressing H2B-mCherry/EGFP-Kleisin-γ were imaged 72 h after siKi-67#1 or control siRNA transfection and d, mean and SD of sisterchromosome axis distance were quantified (n = 508 from 3 experiments(control RNAi), n = 380 from 4 experiments (Ki-67 RNAi), n = 398 from 3experiments (no RNAi)). e, Live HeLa cell stably expressingH2B-mCherry/CENP-A-EGFP were imaged ~60 h after siKi-67 #1 or controlsiRNA transfection and f, mean and SD of interkinetochoredistance were quantified (n = 875 from 3 experiments (control RNAi), n =1489 from 5 experiments(Ki-67 RNAi), n = 818 from 3 experiments (no RNAi)).g, Centromere elasticity assay. Metaphase HeLa cells stablyexpressing H2B-mCherry/CENP-A-EGFP were imaged with 10 s time-lapse beforeand after nocodazole addition. The interkinetochore distance measurementover time in 12 control cells (control siRNA = light gray and untransfectedcells = dark gray) and 10 cells transfected with siKi-67 #1 (gray lines)demonstrated that kinetochores were under tension in Ki-67 depleted cells.The red line indicates the mean. h, Chromosome spreads ofuntransfected or Ki-67 depleted HeLa cells appeared similar. Representativeexamples of 35 chromosome spreads from 2 independent experiments aredepicted. i, Nuclei from control or Ki-67 depleted HeLa cellswere treated with titrated amounts of MNase and DNA was subjected to 1.5%agarose electrophoresis. No detectable difference was seen in twoindependent experiments. Bars, 10 µm in a, h, 5 µm in c,e.

Extended Data Figure 4

Chromosome clustering and motility decrease are not caused by spatialconfinement.

a-b, Representative example of kinetochore trackingperformed in interphase (quantified in Fig.2e). a, Live interphase HeLa cells stably expressingH2B-mCherry/CENP-A-EGFP were imaged in presence of nocodazole andb, kinetochores were tracked in time-lapse videos.c, Representative MSD analyses of kinetochore tracks(CENP-A-EGFP) of mitotic HeLa cells (from Fig.2c, d) and of the interphase cell in panel a. d,Western blot analysis of HeLa wildtype cells, performed at indicated timepoints after siKi-67 #1 or #2 transfection confirmed that both siRNAsdeplete Ki-67 protein with similar efficiency. e, Interphasecells expressing H2B-mCherry 48 h after indicated siRNA transfectionsf, were quantified with regard to nuclear size by automatedsegmentation of H2B-mCherry (bars indicate mean ± SD, **** indicatesp < 0.0001 by Kolmogorov-Smirnov test, data from 25 random positions:n = 303 (siControl), n = 303 (siKi-67 #1), n = 246 (siKi-67 #2)). OnlysiKi-67 #1 caused a significant decrease in nuclear size while siKi-67 #2,which was used for kinetochore tracking in Fig. 2c-e, had no effect on nuclear size. As Ki-67 knockoutcells also had a normal nuclear size (Extended Data Figure 6f, g), we conclude that the effect ofsiKi-67 #1 on nuclear size is likely due to off-target protein depletion.g, Quantification of mitotic cell size of control or Ki-67depleted cells demonstrated that similar to f, only siKi-67 #1 led to asignificant decrease in cell size consistent with an off-target effect (barsindicate mean ± SD, **** indicates p < 0.0001 by Mann-Whitneytest, total sample numbers: n = 74 (siControl), n = 100 (siKi-67 #1), n = 95(siKi-67 #2)). As kinetochore tracking was based on siKi-67 #2, the reducedmotility of mitotic chromosomes cannot be attributed to cell size changes.h, Live HeLa cells stably expressingH2B-mRFP/LAP2β-EGFP were imaged 48 h after siRNA transfection as theyentered mitosis in the presence of nocodazole (n = 15 per condition). Bars,2 µm in a, b, 10 µm in e, h.

Extended Data Figure 5

Ki-67 depletion causes a prolonged mitosis and impairs access of spindlemicrotubules to chromosomes.

a, Mitotic progression determined by live-cellmicroscopy of HeLa cells expressing H2B-Aurora B FRET biosensor, after siRNAtransfection as indicated. 3 independent experiments with total samplenumbers of n = 145 (siControl), n = 97(siKi-67 #2), n = 134 (siKi-67 #3),n =164 (siKi-67 #4). b, Live HeLa cells stably expressing EB3-EGFPwere imaged 2 minutes after release from a 2 h nocodazole treatment,chromosome areas were identified (yellow lines) and c, EB3-EGFPmean fluorescence was measured in chromosome areas (bars indicate mean andSEM from 50 cells per condition, ** indicates p < 0.01 byMann-Whitney test). Bar, 10 µm.

Extended Data Figure 6

Generation of a Ki-67 knockout cell line.

a, CRISPR/Cas9 was used to generate a HeLa cell linewith indicated deletions on exon 5 of the Ki-67 allele. b, DNAsequencing chromatogram confirmed that no further alleles are present.c, Metaphase plates of live HeLa wildtype and Ki-67knockout cells stained with Hoechst (n = 30 per cell line). d,Western blot performed on whole cell lysates of wildtype or Ki-67 knockoutcells. The two high molecular weight bands labeled by anti Ki-67 antibody inwildtype that correspond to the two Ki-67 isoforms are undetectable forKi-67 knockout cells. Actin was used as a loading control. e,Representative time-lapse image series of a Ki-67 knockout cell proceedingfrom prophase to prometaphase in the presence of nocodazole (n = 12, seeSupplementary Video3). Chromosomes are labeled with SiR-Hoechst and two regions wereselected to exemplify coalescence of chromosomes upon their close approach.Arrows mark regions just before their coalescence. f, Automatedsegmentation of SiR-Hoechst-labeled interphase nuclei of wildtype and Ki-67knockout cells confirmed a normal nuclear size of Ki-67 knockout cells (meanand SD of 200 wildtype and 270 Ki-67 KO cells). g,Representative example images of interphase wildtype or Ki-67 knockout cellsstained with SiR-Hoechst of the quantification in f. h, Thesensitivity of Ki-67 knockout cells to low dose nocodazole, caffeine or atopoisomerase II inhibitor (ICRF-193) was compared to wildtype by a colonyformation assay. Representative images from two to three independentexperiments are shown. Bar, 5 µm in c, 10 µm in e, g.

Extended Data Figure 7

Ki-67 has little secondary structure, is highly positively charged, andits absence can be partly compensated by overexpression of corehistones.

a, Folding and charge prediction of full length Ki-67based on FoldIndex and EMBOSS webtools using a sliding window of 100.Unfolded regions are depicted in green, folded regions in orange. Positivecharge is marked in blue, negative charge in red. b,Quantification of overexpressed histone levels in individual cells relatedto the mitotic chromosome morphology phenotype, classified by visualinspection. Note that the mean fluorescence values are not comparable toFig. 3c as different imagingsettings had to be used. Cells are from 4-5 independent experiments.c, Radial localization of overexpressed H2B-mNeonGreen inlive Ki-67 knockout cells (n = 20). Normalized fluorescence intensity alongline profiles across a chromosome arm of live Ki-67 knockout cellstransiently transfected with H2B-mNeonGreen (upper panel) orKi-67-mNeonGreen (lower panel) indicate that overexpressed H2B binds to thesurface as well as internal region within chromosomes. d,e, Stable association of H2B-mNeonGreen with mitoticchromosomes. d, Half of the mitotic chromosomes in Ki-67knockout cells highly overexpressing H2B-mNeonGreen were photobleached andthe recovery of fluorescence was followed by time-lapse recording in animage region (yellow box). Representative example of the quantification ine. e, Curves indicate mean and SD of 20 photobleached and 19unbleached control cells. f, Quantification of mitoticchromosome area relative to total cell area for cells shown in panel b andFig. 3c. Boxes indicate median,quartiles and 1.5 * interquartile range (n = 30 for Ki-67, n = 64 for H2B).g, Live Ki-67 knockout cell transiently transfected with aH2B-mNeonGreen and stained with SiR-Hoechst. While 25 of 64 rescued cellsdisplayed fully separated chromosomes (Fig.3e), a large fraction of cells (39 of 64 rescued cells) showeddetectable chromosome individualization at a lower extent compared towildtype cells. Representative single z-section of the latter is shown.h, Live Ki-67 knockout cells transiently transfected withplasmids for expression of the indicated histone fused to mNeonGreen andstained with SiR-Hoechst. Representative single z-sections of 12-18 cellsfrom 2-3 independent experiments are depicted. Although chromosomeindividualization was restored, chromosomes were not separated to the sameextent as in wildtype cells. Bars, 10 µm.

Extended Data Figure 8

Fluorescence correlation spectroscopy of endogenous Ki-67 tagged withEGFP.

a, Western blot performed on whole cell lysates of aHeLa cell line overexpressing GFP-tagged Ki-67 from a bacterial artificialchromosome (BAC), and of two different clones in which all endogenous Ki-67alleles were N-terminally tagged with EGFP (C1, C2). The two lower bandslabeled by anti Ki-67 antibody in the BAC cell line (black arrows)correspond to the two wildtype Ki-67 isoforms. The EGFP-Ki-67 versionsappear upshifted (green arrows) and the band of the small EGFP-taggedisoform overlaps with the band of the wildtype large isoform.b-h, FCS measurement from 3 independent experiments with111 cells (C1) and 156 cells (C2). b, Imaging and analysispipeline for FCS-calibrated imaging. Metaphase cells were identified basedon Hoechst staining and imaged in 3D (only the central slice is shown). Inthe cytoplasm of the central slice an FCS measurement was performed. Fromthe photon counts (right upper panel) the autocorrelation function (ACF) wascomputed (right lower panel). From the fit of the ACF to Eq. S1 the numberof particles N in the focal volume was obtained. The concentration at theFCS point was computed by dividing N by the effective focal volume Veff andthe Avogadro's constant NA. For details see SupplementaryMethods. c, A calibration curve was obtained by plottingthe concentration computed from FCS against the fluorescence intensity in a5 x 5 pixel region at the FCS measurement point (Eq. S2, dashed line).d, Fluorescence microscopy image of live HeLa cell withendogenous Ki-67, labeled by EFGP, scaled to absolute Ki-67 concentration asdetermined by FCS. e, Example segmentation of chromosomes andthe chromosome surface used for quantification in f-h. Segmentation wasperformed in 3D but for simplification only a single z-section is shown. Achromatin mask was obtained by segmentation of the Hoechst signal. Todetermine Ki-67 concentration on chromosomes the mask was dilated to includeKi-67 signal at the outer chromosome surface (Expanded chromatin mask). Tocalculate Ki-67 molecules on the outer chromosome surface, a rim around thechromatin mask was used (outer rim mask). See Supplementary Methodsfor details. f-h, Quantification of FCS measurements. Boxesindicate median, quartiles and 1.5 * interquartile range. f,Mean Ki-67 concentration in the cytoplasm and on 3D-segmented chromosomesusing the expanded chromatin mask depicted in e. g, Totalnumber of Ki-67 molecules within the whole DNA volume using the expandedchromatin mask depicted in e. h, Density of Ki-67 molecules onthe chromosome surface using the outer rim mask depicted in e. Bars, 10µm.

Extended Data Figure 9

Dual-color labeling of Ki-67’s protein termini indicates anextended conformation oriented perpendicular to the mitotic chromosomesurface.

a, Schematic of Ki-67 labeled at the N-terminus (red)and at the chromatin-binding C-terminus (green). At high densities, Ki-67might acquire extended brush-like conformations, as known for polymericsurfactants. b, Multispectral fluorescent beads with 500 nmdiameter were imaged as reference data to determine dual-color localizationaccuracy. The chromatic register shift in the green and red channel wascorrected by image registration. Line profiles (white line) yieldedfluorescence profiles c, for green and red channels. A sum oftwo Gaussian functions was fitted (solid line) for each channel andpeak-to-peak distances of the corresponding single Gaussian functions(dotted lines) were measured for green (d(G)) and red (d(R)) channels.d, The accuracy of the localization method as illustratedin b, c was determined based on the absolute difference between green andred peak-to-peak distances divided by 2. Mean (black line) and standarddeviation (whiskers) are indicated. e-h, Representative examplecells of the quantification in Fig. 4c.e, Live HeLa wildtype cell expressing EGFP-mCherry-Ki-67.Representative example of a sister chromatid pair oriented perpendicular tothe imaging plane. The line profile (white line) yielded a f,fluorescence profile across one sister chromatid. A sum of two Gaussianfunctions was fitted (solid line) for each channel and peak-to-peakdistances of the corresponding single Gaussian functions (dotted lines) weremeasured for green (d(G)) and red (d(R)), respectively. g, LiveHeLa wildtype cell expressing EGFP-Ki-67-mCherry. Representative example ofa sister chromatid pair oriented perpendicular to the imaging plane. Theline profile (white line) yielded a h, fluorescence profileacross one sister chromatid. A sum of two Gaussian functions was fitted(solid line) for each channel and peak-to-peak distances of thecorresponding single Gaussian functions (dotted lines) were measured forgreen (d(G)) and red (d(R)), respectively. i, Modelillustrating that an increased grafting density of Ki-67 at the chromosomesurface might gradually increase the polymer brush height due to repulsiveforces between the polymers. Bars, 1 µm.

Extended Data Table 1
Proteins ranked by predicted net charge at pH 7.
Rank in human proteomeHugo_IDENSEMBL_IDCharge

Charge of core histones


Supplementary Information

Supplementary Information includes:

Supplementary Figure 1

Supplementary Methods

Supplementary Tables 1-4

Supplementary Videos 1-3

Supplementary References

Video 1
Video 2
Video 3
Figure 1

RNAi screen for mitotic chromosome surface adhesion regulators.

a-c, RNAi screen targeting 1295 genes. a, Expectedchromosome phenotypes in mitotic cells with depolymerized spindles.b, Experimental design to detect chromosome clustering in livemitotic HeLa cells based on the viability marker TO-PRO-3 and a FRET biosensorfor mitotic phosphorylation. c, Individual data points correspondto the median chromosome area of all live mitotic cells per target gene, basedon 2 or 3 different siRNAs. Mean, quartiles, and 1.5 * interquartile range areindicated. d-e, Rescue of RNAi phenotype. d, HeLacells, wildtype or strain mutated in siKi-67 #2 target site, were transfected asindicated and imaged live in presence of nocodazole (n = 9-10 images percondition). e, Quantification of chromosome clustering as in b. Themedian size of the chromosome area was determined in 3 experiments (barsindicate mean ± SD, n > 71 cells per condition and experiment, forexact sample numbers see Methods). Bars, 10µm.

Figure 2

Spatial separation of mitotic chromosomes by Ki-67 is important forchromosome motility.

a, Live HeLa cells, wildtype or siKi-67 #2-resistant strain,transfected as indicated and stained with Hoechst. Quantified in Extended Data Fig. 2a. b,Time-lapse microscopy of HeLa cells expressing fluorescently-tagged H2B,transfected as indicated (NEBD, t = 0 min, n = 16 per condition).c-e, Kinetochore tracking. c, Live mitotic HeLacells stably expressing H2B-mCherry/CENP-A-EGFP transfected with indicatedsiRNAs were imaged in presence of nocodazole and d, kinetochoreswere tracked in time-lapse videos. e, Diffusion coefficientsderived from mean square displacement (MSD) analyses. Bars indicate mean± SD. f, Live mitotic HeLa cells expressingfluorescently-tagged H2B imaged after nocodazole washout (t = 0 min)g, were scored for anaphase entry (cumulative frequency, 3independent experiments with total sample numbers of n = 75 (siControl), n = 51(siKi-67 #2), n = 82 (siKi-67 #3), n = 69 (siKi-67 #4). Bars, 2 µm in c,d, all others 10 µm.

Figure 3

Ki-67's chromosome separation function is not confined within aspecific subdomain.

a, Design of Ki-67 constructs. b, Ki-67 constructs weretransfected in nocodazole-treated Ki-67 knockout HeLa cells (inserts aremagnifications of yellow boxes) and c, analyzed for restoration ofmitotic chromosome separation. Individual cells (dots) were classified bychromosome morphology (clustered: no rescue; dispersed: rescue) and plottedaccording to expression levels. Black bars indicate threshold required formitotic chromosome separation. d, Correlation of Ki-67-truncationexpression levels required for mitotic chromosome separation (as in c, relativeto full length Ki-67) with their size and predicted electrical charge at pH 7.e, Live Ki-67 knockout HeLa cells transfected with indicatedconstructs (quantified in Extended Data Fig.7b). Bars, 10 µm.

Figure 4

Densely grafted Ki-67 molecules have an extended conformation perpendicularto the chromosome surface.

a, Example of a live HeLa cell (from the analysis in c) expressingmCherry-Ki-67-EGFP. Chromosomes oriented perpendicular to the imaging plane(white boxes) were analyzed by line profiles across one sister chromatid (bottompanels; white line) and b, a sum of two Gaussian functions (solidline) was fitted to the line profile to measure peak-to-peak distance of thecorresponding single Gaussians (dotted lines). c, The averagedistance between the labeled Ki-67 polypeptide termini along the axisperpendicular to the chromosome surface was calculated for mCherry-Ki-67-EGFP (n= 53), EGFP-Ki-67-mCherry (n = 52), EGFP-mCherry-Ki-67 (n = 44) andmultispectral fluorescent beads (n = 34). Boxes indicate median, quartiles and1.5 * interquartile range. d, Live nocodazole-treated Ki-67knockout cells expressing different levels of Ki-67-mNeonGreen e,were quantified with reference to chromosome area and Ki-67 levels onchromosomes (each dots represents a cell, 4 independent experiments, dashedlines indicate the median of 39 wildtype and 40 Ki-67 knockout cells).f, Model of Ki-67-mediated mitotic chromosome repulsion. Bars,1 µm in a,10 µm in d.


Author contributions:

D.G. and S.C. conceived the project and designed experiments. B.N. andJ.E generated siRNA library transfection plates. I.P. and A.A.H generatedKi-67-EGFP BAC cell pools. A.Z.P. and J.E. performed FCS measurements. D.G. andT.M.R. performed electron microscopy experiments. S.C. performed all otherexperiments and C.B. assisted in RNAi screen, chromosome spreads, cell linegeneration and cloning. D.G., S.C. and A.A.H. wrote the paper.

Author Information:

Reprints and permissions information is available at The authors declare no competingfinancial interests. Readers are welcome to comment on the online version of thepaper. Correspondence and requests for materials should be addressed to D.G.()


We thank the IMBA/IMP BioOptics microscopy facility, J. Meissner, and M.J. Hossainfor technical support, H. Liu and S. Tietscher for generation of plasmids, C.Haering, M. Samwer, W. H. Gerlich, and O. Wueseke for comments on the manuscript,Life Science Editors for editing assistance, and U. Kutay forLAP2β-GFP/H2B-mRFP-expressing cells. D.G., A.A.H., J.E. have received fundingfrom the European Community’s Seventh Framework Programme FP7/2007-2013 undergrant agreement n° 241548 (MitoSys), and A.Z.P, D.G. and J.E. under grantagreement n° 258068 (Systems Microscopy). D.G. has received funding from anERC Starting Grant under agreement n° 281198 (DIVIMAGE), and from theAustrian Science Fund (FWF) project n° SFB F34-06 (Chromosome Dynamics). S.C.has received funding from a Human Frontier Science Program Long-Term PostdoctoralFellowship and the European Community’s Seventh Framework ProgrammeFP7/2007-2013 under grant agreement n° 330114 (IEF). T.M.R. was supported byDeutsche Forschungsgemeinschaft (DFG): SPP1384 "Mechanisms of GenomeHaploidization" MU 1423/3-2 and grant MU 1423/8-1.


  • 1. KschonsakMHaeringCHShaping mitotic chromosomes: From classical concepts to molecularmechanismsBioessays201537755766[PubMed][Google Scholar]
  • 2. HiranoTCondensin-Based Chromosome Organization from Bacteria toVertebratesCell2016164847857[PubMed][Google Scholar]
  • 3. OhtaSWoodLBukowski-WillsJCRappsilberJEarnshawWCBuilding mitotic chromosomesCurr Opin Cell Biol201123114121[PubMed][Google Scholar]
  • 4. NaumovaNOrganization of the mitotic chromosomeScience2013342948953[PubMed][Google Scholar]
  • 5. Van HooserAAYuhPHealdRThe perichromosomal layerChromosoma2005114377388[PubMed][Google Scholar]
  • 6. OhtaSThe protein composition of mitotic chromosomes determined usingmulticlassifier combinatorial proteomicsCell2010142810821[PubMed][Google Scholar]
  • 7. FullerBGMidzone activation of aurora B in anaphase produces anintracellular phosphorylation gradientNature200845311321136[PubMed][Google Scholar]
  • 8. NeumannBPhenotypic profiling of the human genome by time-lapse microscopyreveals cell division genesNature2010464721727[PubMed][Google Scholar]
  • 9. HericheJKIntegration of biological data by kernels on graph nodes allowsprediction of new genes involved in mitotic chromosomecondensationMol Biol Cell20142525222536[PubMed][Google Scholar]
  • 10. WhitfieldMLGeorgeLKGrantGDPerouCMCommon markers of proliferationNat Rev Cancer2006699106[PubMed][Google Scholar]
  • 11. YerushalmiRWoodsRRavdinPMHayesMMGelmonKAKi67 in breast cancer: prognostic and predictivepotentialThe Lancet Oncology201011174183[PubMed][Google Scholar]
  • 12. SaiwakiTKoteraISasakiMTakagiMYonedaYIn vivo dynamics and kinetics of pKi-67: transition from a mobileto an immobile form at the onset of anaphaseExp Cell Res2005308123134[PubMed][Google Scholar]
  • 13. VerheijenRKi-67 detects a nuclear matrix-associated proliferation-relatedantigen. II. Localization in mitotic cells and association withchromosomesJ Cell Sci198992Pt 4531540[PubMed][Google Scholar]
  • 14. BoothDGKi-67 is a PP1-interacting protein that organises the mitoticchromosome peripheryeLife20143e01641[PubMed][Google Scholar]
  • 15. SobeckiMThe cell proliferation antigen Ki-67 organisesheterochromatineLife20165[PubMed][Google Scholar]
  • 16. VannesteDTakagiMImamotoNVernosIThe role of Hklp2 in the stabilization and maintenance of spindlebipolarityCurr Biol20091917121717[PubMed][Google Scholar]
  • 17. BeaudouinJGerlichDDaigleNEilsREllenbergJNuclear envelope breakdown proceeds by microtubule-inducedtearing of the laminaCell20021088396[PubMed][Google Scholar]
  • 18. CidadoJKi-67 is required for maintenance of cancer stem cells but notcell proliferationOncotarget2016762816293[PubMed][Google Scholar]
  • 19. HofmannKBucherPThe FHA domain: a putative nuclear signalling domain found inprotein kinases and transcription factorsTrends Biochem Sci199520347349[PubMed][Google Scholar]
  • 20. SchluterCThe cell proliferation-associated antigen of antibody Ki-67: avery large, ubiquitous nuclear protein with numerous repeated elements,representing a new kind of cell cycle-maintaining proteinsJ Cell Biol1993123513522[PubMed][Google Scholar]
  • 21. TakagiMMatsuokaYKuriharaTYonedaYChmadrin: a novel Ki-67 antigen-related perichromosomal proteinpossibly implicated in higher order chromatin structureJ Cell Sci1999112Pt 1524632472[PubMed][Google Scholar]
  • 22. TakagiMSueishiMSaiwakiTKametakaAYonedaYA novel nucleolar protein, NIFK, interacts with the forkheadassociated domain of Ki-67 antigen in mitosisJ Biol Chem20012762538625391[PubMed][Google Scholar]
  • 23. RosenMJKunjappuJTSurfactants and Interfacial Phenomena20124thJohn Wiley & Sons
  • 24. MilnerSTPolymer brushesScience1991251905914[PubMed][Google Scholar]
  • 25. HymanAAWeberCAJulicherFLiquid-liquid phase separation in biologyAnnu Rev Cell Dev Biol2014303958[PubMed][Google Scholar]
  • 26. BrangwynneCPMitchisonTJHymanAAActive liquid-like behavior of nucleoli determines their size andshape in Xenopus laevis oocytesProc Natl Acad Sci U S A201110843344339[PubMed][Google Scholar]
  • 27. ZwickerDDeckerMJaenschSHymanAAJulicherFCentrosomes are autocatalytic droplets of pericentriolar materialorganized by centriolesProc Natl Acad Sci U S A2014111E26362645References cited in the method section only[PubMed][Google Scholar]
  • 28. SchmitzMHGerlichDWAutomated live microscopy to study mitotic gene function influorescent reporter cell linesMethods Mol Biol2009545113134[PubMed][Google Scholar]
  • 29. RanFADouble nicking by RNA-guided CRISPR Cas9 for enhanced genomeediting specificityCell201315413801389[PubMed][Google Scholar]
  • 30. GuizettiJCortical constriction during abscission involves helices ofESCRT-III-dependent filamentsScience201133116161620[PubMed][Google Scholar]
  • 31. LukinaviciusGSiR-Hoechst is a far-red DNA stain for live-cellnanoscopyNature communications201568497[Google Scholar]
  • 32. ErfleHWork flow for multiplexing siRNA assays by solid-phase reversetransfection in multiwell platesJournal of biomolecular screening200813575580[PubMed][Google Scholar]
  • 33. HeldMCellCognition: time-resolved phenotype annotation inhigh-throughput live cell imagingNat Methods20107747754[PubMed][Google Scholar]
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