3455Short Report

IntroductionMissegregation of duplicated chromosomes within a bipolar mitosisproduces aneuploid cells, i.e. cells showing a gain or loss of entirechromosomes. One well-known segregation error is chromosomelagging, occurring when a single chromatid remains at the spindleequator after chromosome migration because its kinetochore isconnected to both spindle poles through a merotelic attachment(Cimini et al., 2001). Another segregation error is nondisjunction,i.e. the migration of both sister chromatids to the same pole. Theterm nondisjunction, originally referring to genetically identifiedmeiotic missegregation, has been commonly used to imply a failedseparation of homologous chromosomes or sister chromatids at thecentromere. However, this idea has been brought into question byfluorescence in situ hybridization (FISH) data showing that co-migration of sister chromatids to the same pole occurs in the vastmajority of the cases after centromere separation in somatic culturedcells (Cimini et al., 1999). Furthermore, precocious sister-chromatidseparation due to cohesion defects has been shown to end up assister-chromatid co-migration in yeast (Tanaka et al., 2000; Kitajimaet al., 2004) and premature centromere splitting generates cellaneuploidy in the mosaic variegated aneuploid (MVA) syndrome(Kajii et al., 2001), all implying that there are mechanisms differentfrom retained cohesion in the origin of nondisjunction. In relationto genetic consequences, nondisjunction produces one trisomic

(three copies of a specific chromosome) daughter cell and amonosomic (only one copy of a specific chromosome) one; mostlagging chromosomes will form a micronucleus (a chromatin bodyseparated from the main nucleus) producing a monosomic daughtercell with or without a micronucleus in half of the cases (Ciminiand Degrassi, 2005). Finally, micronuclei can be incorporated inone of the daughter nuclei at the following mitosis producing furtherunbalanced karyotypes (Rizzoni et al., 1989). Monosomic andtrisomic cells might constitute different threats to cell populationsand individuals. All monosomic and most trisomic gametes producenonviable embryos and are, therefore, an important cause ofspontaneous miscarriages and stillbirths in humans. However,chromosome 13, 18 or 21 trisomies are compatible with life andare the cause of severe genetic syndromes (Hassold and Hunt, 2001).In addition, aneuploidy is a nearly ubiquitous feature of humancancers and it has been suggested to play a crucial role both intumour development and progression (Rajagopalan and Lengauer,2004; Weaver and Cleveland, 2006). Most cancer cells display acomplex pattern of chromosomal abnormalities, showing bothchromosome losses and gains, and concomitant structuralaberrations (http://cgap.nci.nih.gov/Chromosomes/Mitelman).Interestingly, recurrent gains or losses of specific chromosomes orchromosome regions (Holloway et al., 1999; Streblow et al., 2007;Vasuri et al., 2008) are frequently observed in tumours, although

Chromosome lagging at anaphase and migration of both sisterchromatids to the same pole, i.e. nondisjunction, are twochromosome-segregation errors producing aneuploid cellprogeny. Here, we developed an assay for the simultaneousdetection of both chromosome-segregation errors in themarsupial PtK1 cell line by using multiplex fluorescence in situhybridization with specific painting probes obtained bychromosome flow sorting. No differential susceptibility of thesix PtK1 chromosomes to undergo nondisjunction and/orchromosome loss was observed in ana-telophase cells recoveringfrom a nocodazole- or a monastrol-induced mitotic arrest,suggesting that the recurrent presence of specific chromosomesin several cancer types reflects selection effects rather thandifferential propensities of specific chromosomes to undergomissegregation. Experiments prolonging metaphase durationduring drug recovery and inhibiting Aurora-B kinase activity

on metaphase-aligned chromosomes provided evidence thatsome type of merotelic orientations was involved in the originof both chromosome-segregation errors. Visualization of mero-syntelic kinetochore-microtubule attachments (a merotelickinetochore in which the thicker microtubule bundle is attachedto the same pole to which the sister kinetochore is connected)identified a peculiar malorientation that might participate in thegeneration of nondisjunction. Our findings imply randommissegregation of chromosomes as the initial event in thegeneration of aneuploidy in mammalian somatic cells.

Supplementary material available online athttp://jcs.biologists.org/cgi/content/full/122/19/3455/DC1

Key words: Chromosome loss, Nondisjunction, Aneuploidy,Kinetochore-microtubule attachments, Merotelic attachments

Summary

Aneuploidy in mitosis of PtK1 cells is generated byrandom loss and nondisjunction of individualchromosomesLiliana Torosantucci1, Marco De Santis Puzzonia1, Chiara Cenciarelli2, Willem Rens3 andFrancesca Degrassi1,*1IBPM Institute of Molecular Biology and Pathology, CNR National Research Council, c/o University ‘La Sapienza’, Via degli Apuli 4, 00185 Rome,Italy2Department of Biology, University Roma Tre, V.le Marconi 446, 00146, Rome, Italy3Centre for Veterinary Science, Department of Veterinary Medicine, University of Cambridge, Cambridge CB3 OES, UK*Author for correspondence (f.degrassi@caspur.it)

Accepted 19 July 2009Journal of Cell Science 122, 3455-3461 Published by The Company of Biologists 2009doi:10.1242/jcs.047944

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the biological significance of these aneusomies in cancer is hard toestablish (Sen, 2000; Albertson et al., 2003). In fact, it is stillunknown whether specific chromosomes have different propensitiesto undergo errors in chromosome segregation or whether theirpresence in cancer cells is the result of cellular selection of cellsbearing random aneuploidy.

In mammalian cell cultures, chromosome loss is observed at lowfrequencies and its frequency is enhanced in cells recovering froma mitotic arrest induced by microtubule-interacting drugs (Ciminiet al., 2001). Much less is known about nondisjunction. Unequivocaldetection of nondisjunction requires a rather complicated assay, inthat segregation of individual chromosomes has to be followed byin situ hybridization on ana-telophase cells. So, very few studieshave investigated nondisjunction in human cells; however, thosethat have nevertheless suggest that nondisjunction occurs atfrequencies higher than chromosome loss (Cimini at al., 1999;Thompson and Compton, 2008). Merotelic kinetochores have beensuggested to produce nondisjunction when more microtubules onthe merotelic kinetochore are connected to the same pole to whichits sister is attached (Salmon et al., 2005). However, due totechnical difficulties in visualizing merotelic attachments, thishypothesis has remained unproven.

To address these issues, we developed an assay for thesimultaneous detection of chromosome loss and nondisjunction forall chromosome pairs in the marsupial PtK1 cell line. This cell linewas chosen because PtK1 cells have been the model system forstudies on chromosome loss at mitosis and possess a very lowchromosome number (2n=12), making easier the analysis ofchromosome segregation for individual chromosomes on anaphasecells. Segregation of individual chromosomes was followed using‘multiplex FISH’ (M-FISH) (Speicher et al., 1996), a special kindof chromosome painting that has been applied to unambiguouslydifferentiate chromosomes in several marsupial species (Rens etal., 2003a; Rens et al., 2006).

Results and DiscussionFor the simultaneous detection of chromosome loss (CL) andnondisjunction (ND) PtK1 chromosome-specific painting probeswere developed from flow-sorted chromosomes. PtK1 chromosomepreparations produced six different peaks in the flow karyotype,corresponding to the six chromosome pairs (Fig. 1A). PtK1chromosome-specific paints with different colours were generatedthrough high purity sorting and degenerate oligonucleotide primed(DOP)-PCR amplification incorporating fluorescence taggeddUTPs. These paints were applied in M-FISH experiments onmetaphase cells to identify each of the six marsupial chromosomepairs in a single metaphase spread by a different colour (Fig. 1B).Furthermore, M-FISH analysis on metaphase cells showed that thesmall chromosome 5 was present in just one copy in this PtK1 cellline, as visible in the reconstructed karyotype (Fig. 1B).

Missegregation events do not occur at a different rate forspecific chromosomesChromosome segregation was then evaluated by M-FISH analysison ana-telophase cells. The high specificity of the chromosomepaints rendered it possible to visualize the position occupied byeach chromosome in the anaphase cell, with the largest chromosome,chromosome 1, protruding towards the periphery of the group ofmigrated chromosomes and the smallest chromosome, chromosome5, in a central position surrounded by the other chromosomes (Fig.2A). Chromosome misdistribution was also easily identified using

M-FISH. The lagging of chromosomes 1 and 3 between the twogroups of chromosomes (Fig. 2B, arrows) as well as the presenceof three copies of chromosome 4 at one pole (Fig. 2B, arrowhead),i.e. an event of ND, could be appreciated by analyzing the imagesfrom the individual filters. With this approach we set out toinvestigate chromosome-specific differences in missegregationrates. ND and CL are rare events in untreated cells; therefore, noclear differences among chromosomes were observed in untreatedcultures, although the extremely low number of events did not allowto draw any statistically sound conclusion (data not shown). Atreatment with the anti-mitotic agents nocodazole (NOC; Fig. 2C)or monastrol (MON; Fig. 2D) was used to increase the frequenciesof ND and CL in ana-telophases harvested after drug release. Toassess whether specific chromosomes were involved at differentrates, we first analyzed the deviation from the expected value ofND or CL calculated by assuming that all chromosomes respondedequivalently (specific chromosome ND = total ND/6). The numberof ND and CL events observed for each chromosome pair was thencompared with the expected value in a χ2 test. This analysis showedno significant deviations from the expected values for both drugs,indicating that no chromosome was preferentially involved in NDor CL. To further investigate this, we performed a different dataanalysis comparing missegregation rates between each chromosome

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Fig. 1. Chromosome characterization of Potorous tridactylus PtK1 cells.(A) Bivariate flow karyotype of PtK1 cells. Chromosomes are separated inindividual peaks corresponding to the six chromosome pairs; each peak isassociated with the corresponding chromosome number (1-5 and X).(B) Example of chromosome painting on a PtK1 metaphase. Chromosomepairs, shown in different colours, are indicated below; the unpairedchromosome 5 is present in single copy. Scale bar: 10 μm.

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and every other one. This analysis revealed that chromosomes 1and 5, which represent the largest and smallest chromosome in PtK1cells, respectively, showed lower ND frequencies compared withthose observed for all other chromosomes after NOC treatment(chromosome 1: P<0.05; chromosome 5: P<0.01). This lowrepresentation of the two chromosomes was not observed for CL:in this case, chromosomes 1 and 5 did not show any significantdifference from the other chromosomes. Analysis of chromosomesusceptibility to missegregate after MON treatment showed thatchromosome missegregation rates were homogeneous for all of thechromosomes, the only exception being that the CL frequency forchromosome 4 was significantly lower than that observed for theother chromosomes (P<0.01). In conclusion, statistically significantdifferences between chromosomes were limited and did not permitthe identification of any chromosome-specific tendency because

they were not homogeneous neither for the chromosome involved,nor for the type of missegregation analyzed (ND vs CL), nor werethey related to a specific inducing drug. Collectively, these dataindicate that the six PtK1 chromosomes undergo ND and/or CL tothe same extent. Furthermore, they suggest that correction ofkinetochore malorientations, which involves the Aurora-B-dependent phosphorylation of the depolymerizing kinesin MCAK(Kline-Smith et al., 2004; Lan et al., 2004; Knowlton et al., 2006;Andrews et al., 2004) together with the activity of the Kif2b kinesin(Bakhoum et al., 2008), and microtubule flux (Ganem et al., 2005),is equally efficient on the different chromosomes, producing randomchromosome missegregation at mitosis.

Among the many factors that have been proposed to influence thesusceptibility of individual chromosomes to missegregate,chromosome size and chromosome position within the mitotic

Fig. 2. Chromosome-segregation errors occur at similar rates for the six PtK1 chromosome pairs. (A) Representative anaphase from untreated PtK1 cells showingproper chromosome segregation. Images from individual filters show that two copies (one copy for chromosome 5) of each chromosome have migrated to eachpole. (B) Example of chromosome missegregation after NOC treatment. M-FISH shows ND for chromosome 4, i.e. three intact copies at one pole and just one atthe other pole (painted blue; arrowhead), and loss of chromosome 1 and 3 (painted red and light blue, respectively; arrows). Scale bar: 10 μm (in A for A and B).(C,D) Frequencies of ND and CL events for individual chromosomes in ana-telophases recovering from (C) a NOC-induced mitotic block or (D) a MON-inducedmitotic block. At least 600 cells in three independent experiments were counted for each treatment. Chromosome-5 missegregation frequencies are presenteddoubled because chromosome 5 is present in one copy in this PtK1 cell line.

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spindle are regarded as crucial. Our data on anaphase cells (Fig. 2A;supplementary material Fig. S1) as well as previous live imagingdata on the same cell line (Cimini et al., 2004) provide evidence thatlarger chromosomes tend to position at the metaphase-plate periphery,possibly owing to a steric hindrance of the large chromosome arms.In this area of the spindle, chromosome oscillations tend to be shorterand less frequent (Khodjakov and Rieder, 1996; Cimini et al., 2004),which might be due to reduced directional instability mediated byaccumulation of the Kif18a kinesin on peripheral kinetochores(Stumpff et al., 2008). Reduced chromosome oscillations couldtheoretically decrease the correction of erroneous kinetochore-microtubule attachments, which requires the release of microtubulesfrom kinetochores and their successive interaction at a different angle(Lampson et al., 2004). Our data indicate that chromosome oscillationsat the metaphase plate do not affect missegregation rates of individualchromosomes but rather suggest a similar correction efficiencyamong the different kinetochores.

It is well known that random chromosome missegregation occursin meiosis. Aneuploidies for nearly all chromosomes are representedamong spontaneous abortions at very early stages of development,but only trisomies for a few chromosomes are vital at birth,demonstrating that meiotic aneuploidies are subjected to prenatalselection (Hassold and Hunt, 2001). Our data suggest a similarscenario for mitotic cells, in which the recurrent presence of specificchromosomes in several cancer types is not the result of a differentpropensity of specific chromosomes to undergo missegregation butis the outcome of a selection process that depends on the identity ofthe extra chromosome. In line with this, a recent study has shownthat aneuploid murine embryonic fibroblasts (MEFs) containing anextra copy of only one chromosome exhibit decreased proliferationrates and decreased cell fitness (Williams et al., 2008). However, thetiming of cell immortalization, one of the early steps in celltransformation in culture, depended on which additional chromosomewas present in the different cell lines (Williams et al., 2008). Thus,it can be hypothesized that, although the presence of an extrachromosome is generally detrimental, a limited number of specificunbalances of gene products caused by individual chromosomeaneuploidy, possibly in conjunction with additional mutations inoncogenes and/or tumour suppressors, might promote tumorigenesis.

Merotelic attachments and nondisjunctionHaving established that no preferential involvement in chromosomemissegregation occurs for specific chromosomes, we then wished toobtain new insights into the mechanism(s) underlying ND. For correctsegregation to occur, sister kinetochores must attach to microtubulesconnected to opposite poles at metaphase. This so-called amphitelicorientation satisfies the mitotic-checkpoint requirements and allowsthe segregation of the two sisters to opposite poles. Other orientationsare also possible and are transiently present during prometaphase,including monotelic (a chromosome with only one kinetochoreattached to microtubules), syntelic (both sister kinetochores connectedto the same pole) and merotelic orientations, the latter being the causefor chromosome lagging when persisting at anaphase (Cimini andDegrassi, 2005). To gain insight into the role of these differentorientations in the generation of ND, we took advantage of thedifferent mechanisms of action of NOC and MON. NOC is a well-known spindle poison capable of disassembling the mitotic spindle.Following NOC removal, the mitotic spindle reassembles throughboth microtubule nucleation from centrosomes and incorporation intothe centrosomal-based microtubule arrays of microtubules nucleatedat kinetochores (Tulu et al., 2006; Torosantucci et al., 2008). During

this process, several extra-centrosomal foci of α-tubulin are presentat very short times after NOC removal (Cimini et al., 2003; Tulu etal., 2006) (supplementary material Fig. S2); these foci favour theformation of high numbers of incorrect kinetochore-microtubuleinteractions, in large part merotelic attachments. MON is a small-molecule inhibitor of the mitotic kinesin Eg5 that is able to arrestcells in mitosis with monoastral spindles; chromosomes in MON-treated cells frequently exhibit sister kinetochores attached tomicrotubules in a syntelic orientation (Kapoor et al., 2000). UponMON removal, syntelic orientations are transformed into amphitelicones through shortage and severing of the syntelic K-fibres andsuccessive attachment of other microtubules at the kinetochores(Lampson et al., 2004) (supplementary material Fig. S2). During thisprocess, several types of mis-attachment are produced.

Missegregation for all PtK1 chromosomes was analyzed inuntreated cells and in cells undergoing ana-telophase 45 or 60minutes after NOC or MON release (Fig. 3). Approximately 6%of untreated PtK1 ana-telophase cells underwent chromosomemissegregation (35 events/569 cells), showing 4.0% ND and 2.1%CL when controls from MON and NOC experiments were pooledtogether. This suggests that ND occurs at higher rates in comparisonwith CL in somatic mammalian cells, as previously observed inhuman primary fibroblasts (Cimini et al., 1999). ND and CL wereefficiently induced over control values in cells harvested 45minutes after NOC release (Fig. 3A) (P<0.005) with anapproximate twofold induction of ND in comparison with CL(28.1% ND vs 12.6% CL). Ana-telophases recovering from MONtreatment exhibited similar rates of missegregation (19.6% ND vs13.1% CL), demonstrating that MON also efficiently induceschromosome missegregation (Fig. 3B) (P<0.001). Cells that wereharvested 60 minutes after drug release displayed lower frequenciesof CL and ND, both after NOC (Fig. 3A) and after MON (Fig.3B) treatment, suggesting that the correction of faulty attachmentsduring metaphase decreases the frequencies of CL and ND for bothdrugs. Merotelic kinetochore attachments occur frequently in earlymitosis and are partially corrected before anaphase onset whenmetaphase is prolonged using the proteasome inhibitor MG132

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Fig. 3. Chromosome-segregation errors are efficiently induced following bothNOC or MON treatment. (A,B) ND and CL frequencies in DMSO-treated ana-telophases (DMSO) and in ana-telophase cells fixed after 45 or 60 minutes ofNOC washout (A; NOC+45 and NOC+60, respectively) or MON washout (B;MON+45 and MON+60, respectively) both without (black bars, –MG132) andwith (grey bars, +MG132) a 2-hour incubation in MG132 after drug release.Between 200 and 400 cells were counted for each treatment from threeindependent experiments.

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(Cimini et al., 2003). We also delayed anaphase onset by MG132treatment to determine whether this had an effect on ND. Thefrequencies of ND were efficiently decreased by incubation inMG132 medium during recovery from either drug (P<0.001 andP<0.05 at 45 and 60 minutes, respectively), although they neverreached control levels (Fig. 3A,B). CL frequencies were alsodecreased (Fig. 3A,B), although to a lesser extent (P<0.05 whendata from both times were pooled together). Altogether, these dataindicate that a fraction of the faulty attachments responsible forND is corrected before anaphase, as already observed for laggingchromosomes (Cimini et al., 2003).

Correction of merotelic attachments before anaphase requiresAurora-B kinase activity as part of an inter-kinetochore tension-sensing mechanism (Kline-Smith et al., 2004; Lan et al., 2004;Knowlton et al., 2006; Andrews et al., 2004; Cimini et al., 2006).We therefore investigated whether Aurora-B activity was responsiblefor the observed time-dependent correction of ND by usinghesperadin, a known Aurora-B inhibitor (Hauf et al., 2003). Theinhibitor was used at a dose (100 nM) that only partially suppressedAurora-B activity, as determined by histone H3 (Ser10)phosphorylation (Fig. 4A,B), because partial inhibition of the kinaseactivity had already been shown to increase merotelic orientations

Fig. 4. Partial inhibition of Aurora-B activity byhesperadin increases missegregation. (A) Inhibitionof Aurora-B activity as assayed by microscopeanalysis of anti-histone-H3 (Ser10) phospho-antibody staining (Phospho-H3). (B) Phospho-H3antibody staining in DMSO-treated prometaphasecells (DMSO) or prometaphase cells after 1-hourincubation in 20 nM or 100 nM hesperadin (hesp)alone, or during the last hour of MG132 incubationof NOC-released cells. At least 100 cells from twoindependent experiments were counted for eachtreatment. (C) ND or CL frequencies in control ana-telophases (DMSO) or in ana-telophases recoveringfrom a NOC-induced mitotic block (NOC) in drug-free medium (black bars, –MG132,), after a 2-hourincubation in MG132 (grey bars, +MG132) or incells incubated in 100 nM hesperadin during thelast hour of MG132 treatment (blue bars,+MG132/hesp). Data are the sum of two-threeindependent experiments. (D) Different types ofmerotelic kinetochores are observed after NOCtreatment. Maximum projections from deconvolvedthree-dimensional (3D) image stacks showing outerkinetochore (Hec1, red), centromere (CREST, blue)and spindle microtubule (α-tubulin, green)immunofluorescence are shown. The reciprocalarrangement of outer-kinetochore and centromeresignals on single z-slices coupled with Hec1distortion was used to identify sister kinetochoresand kinetochore-microtubule interactions.Kinetochore-microtubule interactions were verifiedby rotating reconstructed 3D images. The followingtypes of merotelic orientations (a single kinetochoreattached to two microtubule bundles) wereobserved: mero-syntelic (the thicker microtubulebundle is towards the same pole to which the sisterkinetochore is connected), mero-amphitelic (thethicker microtubule bundle is connected to the poleopposite to that of the sister kinetochore) andbalanced-merotelic (the fluorescence intensity ofthe two microtubule bundles attached at the singlekinetochore is similar). Insets show a twofoldmagnification of the boxed regions. The differentorientations are diagrammed below the insets. Scalebars: 10 μm.

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and lagging chromosomes at anaphase without influencing otheraspects of mitosis such as checkpoint efficiency or cytokinesis (Ciminiet al., 2006). To investigate whether merotelic attachments areinvolved in ND, cells were released from a NOC-induced mitoticarrest in MG132 medium and hesperadin was added 1 hour later. Insuch a way, Aurora-B activity was inhibited when chromosomes hadalready reached the metaphase configuration in the vast majority ofthe cells (supplementary material Fig. S3), a moment when syntelicattachments, which maintain chromosomes close to the poles (Ciminiet al., 2002; Cimini and Degrassi, 2005), were already corrected. Cellswere kept in MG132 and hesperadin for a further 1 hour and thenallowed to progress to anaphase by medium change. Inhibition ofAurora B in this time window very efficiently increased CL frequencyabove the value observed with MG132 alone (Fig. 4C), as alreadyreported (Cimini et al., 2006). Interestingly, correction of improperattachments generating ND was also inhibited, as shown by theincreased frequencies of ND above the MG132 value (Fig. 4C)(P=0.126). These data indicate that improper kinetochore-microtubuleattachments generating ND are present on aligned chromosomes andthat Aurora-B activity is required for their correction.

So, which type of kinetochore-microtubule interactions producesND? Anaphase onset in the presence of syntelic attachments wouldproduce chromosome ND, because both sister kinetochores areconnected to the same pole. However, several lines of evidenceconverge to demonstrate that syntelic attachments activate themitotic checkpoint (Pinsky and Biggins, 2005; Porter et al., 2007),rendering it less likely that syntelic chromosomes will undergo NDin checkpoint-proficient cells. In our data, the high ND frequenciesobtained after inducing large amounts of merotelic orientations(NOC treatment) (Fig. 3A) as compared with preferentially inducingsyntelic orientations (MON treatment) (Fig. 3B), as well as the effectof Aurora-B inhibition on aligned chromosomes (Fig. 4C), suggestthat kinetochore-microtubule interactions that are different fromsyntelic attachments, which localize chromosomes close to thespindle poles, also contribute to ND. Monotelic orientations are veryunlikely to generate ND. Even if monotelic chromosomes were ableto reach the metaphase plate by sliding on already existing K-fibres(Kapoor et al., 2006), the unattached kinetochore would stronglyactivate the mitotic checkpoint, blocking anaphase onset. Merotelicchromosomes congress to the metaphase plate (Cimini et al., 2002).They have been shown to maintain connection to the twomicrotubule bundles during anaphase and lag at the cell equatorwhen the two bundles are of similar size (balanced merotelic) ormove to the pole connected to the thicker microtubule bundle(Cimini et al., 2004). In principle, a merotelically orientedkinetochore, in which the thicker microtubule bundle is connectedto the same pole to which the sister is attached, would segregate tothe same pole as the sister. These so-called ‘mero-syntelic’attachments have been shown to be responsible for ND in live crane-fly meiosis (Janicke at al., 2007), a favourable system to detectkinetochore-microtubule interactions, owing to the large dimensionof the cells. To identify these different merotelic attachments inPtK1 cells, we analyzed Ca2+-resistant kinetochore-microtubuleinteractions after z-stack acquisition and image deconvolution ofprometa-metaphase cells, which were imaged to visualize thecentromere, the outer kinetochore region and microtubules usingCREST, Hec1 and tubulin antibodies, respectively (Fig. 4D). Incultured mammalian cells, clear identification of kinetochore-microtubule interactions is restricted to the peripheral area of thespindle because of the high density of microtubules into the centralarea. Taking this into account, we were nevertheless able to show

that, in addition to syntelic and amphitelic attachments (not shown),both balanced merotelic attachments, mero-amphitelics (a merotelickinetochore with the thicker MT bundle connected to the poleopposite to that of the sister kinetochore) and mero-syntelics (thethicker microtubule bundle towards the pole to which the sisterkinetochore is connected) are present during recovery from NOC(Fig. 4D). In our microscopic analysis, about 10% of aberrantorientations were mero-syntelics (4/45). So, by visualizing mero-syntelic attachments we identified a peculiar kinetochore-microtubule interaction that might be involved in the origin of NDin mammalian somatic cells. However, further high-resolution live-cell studies or electron-microscopy evidence are required tounequivocally identify mero-syntelics as being responsible for ND.

In conclusion, our M-FISH studies in conjunction with the analysisof kinetochore-microtubule interactions provide evidence indicatingthat ND and CL are generated by different types of merotelicattachments. These might originate during the extremely dynamicprocess of centrosome separation and microtubule growth that occursduring recovery from drug treatment. The position of centromeres inrelation to centrosomes during the initial stages of this process seemsto be a stronger determinant for the formation of incorrect interactionswith respect to structural differences at kinetochores among thedifferent chromosome pairs, as we did not observe a preferentialinvolvement of specific chromosomes in segregation errors. Finally,our findings imply random missegregation of chromosomes as theinitial event in the generation of aneuploidy and in the acquisition orloss of specific chromosomes in cancer.

Materials and MethodsCell culture and treatmentsPtK1 cells were grown as described (Rens et al., 1999). NOC, MON and MG132(Sigma-Aldrich) were dissolved in DMSO. To promote chromosome missegregation,cells were incubated in 2 μM NOC for 3 hours or 100 μM MON for 4 hours.Thereafter, cells were washed three times in pre-warmed medium to release the mitoticblock, re-incubated in fresh medium and fixed at the indicated times after drug release.In MG132 experiments, 15-25 minutes after NOC or MON release, cells wereincubated in 7 μM MG132 for 2 hours, then washed three times for 5 minutes inpre-warmed medium and harvested after a further 45 or 60 minutes. 100 nM of theAurora-B kinase inhibitor hesperadin (a generous gift of Tarun Kapoor, TheRockefeller University, NY) was supplied to some cultures for the last hour of MG132treatment. Medium was then exchanged to remove hesperadin and MG132, and cellswere fixed after 45 or 60 minutes.

M-FISH analysisPtK1 chromosome preparation and flow sorting were performed as previouslydescribed (Rens et al., 1999). Flow-sorted chromosomes were used as templates forDNA amplification by DOP-PCR (Telenius et al., 1992) and primary DOP-PCRproducts were used to incorporate biotin-16–dUTP (Roche), FITC-dUTP, Cy5-dUTP,Cy3-dUTP (Amersham), Texas-Red–dUTP (Molecular Probes) and DEAC-dUTP(NEN Life Science Products) by PCR. FISH was performed as previously described(Rens et al., 2003b), except that chromosome paints were denatured at 70°C for 10minutes and slides were denatured at 55°C for 10 seconds. The biotin-labelled probewas visualized by Cy5.5-avidin (Amersham). After detection, slides werecounterstained in 0.08 μg/ml DAPI in 2�SSC for 2 minutes and mounted in ProLongAntifade (Molecular Probes).

ImmunostainingFor the analysis of Ca2+-resistant kinetochore-microtubule interactions, slides werefixed as described (Kapoor et al., 2000) except that they were post-fixed in methanol.Primary antibodies were: CREST serum (Antibodies Incorporated, 1:50), anti-α-tubulin FITC-conjugate (Sigma-Aldrich, 1:100) and anti-Hec1 (Abcam, 1:500). Forphospho-H3 staining, cells were fixed in ice-cold methanol for 5 minutes,permeabilized 5 minutes in PBS/0.5% Triton-X and then incubated with anti-Ser10-phospho-H3 antibody (Upstate Biotechnology, 1:100). DNA was counterstained in0.1 μg/ml DAPI.

Fluorescence microscopy and image acquisitionM-FISH images were captured using Leica QFISH software (Leica Microsystems)and a cooled Sensys CCD camera (Photometrics) mounted on a Leica DMRXAmicroscope equipped with six specific filter sets and a 63� (1.3 NA) objective.

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Lagging chromosomes and ND were scored on anaphases with fully migratedchromosomes (mid-anaphase), late anaphases and telophases. Chromosomes wereconsidered lagging if they were located inside the central 25% of the pole distanceafter all chromosomes had migrated. Between 133-413 ana-telophases were scoredfor each experimental point and data were analyzed using the χ2 test.Immunofluorescence analysis of kinetochore orientation was carried out under anOlympus Vanox microscope equipped with a 100� (1.35 NA) objective and a SPOTCCD camera (Diagnostic Instruments). Colour-encoded images were acquired usingISO 2000 software (Deltasistemi). Three dimensional deconvolution andreconstruction was performed on 0.3-μm-interval z-stacks of optical sections usingAutoDeblur 9.3 (AutoQuant Imaging) software.

We thank Tarun Kapoor for generously providing hesperadin, andDaniela Cimini for many valuable suggestions and comments on thiswork. This work was partially supported by grants from CNR (RTL2007) and from AIRC.

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