mitotic phosphoepitopes are expressed in kc cells ... · were observed with a bio-rad mrc600...

1979Journal of Cell Science 110, 1979-1988 (1997)Printed in Great Britain © The Company of Biologists Limited 1997JCS4361

Mitotic phosphoepitopes are expressed in Kc cells, neuroblasts and isolated

chromosomes of Drosophila melanogaster

Hassan Bousbaa1, Luis Correia1, Gary J. Gorbsky2 and Claudio E. Sunkel1,3,*1Centro de Citologia Experimental da Universidade do Porto, Rua do Campo Alegre 823, 4150 Porto, Portugal2Health Sciences Center, Department of Cell Biology, University of Virginia, Charlottesville, Virginia 22908, USA3Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto, Largo do Prof. Abel Salazar Nº2, 4000 Porto, Portugal

*Author for correspondance (e-mail: [email protected])

The progression of cells from metaphase to anaphase isthought to be regulated by a checkpoint that delays entryinto anaphase until all chromosomes reach a stable bi-polarattachment at the metaphase plate. Previous work hassuggested that the 3F3/2 kinetochore phosphoepitopes areinvolved in this checkpoint system. We show that the 3F3/2centromere phosphoepitopes are present in Kc cells, thirdinstar larval neuroblasts and isolated chromosomes ofDrosophila melanogaster. In tissue culture cells and neuro-blasts isolated from third instar larvae, centromerelabelling is detected from early prophase to the metaphase-anaphase transition but absent once cells enter anaphase.During anaphase, the antibody stains the spindle mid zoneand during telophase the midbody is labelled until cellsseparate. In both cell types, the 3F3/2 antibody stains thecentrosome from prophase to late telophase. The 3F3/2

staining is retained in Kc cells and third instar larvalneuroblasts arrested at the prometaphase state with micro-tubule inhibitors. Also, two mitotic mutants that showabnormal spindle morphology retain the centromerelabelling in a metaphase-like configuration, suggesting thatthey activate the metaphase-anaphase checkpoint. Finally,mitotic chromosomes isolated in the presence of a phos-phatase inhibitor show phosphoepitopes at the primaryconstriction on the surface of each chromatid, however,chromosomes isolated in the absence of a phosphataseinhibitor do not. Incubation of these chromosomes withATP causes the rephosphorylation of the phosphoepitopesat the centromere.

Key words: 3F3 antibody, Centromere, Centrosome, Mitoticcheckpoint, Drosophila

SUMMARY

INTRODUCTION

During early stages of mitosis in higher eukaryotic cells, thefully matured kinetochores capture spindle microtubules. Amajor consequence of this process is that pole-to-kinetochoremicrotubules become stabilised. This is a pre-requisite for theorganisation and maintenance of a proper metaphase configu-ration that is thought to involve the balance of forces appliedto sister kinetochores. Once all the chromosomes have estab-lished a stable bi-polar orientation, cells can proceed intoanaphase. If a single chromosome is unable to establish properbi-polar orientation either by chance or by micromanipulation(Nicklas, 1967), cells arrest at prometaphase until the mono-oriented chromosome assumes an appropriate bi-polar config-uration. Similarly, when microtubule depolymerizing drugs areused prior to the initiation of anaphase, cells arrest atprometaphase with highly condensed chromosomes that aredispersed within the cell. Studies have shown that most, but notall, cell types can arrest for long periods with sister chromatidsremaining paired (Gonzalez et al., 1991; Carmena et al., 1993;reviewed by Rieder, 1995). In many cell types, when the drugis removed spindle microtubules re-polymerise and a normalmetaphase configuration is attained, the metaphase arrest isrelieved and anaphase can proceed. It was proposed that part

of this mechanism might involve a signal emanated from thechromosomes or the spindle preventing cells with an abnormalmetaphase configuration from entering anaphase. The activa-tion of the metaphase-anaphase checkpoint would prolong theprometaphase configuration allowing all chromosomes tobecome properly attached to both spindle poles (Zirkle, 1970).

In a series of micromanipulation experiments in insectmeiosis I spermatocytes, Nicklas and Koch (1969) showed thatkinetochores could sense tension. Applying tension to a freeunpaired X chromosome forces the cell to enter anaphase withimproperly segregated chromosomes (Li and Nicklas, 1995).In mammalian cells in culture, a single mono-oriented chro-mosome also delays anaphase onset. However, this block couldbe removed if the unattached kinetochore is destroyed with alaser microbeam (Rieder et al., 1995), thus providing substan-tial support for the model in which mal-oriented chromosomessend an inhibitory signal that activates the metaphase-anaphasecheckpoint (McIntosh, 1991).

Further evidence that kinetochore components wereinvolved in the metaphase-anaphase checkpoint was obtainedwhen mammalian cells were stained for immunofluorescencewith the 3F3/2 monoclonal antibody (Gorbsky and Ricketts,1993). This antibody recognises the epitopes in the phospho-rylated form (Campbell and Gorbsky, 1995) and can be

1980 H. Bousbaa and others

detected if cells are prepared for immunofluorescence in thepresence of phosphatase inhibitors. Originally, the antibodyhad been prepared against cell extracts treated with ATP-γ-S(Cyert et al., 1988). The 3F3/2 antibody stains preferentiallykinetochores that are not under tension, while the kinetochoresthat are attached to microtubules become progressively lessstained. More direct evidence for involvement of the dephos-phorylation of the 3F3/2 epitopes in the initiation of anaphasewas obtained by injection of the antibody into culture cells.Those results indicated that the antibody did not impair con-gression but delayed entry into anaphase (Cambell andGorbsky, 1995). Since chromosomes that become properlyoriented lose the 3F3/2 kinetochore phosphoepitopes, it wasproposed that the phosphorylated epitopes of misaligned chro-mosome participate in a signalling pathway that activates thecheckpoint inducing the delay at metaphase. A direct relation-ship between tension at the kinetochore and dephosphorylationof the 3F3/2 epitopes was established by combining microma-nipulation of meiotic chromosomes in spermatocytes andimmunofluorescence to detect the kinetochore phospho-epitopes (Nicklas et al., 1995). Mono-oriented chromosomeswere shown to retain the 3F3/2 phosphoepitopes on the kine-tochores and delay anaphase onset. However, if tension isapplied to the mono-oriented chromosome, the 3F3/2 phos-phoepitope gradually diminishes from the kinetochores andafter some time anaphase starts. These results strongly suggestthat tension alters the phosphorylation state of kinetochorecomponents and this in turn, removes the inhibitory signal thatactivates the metaphase-anaphase checkpoint.

The activation of the metaphase-anaphase checkpoint is likelyto involve a number of other gene products. In Saccharomycescerevisiae, the bub (budding uninhibited by benzimidazole; Hoytet al., 1991) and mad (mitotic arrest deficient; Li and Murray,1991) mutants were isolated by their inability to delay celldivision in the presence of microtubule depolymerizing agents.Both mad and bub genes are expected to encode components ofa signalling mechanism that detects abnormal spindle structure,ultimately inhibiting the machinery that initiates sister chromatidseparation and anaphase. Recently, the frog (XMAD2) andhuman (hsMAD2) homologues of MAD2 were isolated andshown to be present on unattached kinetochores even though alarge soluble pool persists in the cytoplasm (Chen et al., 1996;Li and Benezra, 1996). In both organisms, MAD2 is animportant component of the spindle assembly checkpoint.

In this study, we used the 3F3/2 antibody to stain DrosophilaKc cells in culture and third instar larval neuroblasts. Theantibody specifically recognised components of the centromerefrom prophase to early metaphase, and the centrosome fromearly prophase to late telophase. The immunofluorescencepattern of 3F3/2 phosphoepitopes in the neuroblasts of twoDrosophila mitotic mutants that alter chromosome segregationis also described. Finally, we showed that dephosphorylated3F3/2 centromere epitopes can be rephosphorylated on isolatedchromosomes.

MATERIALS AND METHODS

Kc cell culture and indirect immunofluorescenceDrosophila Kc cells were cultured at 25°C in D-22 insect medium(Sigma, USA) supplemented with 5% FCS (Gibco BRL), penicillin,

streptomycin and fungycin (Gibco BRL). For immunofluorescence,cells were grown on glass coverslips at a density of 106 cells/ml. Toarrest cells in mitosis, either 50 µM taxol or 25 µM colchicine (Sigma,USA) were added to cells during the late exponential growth phaseand incubated for a further 16 hours. For immunofluorescence, ascultured Kc cells do not adhere to coverslips, they were treated as pre-viously described (Gorbsky and Ricketts, 1993), except that CHAPSdetergent was substituted by Triton X-100. Briefly cells were simul-taneously lysed and fixed in lysis/fixative buffer (1.5× PHEM, 2%Triton X-100, 0.15% glutaraldehyde, 2% formaldehyde, 10 µMmicrocystin LR (Sigma, USA) by adding it directly to the culturedishes. Unless otherwise stated all immunofluorescence staining wasdone in the presence of microcystin. Coverslips were washed inMBST (10 mM MOPS, pH 7.4, 150 mM NaCl, 0.05% Tween-20) andafter 30 minutes blocking in 10% FCS in MBS, were incubated for 1hour with the 3F3/2 monoclonal antibody diluted 1:1,000 in MBSwith 10% boiled FCS. Coverslips were washed in MBST andincubated with fluorescein-conjugated anti-mouse IgG antibody(Amersham, UK) at 1:200 in MBS, containing 10% FCS, for 45minutes at 25ºC. The coverslips were washed in MBST, followed byMBS alone and the DNA labelled with 1 µg/ml propidium iodide.Coverslips were mounted in vectashield (Vector Labs, UK). All incu-bations were performed at 25ºC. For immunofluorescence, freshlyprepared isolated mitotic chromosomes (see below) were allowed toadhere to the slide for 10 minutes in the presence of 1% bovine serumalbumin (Sigma, USA) at 25ºC. After washing in PHEM (60 mMPipes, 25 mM Hepes, 10 mM EGTA, 4 mM MgSO4), chromosomeswere fixed for 10 minutes in PHEM containing 1% formaldehyde,0.075% glutaraldehyde and 0.1 µM microcystin LR. Immunofluor-escence was done as described for whole cells.

Neuroblast preparations and immunostainingThird-instar larvae, grown at 25°C, were washed in saline (0.7%NaCl) and dissected in NTM (0.7% NaCl, 5 µM taxol, 10 µM micro-cystin). Isolated brains were permeabilized for 15 seconds in NTMT(NTM and 0.3% Triton X-100) and fixed for 15 minutes in 3.7%formaldehyde in NTM, from a 37% stock solution. Brains were rinsedrapidly in 0.7% NaCl, and blocked in NFT (0.7% NaCl, 10% boiledFCS, 0.1% Triton X-100). Immunostaining was done with 3F3/2antibody at 1:1,000 dilution in NFT for 16 hours at 4°C in the presenceof 2.5 µg/ml of DNase free RNase. Whole brains were washed 4 timesfor 15 minutes in NFT at 25ºC and incubated for 2 hours in the secondantibody diluted 1:200 in NFT at 25ºC. The tissue was washed 4 timesfor 15 minutes in NFT, once in 0.7% NaCl for 5 minutes and stainedfor DNA for 2 minutes in 1 µg/ml propidium iodide in 0.7% NaCl.Finally, the tissue was transferred to a drop of Vectashield containing1 µg/ml propidium iodide. All preparations for immunofluorescencewere observed with a Bio-Rad MRC600 confocal laser microscopeand image analysis was done using the COMOS software.

Isolation of mitotic chromosomesChromosomes were prepared following the method of Gasser andLaemmli (1987) as modified by Taagepera et al. (1993). Briefly, 50ml Kc cell culture (106/ml) were incubated for 16 to 48 hoursdepending on individual cultures, with 25 µM colchicine. Cells werecollected by centrifugation at 2,000 rpm for 5 minutes and resus-pended in swelling buffer (15 mM Tris-Cl, pH 7.4, 80 mM KCl, 5mM EGTA, 2 mM K-EDTA, 1 mM spermidine, 0.5 mM spermine,40 nM microcystin LR, 0.1 mM PMSF, 1 µg/ml each of aprotinin andleupeptin). After 5 minutes incubation at room temperature, 0.1%digitonin was added and cells were lysed with a Dounce hom-ogenizer. Nuclei and cell debris were pelleted by centrifugation at1,000 rpm for 5 minutes and the supernatant layered over a glycerolgradient of 40, 60 and 80% in 5 mM Tris-Cl, pH 7.4, 2 mM KCl, 2mM K-EDTA, 2 mM spermidine, 100 nM microcystin LR, PMSF andprotease inhibitors. The gradient was centrifuged in a swinging bucketrotor at 4,000 g for 20 minutes. Chromosomes were collected from

19813F3 phosphoepitopes in Drosophila

within the 60% glycerol gradient fraction, used immediately or frozenin liquid nitrogen and stored at −80ºC.

Rephosphorylation of the 3F3/2 centromere epitopeChromosomes were isolated as described above except that micro-cystin was removed from all buffers. They were washed in glycerolgradient buffer and resuspended in kinase buffer (10 mM Hepes, pH7.5, 75 mM KCl, 5 mM MgCl2, 2 mM DTT, 1 mM EGTA). To testfor phosphorylation, a sample of the chromosome suspension wasincubated with 1 mM ATP in the presence or absence of 10 µM micro-cystin for 20 minutes or 60 minutes at 25ºC. In some cases, 2 mM ofN-ethylmaleimide (NEM), a sulfhydryl reactant that interferes withkinase activity, were added to the rephosphorylation assay. Chromo-somes were then immunostained with the 3F3/2 antibody as describedabove.

RESULTS

Detection of 3F3/2 phosphoepitope in Kc cellsIn mammalian cells and grasshopper spermatocytes, the 3F3/2antibody recognises several cellular phosphoproteins thatcontain the 3F3/2 epitope. Therefore, high detergent extractionwas needed to visualise cellular structures such as centromeresthat contain 3F3/2 phosphoepitopes. This extraction was donesimultaneously with fixation to allow adherence of Kc cells tocoverslips. This procedure did not appear to affect cell mor-phology and structure. Immunostaining with anti-β-tubulinantibody showed normal spindle morphology in cells under-going division (Fig. 1A-C).

The full preservation of the 3F3/2 epitopes required the

Fig. 1. Immunolabelling of Kc cells inexponential cultures with an anti-β-tubulinantibody (A-C) or 3F3/2 antibody (D-F).All preparations were counterstained withpropidium iodide to visualize DNA which isshown in red. Cells labelled with β-tubulin(A-C) in green, show a normal metaphasespindle configuration with all chromosomesaligned at the metaphase plate (A), theelongated spindle microtubules during lateanaphase (B) and the microtubule stainingat either side of the midbody in latetelophase (C). Cells prepared in the absenceof microcystin and labelled with the 3F3/2antibody show a weak staining (green) atprophase (D), prometaphase (E) andmetaphase (F). Bars, 5 µm.

presence of the phosphatase inhibitor microcystin duringextraction and fixation. However, unlike mammalian cells, inKc cells a low level of staining was still detected in mostmitotic cells (>80%) independently of their mitotic stage (seeFig. 1D-F and compare with Fig. 2), even in the absence ofmicrocystin. This residual staining was also described forgrasshopper spermatocytes (Nicklas et al., 1995).

Cell cycle distribution of 3F3/2 phosphoepitopes inKc cellsIn cells treated with the phosphatase inhibitor, the 3F3/2antibody labelled the centromeres, centrosomes and midbodyduring mitosis. The staining pattern of the 3F3/2 antibodyduring different cell cycle stages can be seen in Fig. 2 (seeTable 1 for quantification). Most interphase cells did not showany labelling, however, a small proportion showed diffuseoverall staining of the nucleus (Fig. 2A). During prophase,3F3/2 staining was seen over discrete areas of the chromo-somes (Fig. 2B). In prometaphase cells, the two sister chro-matids of each chromosome were rarely individualized butcentromere staining of the 3F3/2 phosphoepitopes was seenassociated with the primary constriction of most chromosomes(Fig. 2C). During metaphase, most cells (75%) did not show astrong and well defined staining of the centromeres, instead, aweak and diffuse staining was seen over the central part of themetaphase plate (Fig. 2D). The staining level was comparableto cells prepared without microcystin (Fig. 1D-F). The cen-tromere labelling disappeared completely during anaphase anda proportion of cells (22%) showed staining of the central partof the spindle (Fig. 2E). Finally, during telophase the staining


Table 1. Quantification of 3F3/2 staining of Kc cells and neuroblasts1 or 2 dots stained 2 dots stained Diffuse labelling Spindle

No staining per centromere per centromere of chromosome midzone or Cell cycle stage of the centromere in some chromosomes in all chromosomes or nuclear region midbody labelling

Kc cells*Prophase 0 9 81 29 NAPrometaphase 0 33 67 10 NAMetaphase 16 5 13 75 NAAnaphase 45 4 0 41 22Telophase 2 0 0 85 98

Colchicine-arrested 0 15 85 0 NA

Neuroblasts*Prophase 2 21 70 17 NAPrometaphase 9 43 53 8 NAMetaphase 70 15 19 60 NAAnaphase 12 2 0.5 13 29Telophase NA NA NA 73 65

Z section confocal series were made for each cell to determine single or double centromere labelling in all chromosomes. *Numbers are percentage over total. At least 100 cells counted for each phase of the cell cycle; because a cell can show mixed immunostaining pattern the

number will not add up to 100.NA, none applicable.

localized to the midbody, and a diffuse speckle pattern ofnuclear staining reappeared over the decondensing chromo-somes (Fig. 2F).

Throughout mitosis, the 3F3/2 antibody recognised two

Fig. 2. Cell cycle distribution of 3F3/2reactive epitopes in Kc cells preparedin the presence of microcystin (A-F).Either interphase or very earlyprophase nuclei have a diffusestaining (A). During prophase, the3F3/2 staining is concentrated to adiscrete region of each chromosome(B) and is present at the centrosomes(arrowheads). By prometaphase,chromosomes are now fullycondensed and the 3F3/2 labelling canbe detected over the primaryconstriction of most chromosomesand the two centrosomes (arrowheads)can also be seen (C). Once cells gointo metaphase, the two centrosomeslocated at either side of the metaphaseplate are clearly observed and there isa dim diffuse 3F3/2 staining at thecentromere regions of thechromosomes (D). As anaphaseproceeds, the labelling on thechromosomes disappears, thecentrosomes remain strongly stainedand a band of labelling can be seenrunning across the central part of thespindle (E). Later, in telophase, thetwo centrosomes remain labelled(arrowheads), although much moreweakly, there is a diffuse staining overthe decondensing chromosomes andthe midbody can be seen as a singleband between the two separatingnuclei (star) (F). Bars, 5 µm.

round structures that from their position and size could corre-spond to the centrosomes (Fig. 2D-F). In order to determinewhether these structures corresponded to the centrosomes, Kccells were simultaneously labelled with the 3F3/2 antibody and


Fig. 3. Colocalization of the3F3/2 phosphoepitopes withCP190 antigen at thecentrosomes. A cell intelophase with 3F3/2 stainingrevealed by a FITC-conjugated secondaryantibody (A) and CP190staining revealed by a TexasRed-conjugated secondaryantibody (B). C is the mergedimage of A and B showingoverlapping of the two colorsat the centrosome (shown inyellow). Bar, 5 µm.

the centrosome associated antigen CP190 (Whitfield et al.,1988). The results indicated that there was perfect co-localiza-tion between the staining patterns of 3F3/2 and CP190 (Fig.3). The centrosome was stained by 3F3/2 from early prophaseto late telophase (Fig. 2).

Cell cycle distribution of 3F3/2 phosphoepitopes inwild-type neuroblastsWe developed a protocol to immunostain third instar wholebrains with the 3F3/2 antibody. The results showed the samemicrocystin-dependent labelling of the centromeres and thecentrosome as shown for Kc cells (Fig. 4). During interphase,neuroblasts showed a very light overall nuclear staining, but byearly prophase the staining became more punctate (Fig. 4A).As cells entered prophase, a discrete labelling over condensingchromosomes was visible and centrosomes were also stained(Fig. 4B). When cells reached a prometaphase configuration,centromere staining became stronger, chromosomes began toalign at the metaphase plate and the centrosomes can be seenat either side (Fig. 4C). During metaphase two types of stainingpattern were found. A small proportion of the cells showed avery clear dot-like labelling of paired centromeres (19%)located at the center of the metaphase plate and the centro-somes at either pole (Fig. 4D). However, most well organisedmetaphase plates showed a significantly lower level of stainingthat became diffuse over the metaphase plate (60%) while cen-trosomes remained labelled (Fig. 4E). As anaphase started,most cells only showed centrosome staining and no centromerelabelling can be found (Fig. 4F). Occasionally, we have beenable to observe anaphase cells with centrosome staining, aswell as irregular labelling of the spindle midzone (Fig. 4G).Later, during telophase, cells showed both centrosome andmidbody staining (Fig. 4H-I) that remained until chromosomeswere completely decondensed and then gradually decreased inintensity.

3F3/2 phosphoepitope is retained at the centromereof chromosomes from cells arrested in mitosisIt has been shown previously that 3F3/2 kinetochore stainingof mitotic chromosomes could be strongly enhanced if cellswere arrested in prometaphase with microtubule poisoningagents (Campbell and Gorbsky, 1995). In order to study thiseffect in Kc cells, an asynchronous culture was treated witheither colchicine or taxol. Cells were incubated for 16 hours

with either drug, fixed in the presence of microcystin andstained with the 3F3/2 antibody as described above (Fig. 5;see Table 1 for quantification). In cells arrested inprometaphase with colchicine, chromosomes remained highlycondensed and dispersed within the cell, and all centromereswere stained (Fig. 5A). Although staining intensity was notquantified, it seemed that the differential 3F3/2 stainingnormally observed in exponentially growing cells disappearedand all centromeres exhibited the same bright labelling in amanner similar to that of a normal prophase cell (Fig. 5C).The centrosome staining remained strong in all treated cells.Identical results were obtained when taxol was used, with thedifference that in cells treated with this drug prometaphasechromosomes remained tightly packed at the center of the cell(Fig. 5B).

The centromere staining was also retained in third larvalinstar neuroblasts after incubation of wild-type tissue incolchicine or by immunostaining neuroblasts isolated fromlarvae homozygous for mutations that are known to affectmetaphase-to-anaphase progression. Wild-type third instarlarval neuroblasts incubated in colchicine prior to fixation inthe presence of microcystin showed strong centromerelabelling, as well as centrosome staining (Fig. 6A).

A mitotic mutation that has been well characterised andwhich causes a strong metaphase arrest is asp (abnormalspindle) (Ripoll et al., 1985). This mutation appears to affectspindle behaviour in that chromatids never segregate but anirregular metaphase plate is usually observed associated withvarious types of abnormal bipolar spindles (Fig. 6B). In thiscell, a well organised metaphase plate can be seen in closeassociation with a bipolar spindle that contains more micro-tubules at one side. Immunostaining of asp homozygous neuro-blasts showed metaphase arrested cells with a highlycondensed mass of chromosomes and strong centromere andcentrosome labelling (Fig. 6C). It is significant that in mostcells that displayed a bipolar centrosome arrangement, thekinetochores never align across the metaphase plate. Thestrong metaphase arrest phenotype caused by this mutation andthe centromere-associated staining of most of these cells canbe seen at a lower magnification (Fig. 6D).

Neuroblasts homozygous for a mutation in the γ-tub23Cgene were also studied for 3F3/2 immunolabelling. A mutationin this gene (γ-tub23CPI) was reported to affect the function ofmicrotubule organizing centres and results in various degrees


Table 2. Quantification of 3F3/2 staining in isolated chromosomesNo 1 dot stained 2 dots stained Brightness of

staining per centromere per centromere the staining*

Isolated chromosomes†Without microcystin 98 2 0 −With microcystin 17 18 65 ++

Rephosphorylated chromosomes†Without microcystin 36 39 25 +With microcystin 3 7 90 +++

*For each experiment, the brightness of fluorescent centromeres was classified as no staining (−), weak (+), medium (++) or strong (+++).†Numbers are percentage over total. At least 200 chromosomes were counted in each case.

Fig. 4. Distribution of 3F3/2reactive phosphoepitopesduring the mitotic cycle inwild-type third instar larvalneuroblasts. In all figures3F3/2 staining is shown ingreen and DNA staining withpropidium iodide in red.(A) Very early prophase cellshowing condensing chromatinand a light 3F3/2 staining overthe whole nucleus.(B) Prophase cell showing amore discrete staining overcondensing chromosomes andthe two centrosomes.(C) Prometaphase cell withstrong labelling of 3F3/2 overthe chromosomes and the twocentrosomes located at eachside of the metaphase plate.(D) Cell at metaphase showingvery discrete centromerelabelling organised in pairsrunning across the metaphaseplate with the two centrosomeslocated at each side. (E) Mostcells at metaphase show a lowlevel of diffused centromerelabelling at the center of themetaphase plate and two welldefined centrosomes. (F) Acell undergoing anaphase Bshowing no 3F3/2 staining ofthe centromeres and welldefined centrosomes.(G) Occasionally, cells inanaphase also show 3F3/2staining of the central spindlezone. (H) Early telophase cellsshowing light staining of thecentrosomes and strongmidbody staining. (I) At laterstages of telophasecentrosomal staining becomeseven lighter but midbodystaining remains labelled. Bar,5 µm.

of aberrant spindle morphology (Sunkel et al., 1995). Asshown in Fig. 6E, 3F3/2 centromere staining was retained in

metaphase-arrested cells while no labelling was observed at thecentrosome.


Fig. 5. 3F3/2 staining inmitosis-arrested cells. Kccells were arrested witheither colchicine (A) or taxol(B) and labelled with 3F3/2(green) and counterstainedfor DNA with propidiumiodide (red). Drug-arrestedcells show 3F3/2 staining inall centromeres of thecondensed chromosomes.The staining pattern can becompared with a normalprophase cell (C). Bar, 5 µm.

Fig. 6. Immunostaining with3F3/2 antibody of wild-type andmutant neuroblasts arrested atmetaphase. Third larval instarneuroblasts with 3F3/2 (A,C-E) orβ-tubulin (B) are seen in green andDNA stained with propidiumiodide in red. Wild-type neuroblastarrested with colchicine (A), aspmutant (B-D) and (γ-tub23CPI)mutant (E) neuroblasts. (A) Cellshowing highly condensedchromosomes arranged in anirregular metaphase plate withstrong centromere labelling aswell as two centrosomes (stars).(B) Mutant neuroblast showing anabnormal metaphase plate andassociated spindle. (C) Mutantneuroblast arrested in metaphaseshowing strong centromerelabelling and two centrosomeslocated at either pole. (D) Lowmagnification of a field of mutantneuroblasts showing many cellsarrested at metaphase with strongcentromere and centrosomelabelling. (E) γ-tub23CPI mutantneuroblast stained with 3F3/2antibody showing consistentstaining at the centromeres of ametaphase arrested cell, note theabsence of centrosome staining.Bars: 10 µm (A-C,E); 15 µm (D).

Rephosphorylation of isolated mitotic chromosomesfrom Kc cellsThe protocol we described here to isolate chromosomes fromDrosophila culture cells allowed a good preservation of the3F3/2 phosphoepitope providing that microcystin was presentin all extraction buffers. Other more lengthy protocols for chro-mosome isolation resulted in a significant loss of the phos-phoepitope even in the presence of high concentrations ofmicrocystin (H. Bousbaa, L. Correia and C. E. Sunkel, unpub-lished results).

Chromosomes isolated with or without microcystin were

spread over microscope slides and fixed. Staining with the3F3/2 antibody showed that if chromosomes were isolated inthe absence of microcystin few (2%) showed centromerestaining (Fig. 7A, see Table 2 for quantification). However,when isolated in the presence of microcystin the centromerephosphoepitopes can be readily detected at the primary con-striction as either one (Fig. 7B) or two dots in most chromo-somes (83%) (Fig. 7C). Chromosomes that showed stainingwere studied further by serial optical sectioning using a laserconfocal microscope to determine whether the centromeres ofboth sister chromatids were stained. A single dot labelling was


Fig. 7. Detection of 3F3/2centromere phosphoepitopeson isolated chromosomes andafter their rephosphorylationin vitro. Mitoticchromosomes were isolatedin the absence (A,D-F) or inthe presence (B,C) ofmicrocystin and directlyfixed and immunostainedwith the 3F3/2 antibody (A-C) or incubated with ATP for20 (D,E) or 60 minutes (F) inthe absence (D) or in thepresence (E,F) of microcystinbefore fixation andimmunostaining. In allfigures 3F3/2 staining isshown in green and DNAstained with propidiumiodide in red.(A) Chromosomes isolated inthe absence of microcystindo not show 3F3/2 labelling.(B) Chromosomes isolated inthe presence of microcystinviewed from the sideshowing labelling of a rounddisk-like structure locatedover the primary constriction.(C) Isolated chromosomeviewed from the sideshowing that the 3F3/2labelling is located at thesurface of each chromatid over the primary constriction. Centromere epitopes of isolated chromosomes rephosphorylated in vitro in the absenceof microcystin show a low level of 3F3/2 staining (D), while in the presence of the phosphatase inhibitor they show a very strong level ofstaining by 20 minutes (D) or 60 minutes (E) of incubation. Note the centromere pairs on two of the chromosomes (arrowheads). Bar, 5 µm.

observed at the centromere of 18% of chromosomes, whilemost chromosomes (65%) showed two dots located at theprimary constriction. The structures labelled by the 3F3/2antibody localized at the centromere on the surface of eachchromatid (Fig. 7C).

Since most of the 3F3/2 centromere staining was retainedduring chromosome isolation in the presence of microcystin andabsent when chromosomes were isolated without microcystin,we tested whether the phosphoepitope could be rephosphory-lated in situ without the addition of either cellular or chromo-some protein extracts. For this, chromosomes isolated in theabsence of microcystin were incubated in the presence of ATPwith or without microcystin for either 20 or 60 minutes beforefixation and immunostaining. Due to the difficulty of definingthe area of the centromere that was labelled, the intensity of thestaining was appreciated semi-quantitatively (Table 2). Theresults showed that if isolated chromosomes were incubated withATP for either 20 or 60 minutes in the absence of microcystin,a low level of centromere staining was observed (Fig. 7D; seeTable 2 for quantification). This level of staining was the highestencountered and in many chromosomes (36%) no staining wasobserved. However, if isolated chromosomes were incubatedwith ATP for either 20 (Fig. 7E) or 60 minutes (Fig. 7F) in thepresence of microcystin, strong centromere staining wasobserved in most chromosomes (97%). In these preparations,

most chromosomes (90%) showed labelling of both centromeresand the intensity of the labelling was greater than that of chro-mosomes isolated in the presence of microcystin (Table 2).

In order to demonstrate that the rephosphorylation ofisolated chromosomes was not due to cytoskeletal contami-nants carried through during chromosome isolation, weprepared chromosomes using a sucrose and Percoll gradientprotocol (Lewis and Laemmli, 1982). This protocol allowedthe removal of cytoskeletal contaminants as determined byactin and tubulin immunodetection (data not shown). However,the chromosome yield was much lower than that of theprevious protocol. The results obtained were essentially thesame as those described above. Addition of ATP to these chro-mosomes resulted in the rephosphorylation of the 3F3/2 cen-tromere epitopes. The efficiency of the rephosphorylationdepended on whether microcystin was absent or present duringthe assay (data not shown). In situ rephosphorylation of chro-mosomes isolated with either protocol was prevented if NEMwas present during the reaction (data not shown).

DISCUSSION

The 3F3/2 phosphoepitope is present in DrosophilaIn this study we showed that the 3F3/2 phosphoepitopes are


expressed in Drosophila with a similar cell cycle distributionas described for mammalian cells (Gorbsky and Ricketts,1993) and grasshopper spermatocytes (Nicklas et al., 1995).These results suggest that the biochemical components recog-nised by the 3F3/2 antibody that are associated with the spindleassembly checkpoint, are evolutionary conserved. The descrip-tion of the 3F3/2 phosphoepitopes in Drosophila is of greatinterest since this organism has been extensively used for thegenetic and molecular analyses of proteins required for mitosis.

The centromere of mitotic chromosomes containsthe 3F3/2 phosphoepitopesWe have shown that in mitotic Kc cells, 3F3/2 phosphoepitopeslocalise at the primary constriction of condensed chromo-somes. At this level of analysis, it is not possible to determinethe precise localisation of the reactive phosphoepitopes.Therefore, isolated mitotic chromosomes were immunostainedwith the 3F3/2 antibody to obtain a better definition of the cen-tromeric labelling. Our results show that the 3F3/2 labellingoccupies a discrete round area on both sides of the primary con-striction excluding the pairing domain of the centromere ofsister chromatids. Taken together with the previous descrip-tions of 3F3/2 labelling of the kinetochore (Gorbsky andRicketts, 1993; Campbell and Gorbsky, 1995), our resultsstrongly suggest that the reactive phosphoepitopes localise tothe kinetochore of Drosophila mitotic chromosomes. However,and in the absence of a kinetochore marker in Drosophila, onlyultrastructural analysis will provide a definite localisation ofthe phosphoepitopes.

Drosophila cells show a conserved cell cycledistribution of 3F3/2 phosphoepitopesThe 3F3/2 monoclonal antibody was shown to stain compo-nents of the kinetochore in mammalian culture cells andgrasshopper spermatocytes. It recognises kinetochore phos-phoepitopes from prophase through prometaphase and whenchromosomes establish stable bipolar spindle attachments, thekinetochore staining is dramatically reduced (Gorbsky andRicketts 1993). Our results show that both Kc cells and thirdinstar neuroblasts display a very similar 3F3/2 centromerestaining pattern. Centromere staining can be observed duringearly stages of mitosis, is reduced at metaphase and absentduring anaphase. Therefore, it is reasonable to propose that thesame mechanism described for the metaphase-anaphase kin-etochore checkpoint control in other cell types (Gorbsky, 1995)is present in Drosophila cells. However, due to the small chro-mosome size, the very tight metaphase plate organisation andthe difficulty of distinguishing mono-oriented chromosomes inKc cells and wild-type neuroblasts, we were unable todetermine whether kinetochores that are attached to micro-tubules show a reduced staining as compared to unattachedkinetochores.

Apart from the staining at the centromere and centrosome,we showed that the 3F3/2 monoclonal antibody also labels thecentral spindle zone during late stages of anaphase and themidbody during telophase. Midbody staining by the 3F3/2antibody has been seen before in mammalian cells (G. J.Gorbsky, unpublished results). Other proteins are known tofollow the same pattern of relocalization during mitosis. Chro-mosomal passenger proteins like the INCENPs localise to aregion adjacent to the kinetochores at prometaphase, move to

the spindle during anaphase and are concentrated at themidbody during telophase (Earnshaw and Bernat, 1991). It isyet unclear whether the localisation of 3F3/2 reactive phos-phoepitopes at the midbody is of any functional significance.

Cells that are prevented to enter anaphase retain thecentromere stainingCells arrested at prometaphase have been shown to retain astrong kinetochore staining with the 3F3/2 antibody (Campbelland Gorbsky, 1995). Incubation of Kc cells with drugs thataffect microtubule dynamics shows the same effect. As per-turbations in the mitotic spindle formation are thought toactivate the spindle assembly checkpoint, our results suggestthat such a checkpoint also functions in Drosophila. Theretention of 3F3/2 centromere phosphoepitopes is not onlyobserved after chemical modification of microtubules but isalso seen in neuroblasts homozygous for mitotic mutations thatare known to cause alteration in spindle behaviour. Themutation asp has been shown to cause a complete metaphasearrest with most cells having a 4n chromosome contentorganised in irregular metaphase plates (Ripoll et al., 1985).Most of these cells have abnormal bipolar spindles (Carmenaet al., 1991). Also, a mutation in one of the two γ-tubulin genes(γ-tub23C) was reported to affect microtubule nucleation andinduce the formation of disorganised spindles (Sunkel et al.,1995). In mutant neuroblasts for either of these mutations,3F3/2 staining still persists in cells that have a metaphase-likeconfiguration, suggesting that either spindle microtubules areunable to make proper contact with the kinetochores or thattension is not applied through stably attached microtubules. Asa consequence, it is likely that in these mutant cells themetaphase-anaphase kinetochore checkpoint is fully activatedand entry into anaphase is delayed.

3F3/2 centromere reactive epitope can berephosphorylated on isolated chromosomesWe showed that if chromosomes are isolated in the presenceof microcystin, centromere staining is present in most sisterchromatids. However, if chromosomes are isolated in theabsence of phosphatase inhibitors, the 3F3/2 centromereepitopes are dephosphorylated and no labelling is detected.Similarly, if mammalian cells are fixed in the absence of phos-phatase inhibitors most 3F3/2 kinetochore reactivity is lost(Gorbsky and Ricketts, 1993). Since the 3F3/2 antibodyappears to recognise kinetochore antigens only in their phos-phorylated form (Gorbsky and Ricketts, 1993; Campbell andGorbsky, 1995), the absence of labelling does not necessarilyindicate that the phosphoproteins are gone. They are likely tobe still located at the kinetochore but the antibody cannotrecognise them. Indeed, if mammalian cells are treated so thatkinetochore phosphoepitopes are dephosphorylated, additionof ATP will cause their rephosphorylation (M. S. Campbell andG. J. Gorbsky, unpublished work). Therefore, we wanted todetermine whether the same effect could be observed withisolated Drosophila chromosomes. For this, chromosomeswere isolated in the absence of microcystin and then incubatedwith either ATP alone or with ATP and microcystin. Our resultsshow that if chromosomes are incubated with ATP and micro-cystin, the vast majority of centromeres are rephosphorylated.The rephosphorylation of centromere phosphoepitopes isunlikely to be due to cytoskeletal contaminants since chromo-


somes isolated using a sucrose/Percoll gradient protocol alsoshow a similar pattern of rephosphorylation. These results donot allow us to determine whether the ‘3F3/2-kinase’ is a chro-mosome associated protein or whether it is a solublecomponent that becomes tightly associated with the chromo-somes during the isolation protocol. Up to now, there have beenno reports of immunolocalization of mitotis associated kineto-chore kinases. The only data available come from studies in S.cerevisiae in which the MCK1 gene was described (Shero andHieter, 1991). This gene encodes a putative Ser/Thr proteinkinase that might have a role in regulating the activity of cen-tromere-associated proteins by phosphorylation. It is also sig-nificant that a yeast strain carrying a null MCK1 mutation isunable to arrest at metaphase in the presence of Benomyl (amicrotubule depolymerizing agent) and shows a dramaticincrease in the rate of mitotic chromosome loss (Shero andHieter, 1991). This result would suggest that in the absence ofMCK1 yeast cells are unable to activate the metaphase-anaphase checkpoint. The presence of the ‘3F3/2-kinase’ at thekinetochore could lead to differential phosphorylation betweensister kinetochores that differ in their attachment state to micro-tubules, therefore we favour the hypothesis that a chromosomeassociated kinase is responsible for the phosphorylation ofthese epitopes.

Finally, when rephosphorylation of isolated chromosomes iscarried out in the absence of microcystin the number of chro-mosomes showing 3F3/2 centromere staining is significantlyreduced. These results suggest that either a contaminatingphosphatase is purified together with the isolated chromo-somes, or that a phosphatase is part of the mitotic chromosome.However, protein phosphatases like PP1 have been shown tobe present in mitotic chromosomes (Fernandez et al., 1992;Paulson et al., 1996), suggesting that the phosphatase respon-sible for the dephosphorylation of 3F3/2 reactive epitopes isalso associated to the mitotic chromosomes.

We thank A. Moreira for her early work on Kc cell synchronisa-tion. We are in debt to all the members of the lab in Porto for all theirsupport and encouragement during this work. H. Bousbaa holds aHCMP fellowship from the European Union and L. Correia a schol-arship from the PRAXIS XXI program of the EU. The work in Portois financed by grants from the EU, PRAXIS XXI and JNICT ofPortugal. G.J.G. is supported by a grant from the National Institute ofGeneral Medical Sciences.

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(Received 12 October 1996 - Accepted 23 June 1997)

mitotic phosphoepitopes are expressed in kc cells ... · were observed with a bio-rad mrc600...

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