a monoclonal antibody recognizing nuclear matrix ... · chicken hepatoma). t24, hela, caco, mcf7,...

Journal of Cell Science 101, 773-784 (1992)Printed in Great Britain © The Company of Biologists Limited 1992

773

A monoclonal antibody recognizing nuclear matrix-associated nuclear

bodies

NICO STUURMAN1, ARJAN DE GRAAF2, ARNO FLOORE1, ARTHUR JOSSO1, BRUNO HUMBEL2,

LUITZEN DE JONG1 and ROEL VAN DRIEL1*J £ . C. Slater Institute for Biochemical Research, University of Amsterdam, Plantage Muidergracht 12, 1018 TV Amsterdam, The Netherlands''•Department of Molecular Cell Biology, State University of Utrecht, Padualaan 8, 3584 CH Utrecht, The Netherlands

*Author for correspondence

Summary

We have isolated a monoclonal antibody, 5E10, thatlabels discrete spots in the interphase nucleus. Byimmunoblotting mAb 5E10 recognized predominantly a126 kDa polypeptide with an isoelectric point of 5.5.Indirect immunofluorescence showed that mAb 5E10labeled spots in many cell lines and tissues from rat orhuman origin, but not in cells from mouse, chicken,African green monkey, or the lower eukaryotes Sacchar-omyces and Dictyostelium. In the human bladdercarcinoma cell line T24 the number of nuclear spots wasfound to be 21 ± 10 (n=132). In many cells spots werefound also in the cytoplasm. In a small fraction of T24cells the mAb revealed thread-like structures in additionto spots. Throughout mitosis the antigen was found to beclustered in the cytoplasm, not associated with meta-phase chromosomes. The spherical structures that

contain the antigen were tightly bound to the nuclearmatrix. Immunogold labeling with mAb 5E10 showedthat the antigen is localized in 0.3 pan diameterspherical, electron-dense structures, reminiscent ofnuclear bodies. Double-labeling experiments showedthat these spots do not colocalize with Ul snRNPs andcentromeres. The spots did colocalize with nuclearspeckles recognized by a primary biliary cirrhosisautoimmune serum, which is thought to recognizenuclear bodies. On the basis of these observations weconclude that mAb 5E10 recognizes discrete nuclearsubstructures, most likely nuclear bodies.

Key words: nuclear body, nuclear matrix, nuclearcompartmentation, monoclonal antibody, cell cycle.

Introduction

In recent years evidence has accumulated showing thatthe nucleus contains a variety of domains that can bedistinguished on the basis of morphological and func-tional criteria. Examples are: the nucleolus, being themost conspicuous nuclear substructure; individualchromosomes, which occupy discrete nuclear territoriesin interphase nuclei (Manuelidis, 1985); replicationdomains (Nakamaru et al., 1986; Nakayasu and Berez-ney, 1989); snRNP (small nuclear RNP) clusters, whichare likely to be involved in RNA processing (Fu andManiatis, 1990; Potashkin et al., 1990; Spector, 1990;Carmo-Fonseca et al., 1991); and RNA tracks, possiblyreflecting RNA transport routes (Lawrence et al.,1989). These domains reveal a nuclear infrastructureabout which little is known.

A useful concept for understanding this nuclearinfrastructure is the nuclear matrix. This matrix isoperationally defined as the nuclear substructure thatremains after removing soluble proteins and chromatin.It consists of a nuclear lamina, which completely

encloses the nucleus and lines the inside of the doublenuclear membrane (Gerace and Burke, 1988; Nigg,1989), and an internal fibro-granular network (Capco etal., 1982; Verheijen et al., 1988; He et al., 1990). It hasbeen shown that many key processes in the nucleus, likeRNA synthesis (Jackson and Cook, 1985), RNAprocessing (Smith et al., 1989; Zeitlin et al., 1989),RNA transport (Schroder et al., 1987a, b; Xing andLawrence, 1991) and DNA replication (Jackson andCook, 1986; Nakayasu and Berezney, 1989), are matrix-associated. Interestingly, many of these activities arelocated in specific nuclear domains. In addition to thekey processes listed above, chromatin interacts with thenuclear matrix via specific sequences, called matrix-associated regions or MARs (Mirkovitch et al., 1984;Gasser et al., 1989; Garrard, 1990). In this way thenuclear matrix organizes the chromatin in loops with anaverage size of 80-90 kbp (Jackson et al., 1990). Thestructure of the nuclear matrix is still poorly under-stood. Its most likely function is to maintain a correctspatial nuclear organization.

The protein composition of the nuclear matrix is

774 N. Stuurman and others

complex (Fey et al., 1986; Kaufmann et al., 1986;Stuurman et al., 1990). It is still not evident whichmatrix components have a structural role and which areaccessory proteins (see, for a discussion, De Jong et al.,1990). Recently, Stuurman et al. (1990) have made acomparison of the protein composition of nuclearmatrices isolated from different cell lines and tissues ofmouse and some other mammals, based on 2-dimen-sional gel electrophoresis. They show that a specific setof polypeptides is shared by all the matrices that wereanalyzed. It is speculated that these conserved, so-called 'minimal matrix' polypeptides have an import-ant, possibly structural, function in the nucleus.

We set out to generate monoclonal antibodies thatrecognize ubiquitous components of the nuclear matrix.One of the isolated antibodies is described here. Themonoclonal antibody 5E10 predominantly recognizes a126 kDa antigen that is present in most rat and humancells tested so far. The antigen is a nuclear matrixprotein. Ultrastructural studies, using immunofluor-escence microscopy and immunogold electron mi-croscopy, showed that the antigen is located in sphericaldomains with a diameter of about 0.3 ptm. Transmissionelectron microscopy identifies the immunolabeled nu-clear domains as nuclear bodies. Nuclear bodies areelectron-dense nuclear structures of diverse mor-phology (Bouteille et al., 1974; Chaly et al., 1983a).Evidently, the antibody labels nuclear bodies that aretightly associated with the nuclear matrix.

Materials and methods

Cell cultureThe following cell lines were used: T24 (human bladdertransitional carcinoma), HeLa (human cervix carcinoma),SK-N-MC-IX-C (human neuroblastoma), CaCo (humancolon carcinoma), MCF7 (human breast adenocarcinoma),CV1-HP8 (African green monkey kidney), NRK (newbornrat kidney), FTO-2B (rat hepatoma), NS-1 (mouse myel-oma), P19EC (mouse embryonal carcinoma), P19MES-1(mouse mesodermal cell line derived from P19EC), NIH 3T3(mouse fibroblast) and CH249 (virus MC29-transformedchicken hepatoma). T24, HeLa, CaCo, MCF7, NIH 3T3, NS-1 and CH249 were maintained in DME (Gibco, Paisly, UK);CV1-HP8, P19EC, P19MES-1 and FTO-2B in a 1 to 1 mixtureof DME and Ham's F12 nutrient mixture (Gibco); NRK andSK-N-MC-IX-C in RPMJ 1640 (Gibco). All media weresupplemented with 10% (v/v) FCS (Gibco).

Isolation and fractionation of nucleiRat liver nuclear matrix was isolated using a slight modifi-cation of published procedures (Kaufmann and Shaper, 1984;Stuurman et al., 1990). All steps were performed on ice or at4°C. Rat liver was minced in STEM (0.25 M sucrose, 50 mMTris-HCl (pH 7.4 at 4°C), 0.2 mM EGTA, 5 mM MgSO4, 1mM PMSF), washed three times in the same buffer bydecantation and subsequently homogenized using a motor-driven Potter-Elvehjem homogenizer. The homogenate waswashed three times by centrifugation at 1,000 g. The crudenuclear pellet was resuspended in DES (2.1 M sucrose, 50mM Tris-HCl (pH 7.4 at 4°C), 0.2 mM EGTA, 5 mM MgSO4,1 mM PMSF), layered on a DES cushion and spun at 73,000 gfor 60 min. The pellet was resuspended in STEM, layered on a

DES cushion and centrifuged for 30 min at 73,000g. Resultingpurified nuclei were incubated for 60 min with 0.5 mM sodiumtetrathionate (Pierce, Oud Beijerland, The Netherlands) inSTEM at a density of 108 nuclei/ml. This oxidative procedurestabilizes the nuclear matrix. After three washes with STEM(10 min, 1,000g followed by resuspension in STEM), nuclei ata density of 5 x 108 nuclei/ml were digested with 250 //g/ml(2000 Kunitz units/mg) DNase I (Sigma, St. Louis, USA) and250 /ig/ml (48 units/mg) RNase A (Sigma) in STEM for 60min. Nuclei were centrifuged for 10 min at 1,500 g andresuspended at a density of 5 x 108 nuclei/ml in LES (10 mMTris-HCl (pH 7.4 at 4°C), 0.2 mM EGTA, 0.2 mM MgSO4, 1mM PMSF). HES (2 M NaCl in LES) was slowly added up toa concentration of 1.6 M NaCl. After 10 min the suspensionwas centrifuged at 5,000g. This extraction was repeated once.The resulting nuclear matrix was further fractionated byextraction with 20 mM dithiothreitol (DTT) in 1 M NaCl inLES for 20 min. The material sedimented after centrifugationfor 10 min at 10,000 g is enriched in nuclear envelopes(Kaufmann and Shaper, 1984).

To prepare the fraction used for immunization the nuclearenvelope-enriched fraction was further extracted with 10%(w/v) sucrose, 2% (w/v) Triton X-100, 20 mM MES-KOH, pH6.0, 300 mM KC1, 2 mM EDTA, 1 mM DTT and subsequentlywith 2% (w/v) Triton X-100, 20 mM Tris-HCl, pH 9.0, 500mM KC1, 2 mM EDTA, 1 mM DTT (Aebi et al., 1986). Thisprocedure results in a lamin-depleted residue, enriched insome nuclear matrix proteins.

Production of monoclonal antibodiesOur initial goal was to prepare antibodies against minimalmatrix proteins (Stuurman et al., 1990) other than lamins. Tothis end mice were immunized with a lamin-depleted matrixsub fraction (see above). This paniculate material was resus-pended in PBS (150 mM NaCl, 6.7 mM sodium phosphate,pH 7.4). Primary immunization was with 750 ,ug protein inFreund's complete adjuvant, Balb/c mice were boosted 4times with 300 /ig protein in Freund's incomplete adjuvant at4-week intervals. Three days after the last booster, spleencells were fused to NS-1 cells by electrofusion according to theprotocol of Van Duijn et al. (1989). Hybridomas secretingrelevant immunoglobulins were selected by ELISA (Tijssen,1985), with detergent-extracted rat liver nuclear envelopes(see above) as antigen. Culture supernatants positive in thisELISA were subsequently screened by immunofluorescenceon methanol-fixed T24 cells (see below) and by immunoblot-ting using detergent-extracted rat liver nuclear envelopes assource of antigen. Cells from wells with culture supernatantthat was positive in all tests were subcloned three times bylimited dilution.

In situ preparation of nuclear matricesPreparation of nuclear matrices from HeLa cells for in situlocalization of antigens was carried out essentially asdescribed by Fey et al. (1986). Briefly, HeLa cells oncoverslips were washed in PBS and permeabilized for 3 min at4°C by incubation with 0.5% (w/v) Triton X-100 in cytoskel-eton-stabilizing buffer (CSK: 10 mM Pipes (pH 6.8), 0.1 MKC1, 0.3 M sucrose, 3 mM MgCl2,1 mM EGTA, and 1.2 mMPMSF). To reveal nuclear matrices, chromatin was digestedwith 1000-2000 Kunitz units/ml of RNase-free DNase I(Boehringer, Mannheim, FRG) in digestion buffer (10 mMPipes (pH 6.8), 50 mM KC1, 0.3 M sucrose, 3 mM MgCl2, 1mM EGTA, 1.2 mM PMSF and 0.5% (w/v) Triton X-100) for30 min at room temperature. Subsequently, ammoniumsulfate was added from a 1 M stock solution in digestionbuffer lacking KC1 to a final concentration of 0.25 M. After 3

Nuclear matrix-associated substructures 775

min the extracted components were removed by washing inCSK buffer.

Pre-embedment immunogold labeling and electronmicroscopyThe procedure used for pre-embedment labeling has beendescribed by De Graaf et al. (1991). HeLa cells were grownon Thermonox (Miles Lab Inc, Naperville, USA) up to 80%confluency. For in situ isolation of nuclear matrices cells wereextracted as described above. Nuclear matrix preparations(or, alternatively, cells permeabilized with 0.5% (w/v) TritonX-100 in CSK buffer as described above) were fixed with0.25% (v/v) glutaraldehyde in CSK buffer for 30 min. Freealdehyde groups were inactivated by incubation with a freshlyprepared solution of 50 mM glycine in PBS (pH 7.4) for 10min. The fixed preparation was washed several times withPBG (0.2% (w/v) gelatin, 0.5% (w/v) BSA in PBS).Extracted cells, still on Thermonox, were incubated withundiluted 5E10 culture supernatant for 1 to 2 h at roomtemperature. Subsequently, the samples were washed in PBGand incubated overnight with a 100-fold dilution of goat anti-mouse antibody conjugated to 1 nm diameter gold particles(Janssen Pharmaceutica, Beerse, Belgium; a gift from Dr.J.L.M. Leunissen). Preparations were washed extensivelywith PBG and PBS and were post-fixed in 1% (v/v)glutaraldehyde in PBS for 30 min at room temperature.Silver-enhancement of the 1 nm colloidal gold particles wascarried out according to Danscher and Norgaard (1983).Preparations were cryoprotected with 30% (v/v) N,N-dimethylformamide (Meissner and Schwarz, 1990) and fastfrozen in liquid propane in a KF80 rapid-freeze apparatus(Reichert-Jung, Wien, Austria). The samples were freeze-substituted in methanol containing 0.5% (w/v) uranyl acetateand 0.1% (v/v) glutaraldehyde, according to Humbel andMiiller (1986). Finally, preparations were embedded in Epon.Labeled material was cut in 0.2-0.25 jim thick sections,parallel to the substratum, using an Ultracut E (Reichert-Jung). Sections were examined and photographed in a PhilipsEM 420 electron microscope operated at 120 kV.

ImmunofluorescenceCells grown in monolayer on glass coverslips were washedonce with PBS and fixed by incubation in methanol for 5 minat —20°C. Alternatively, cells were fixed by a 20 minincubation in freshly prepared 2% (w/v) p-formaldehyde,0.5% (w/v) Triton X-100 in PBS, followed by a 5 min wash in0.5% (w/v) Triton X-100 in PBS and a 10 min incubation in 0.1M NH4CI in PBS to inactivate any free aldehyde groups.After three washes with PBS, coverslips were incubated for 45min at room temperature upside down on 40 /A of hybridomatissue culture supernatant placed on parafilm. The coverslipswere washed three times with PBS and incubated for 45 minwith affinity-purified goat anti-mouse IgG (heavy and lightchain) conjugated to FITC (Cappel) diluted 500 times in PBSwith 1% (w/v) BSA. The coverslips were washed with PBS,incubated for 5 min in 0.4 ,ug/ml Hoechst 33258 to label DNA,washed three times with PBS and mounted with 0.1% (w/v) p-phenylenediamine in 10% (v/v) PBS, 90% (v/v) glycerol, pH8.0 (Krenik et al., 1989).

For double-labeling experiments four human autoimmunesera were used: G57 serum, specific for Ul snRNP-specificproteins (Van Venrooij, personal communication; see alsoVan Venrooij and Stillekens, 1989), H33 serum, specific forcentromeres (Van Venrooij, personal communication), serum1745, recognizing "nuclear dots" (Ascoli and Maul, 1991; andMaul, personal communication) and SUN-3 serum (from aprimary biliary cirrhosis patient), which binds a 100 kDa

nuclear protein (Szostecki et al., 1987). The sera were dilutedin 5E10 tissue culture supernatant (G57 serum, 3,000 times;H33 serum, 10,000 times; serum 1745, 5 times; SUN-3 serum,100 times) and used for the primary incubation. The secondincubation was in PBS with 1% (w/v) BSA and a mixture ofaffinity-purified goat anti-mouse IgG (heavy and light chain)conjugated to rhodamine (Cappel) (final dilution 100-300times) and either sheep anti-human Ig conjugated to biotin(Amersham, Buckinghamshire, UK) (final dilution 200 times)or goat anti-human IgG conjugated to biotin (BRL) (100times). After three washes in PBS the coverslips wereincubated for 45 min with streptavidin conjugated to FITC(Amersham) diluted 250 times in PBS with 1% (w/v) BSA.The cells were stained for DNA and mounted as describedabove. Cells were viewed using a Leitz Orthoplan fluor-escence microscope and photographed on Kodak Tri-X Pan orTmax 100 film.

Gel electrophoresis and immunoblottingSamples originating from equivalent amounts of nuclei wereseparated on 8% (w/v) polyacrylamide/SDS gels according toLaemmli (1970), or by isoelectrofocusing followed bySDS/polyacrylamide gel electrophoresis (O'Farrell, 1975).Protein was blotted to nitrocellulose using the method ofTowbin et al. (1979). Blots were stained for total protein with0.1% Fast Green in 10% methanol, 10% acetic acid for 30 sand subsequently destained in the same solution without dye.The blots were blocked with 1% (w/v) blocking reagent(Boehringer) in PBS for 2 h at room temperature, washed 2times with PBGT (0.1% (w/v) gelatin, 0.5% (w/v) BSA,0.05% (w/v) Tween-20, 450 mM NaCl, 6.7 mM sodiumphosphate, pH 7.4), and incubated with undiluted hybridomatissue culture supernatant. Binding of the antibody wasdetected by subsequent incubation with biotinylated sheepanti-mouse Ig (Amersham) and streptavidin conjugated tohorseradish peroxidase (Amersham), both diluted in PBGT.Peroxidase activity was visualized with HRP color (BioRad,Richmond, USA), according to the manufacturer's instruc-tions.

Results

Specificity of monoclonal antibody 5E10We have selected a number of hybridomas that secretemonoclonal antibodies that recognize nuclear matrixcomponents. Antibodies were screened for reactivitywith rat liver nuclear matrix by ELISA and immuno-blotting after SDS/polyacrylamide gel electrophoresis.Positive clones were tested by indirect immunofluor-escence for reaction with methanol-fixed T24 humanbladder carcinoma cells. One of the ensuing antibodies,5E10, gave a typical speckled pattern in indirectimmunofluorescence. The antibody was of the IgGtype. The specificity of the monoclonal antibody wastested by immunoblotting using rat liver nuclei as asource of antigen. The mAb 5E10 reacted with at leastfour protein bands, with apparent molecular masses of149,126, 95 and 63 kDa (lane 2 in Fig. 1, the four bandsare indicated by arrows). These protein bands were notlabeled on blots incubated without primary antibody orprobed with other mAbs specific for nuclear proteins(e.g. mAb 41CC4, which is specific for the nuclearlamins; data not shown). The 126 kDa band was themost prominent one. The bands at lower molecular


1 2 3 4 5 6 7

rnkDa

— 94

IP— 67

. ' ' ^ — 4 3

Fig. 1. SDS-PAGE, followed by immunoblotting with mAb5E10, of rat liver nuclei and nuclear fractions and of HeLacells. Rat liver was fractionated as described in Materialsand methods. HeLa cells were cultured as described inMaterials and methods. Cells were washed twice at 0-4cCin PBS by centrifugation at 250 g for 10 min andresuspended in a medium (pH 7.4) containing 50 mMNaCl, 10 mM MgCl2) 10 mM NaP,, 1% (w/v) Triton X-100and 1 mM PMSF. The suspension of lysed cells was treatedwith 0.25 mg/ml DNase I for 30 min at 0°C and thendiluted 5 times in sample buffer (Laemmli, 1970). Numbersand arrows on the left indicate four rat liver polypeptidesrecognized by 5E10 antibody. Molecular masses of markerproteins are indicated on the right. 2X106 nuclearequivalents were loaded on the following lanes: 1, post-nuclear supernatant; 2, purified nuclei; 3, high salt extract:proteins extracted from DNase- and RNase-treated nucleiwith buffer containing 1.6 M NaCl; 4, nuclear matrix:residue after extraction with 1.6 M NaCl; 5, proteinsextracted from the matrix with buffer containing 20 mMDTT and 1 M NaCl; 6, residue after extraction of thenuclear matrix with 20 mM DTT and 1 M NaCl, enrichedin nuclear envelopes. Lane 7 was loaded with HeLa cellproteins from 2X105 cells.

B t

kDa

-94

-67

-43

. * - •

•»*«*T- 4 3

Fig. 2. Two-dimensional gel-electrophoresis of rat livernuclear envelopes and detection of the 5E10 antigen byimmunoblotting. Upper: immunoblot labeled with mAb5E10. Lower: silver-stained gel of the same preparation.Molecular masses of marker proteins are indicated on theright. Thick arrows indicate positions of lamins A, B and Cand an unknown, abundant matrix protein. These proteinswere visualized by staining of the blot for total proteinwith fast green prior to immunoreactions. The thin arrowindicates the position of polypeptides recognized by mAb5E10 on the immunoblot and also the correspondingposition on the silver-stained gel. On the originalimmunoblot additional 5E10-labeled spots were visible at api value between those of lamins B and A/C withmolecular masses corresponding to the proteins indicatedin Fig. 1 by arrows 1 and 3.

mass may represent proteolytic breakdown products ofthe 149 and/or 126 kDa proteins. Other tissues like ratspleen and rat brain, and cell lines like HeLa and T24contained the same major 126 kDa antigen, in additionto small quantities of higher and lower molecular masspolypeptides recognized by the mAb. This is shown inFig. 1 (lane 7) for HeLa cells.

A nuclear fraction of rat liver cells comprising about0.25% of total cellular protein (lane 4 in Fig. 1,described in more detail below) and highly enriched in5E10 antigen, was used for 2-D gel electrophoresis,blotted onto nitrocellulose and probed with 5E10antibody. The major protein spot recognized by mAb5E10 (thin arrow in Fig. 2A) appeared to consist of atleast three polypeptides with the same apparentmolecular mass (126 kDa), but different pi values(around 5.5). This suggests differential post-trans-lational modifications such as phosphorylation. At thesame position on the corresponding silver-stained 2-Delectropherogram no protein staining was visible (thin

arrow in Fig. 2B). Evidently, 5E10 recognizes aquantitatively minor protein.

Distribution of the 5E10 antigen in interphaseIndirect immunofluorescence revealed the presence of anumber of brightly labeled spots in the nuclei of T24cells (Fig. 3). No such labeling was observed aftercontrol incubations without primary antibody or withmAbs specific for other nuclear proteins (e.g. mAb41CC4 and mAb AM88; De Graaf et al., 1991; data notshown). The spots could not be recognized using phase-contrast optics. The spots were located throughout thenucleoplasm. The number of spots per T24 nucleus washighly variable and ranged from 3 to 58. The averagenumber of nuclear spots was 21 ± 10 (n=132). In mostcells a small number of fluorescently labeled spots wasobserved also in the cytoplasm (2 ± 3 spots per cell;n=76). No significant fluorescent labeling was observedoutside the spots (Fig. 3). No spots were detected innucleoli. Identical labeling patterns were observed for

Phase-contrast


5E10

• ' t

Fig. 3. Localization of 5E10 antigen in interphase cells by indirect immunofluorescence. T24 cells were grown on coverslips,fixed with p-formaldehyde and fluorescently labeled with mAb 5E10. Bar, 10 /an.

Phase-contrast Hoechst 33258 5E10

Fig. 4. Thread-like structures labeled by mAb 5E10 in nuclei of T24 cells. T24 cells were fixed with methanol andfluorescently labeled with mAb 5E10, and DNA was stained using Hoechst 33258. A selected cell is shown that displays alarge array of threads. Bar, 10 /jm.

T24 cells fixed with either 0.1% (v/v) glutaraldehyde ormethanol, instead of 2% (w/v) p-formaldehyde (notshown). Evidently, the labeling pattern was indepen-dent of the fixation procedure. The same speckled pat-tern was also found in HeLa cells (see Fig. 7B and D).

In a small percentage of T24 cells (less than 5% of thetotal cell population) large thread-like structures in theinterphase nucleus were labeled with mAb 5E10, inaddition to spots (Fig. 4). The maximum number of

threads per nucleus was about ten. Their length variedconsiderably. Some were as long as the diameter of thenucleus, i.e. about 10 /an. The threads were foundreproducibly and relatively frequently in T24 cells.Similar structures were found in HeLa cells and NRKcells, albeit with lower frequency. Generally, in thosecell types the structures were shorter. Like the spots,the threads were observed irrespective of the fixativeused (methanol, p-formaldehyde or glutaraldehyde;


data not shown). Clearly, the threads are not fixationartifacts.

To analyze the localization of the antigen at theelectron microscopic level, HeLa cells were labeledusing a pre-embedment immunogold labeling method.This procedure ensures a high labeling efficiency (DeGraaf et al., 1991). The immunogold label was found indiscrete, electron-dense, roughly spherical structureswith a diameter of about 0.3 /im (Fig. 5A and C). The5E10-labeled structures were rather uniform in size andshape. Very few gold particles (not more than aftercontrol labeling procedures omitting the first antibody)were found outside the clusters. As was observed afterimmunofluorescent labeling, 5E10 clusters were scat-tered throughout the nuclear interior. The clusters weresurrounded by regions containing low concentrations ofuranyl-stained material. This strongly suggests that the5E10 clusters are located in interchromatin areas. Oftenthe 5E10-labeled structures were connected to thesurrounding material via thin fibres (Fig. 5A and C).Immunogold labeling coincided with electron-denseparticles that may be nuclear bodies.

Distribution of the 5E10 antigen in mitosisMitotic cells in T24 cultures were identified on the basisof chromatin morphology visualized by labeling withHoechst 33258. Different mitotic stages were dis-tinguished according to criteria used by Chaly et al.(1984). Surprisingly, 5E10-labeled spots were found tobe present in all mitotic phases. In prophase the numberof spots per nucleus was generally less than ininterphase (Fig. 6), indicating that spots fused ordisassembled. In prometaphase cells the number ofspots had decreased even further. Many prometaphasecells had short, 5E10-labeled threads. In metaphase5E10-labeled spots were always dispersed throughoutthe whole cell volume. The antigen was never foundassociated with metaphase chromosomes. In metaphaseand anaphase no more than ten spots of unequal sizeand staining intensity were visible. Often the brighterlabeled spots in anaphase cells were located in thevicinity of the metaphase plate. In telophase thenumber of spots seemed to increase somewhat. At thatpoint in the cell cycle many of the spots were stilllocated outside of the reassembling nucleus. In early Gjmost spots were found again inside the nucleus.Obviously, throughout the mitotic cycle the 5E10antigen is found in discrete spot-like structures. We arenot aware of other antigens with a similar distributionduring mitosis.

Cell type- and species-specific occurrence of the 5E10antigenThe monoclonal antibody reacted with all rat cell lines(NRK and FTO-2B) and human cell lines (HeLa, T24,SK-N-M-IX-C, CaCo and MCF7) tested by indirectimmunofluoresence, but not with a variety of cellsoriginating from mouse (P19EC, P19MES-1 and 3T3),chicken (CH249) or African green monkey (CV1-HP8).The antibody did not react with the lower eucaryotes

Dictyostelium and Saccharomyces. In all positive cellsthe same speckled distribution of the 5E10 antigen wasobserved as shown in Fig. 3 for T24 cells. (See also Fig.7, lanes B and D, for the same pattern in HeLa cells.)

Indirect immunofluorescence of nuclei isolated fromrat tissues showed that not every individual nucleusfrom these tissues bound the 5E10 antibody. Most livernuclei (±90%) reacted with the 5E10 antibody; how-ever, only a limited fraction of the kidney cell nuclei(±10%) did. Evidently, both tissues contain cell typesthat are 5E10 negative. No reaction at all was observedwith nuclei obtained from murine liver, brain andkidney. These observations were confirmed by westernblotting. Nuclei from rat liver, brain and kidney showedthe presence of the same major 126 kDa antigen asfound in the human cell lines T24 and HeLa. Noreaction was found with nuclei from the murine tissues.Clearly, the antigen is present in most, but not all, ratand human cells.

Association of the 5E10 antigen with the nuclearmatrixWe have investigated whether the 5E10 antigen is asoluble protein, chromatin-associated, or a nuclearmatrix-bound protein. To this end we fractionated ratliver cells as follows. The tissue was homogenized andnuclei were isolated by centrifugation. The 5E10antigen was present in the nuclear fraction, butcompletely absent from the post-nuclear supernatant(Fig. 1, lanes 1 and 2). Subsequently, nuclei weretreated with DNase I, RNase A and 1.6 M NaCl toextract essentially all the chromatin and RNA. Thisprocedure released only a small fraction of the 5E10antigen (Fig. 1, lane 3). Evidently, the antigen wasassociated with the residual fraction (Fig. 1, lane 4),called nuclear matrix (Stuurman et al., 1990). Thismatrix was further fractionated by incubation withreducing agent, i.e. DTT (20 mM). In this way part ofthe internal fibro-granular structure that is presentthroughout the nucleoplasm is extracted. The residualsedimentable fraction is enriched in nuclear envelopes(Kaufmann and Shaper, 1984). About one-third of the5E10 antigen was released after reduction (Fig. 1, lane5). The other two-thirds was still associated with thenuclear envelope-enriched fraction (Fig. 1, lane 6). Weconclude that the 5E10 antigen is tightly associated withthe nuclear matrix.

To investigate the ultrastructural localization of theantigen associated with the nuclear matrix, we havestudied its distribution in nuclear matrices prepared insitu from HeLa cells according to the method of Fey etal. (1986). These preparations contained 72% ofnuclear RNA and less than 1% of the original amountof DNA. The matrices were processed for electronmicroscopy by pre-embedment labeling with colloidalgold-labeled antibody. Comparison of Fig. 5 A/C andB/D shows that the 5E10 antigen-clusters found inTriton X-100-extracted cells were retained in thesenuclear matrices. Their size and shape had not beenchanged by the matrix isolation procedure. Theseresults showed that the 5E10 antigen clusters are


B^

fDFig. 5. Localization of the 5E10 antigen in nuclei and nuclear matrices by electron microscopy in Epon sections. (A and C)Nuclei of Triton X-100-permeabilized and glutaraldehyde-fixed HeLa cells. (B and D) Nuclear matrices (glutaraldehyde-fixed) of HeLa cells. The preparations were labeled with mAb 5E10 using a pre-embedment immunogold labeling protocol.The ultra-small colloidal gold particles (1 nm) were silver-enhanced. Epon-embedded sections of 250 nm are shown in thisfigure. L and N, nuclear lamina and nucleoli, respectively. D is a higher magnification of B; A and C are derived fromdifferent cells in the same section. Bars: A and B, 1 /xm; C and D, 0.1 /an.

associated with the nuclear matrix, thereby confirmingthe biochemical data presented in the previous section.

Immunofluorescence data showed that the 5E10-

labeled spots remained detectable in the matrix afterextensive RNase and DNase treatment of these nuclearmatrices also (not shown), notwithstanding the removal


Phaso-contrast Hoechst 33258 5E10

Fig. 6. Localization of 5E10 antigen duringmitosis. T24 cells were grown on coverslipsand fluorescently labeled with mAb 5E10.DNA was stained with Hoechst 33258.(A) Interphase. (B) Prophase.(C) Prometaphase. (D) Metaphase.(E) Anaphase. (F) Telophase. (G) Early G!cells. Bar, 10 fixn.

of most of the internal nuclear material by thisprocedure. This strongly suggests that RNA and DNAare not major structural components of the 5E10domains.

Relation of the 5E10 antigen to other nuclearcomponentsThe punctate labeling pattern of 5E10 observed afterindirect immunofluorescence is reminiscent of the


Autoanti bodies 5E10

B

Fig. 7. Double labeling with autoantibodies and mAb5E10. (A) Methanol-fixed T24 cells were labeled withhuman autoimmune serum H33 recognizing centromeres(left panel) and with mAb 5E10 (right panel).(B) p-Formaldehyde-fixed HeLa cells were labeled withhuman autoimmune serum G57 recognizing Ul snRNPs(left panel) and with mAb 5E10 (right panel).(C) p-Formaldehyde-fixed T24 cells were labeled withautoantibody 1745 (left panel) and with mAb 5E10 (rightpanel). (D) p-Formaldehyde-fixed HeLa cells were labeledwith autoantibody SUN-3 (left panel) and with mAb 5E10(right panel). Bar, 10 ^m.

distribution of centromeres and snRNPs (Ringertz etal., 1986; Nigg, 1988). The autoimmune serum H33recognizes centromeres in a number of cultured celllines (Van Venrooij, personal communication). Analy-sis of interphase cells that were double labeled with the

mAb 5E10 and the H33 serum showed no significantcolocalization of centromeres with 5E10 spots (Fig.7A). The autoimmune serum G57 reacts with UlsnRNP-specific proteins (Van Venrooij, personal com-munication). It labeled T24 nuclei in a more or lessdiffuse manner with the exclusion of nucleoli andsomewhat brighter staining in other areas of thenucleus, in agreement with published results (Verheijenet al., 1986; Carmo-Fonseca et al., 1991). Doublelabeling of the anti-snRNP serum and the mAb 5E10showed that most of the 5E10 spots did not colocalizewith the areas of high intensity staining with G57 (Fig.7B). Also no colocalization was observed with anotherantibody (mAb 2.73; Billings et al., 1982) specific forUl snRNPs, and an antibody binding U2 snRNPs(mAb 4G3; Habets et al., 1989) (data not shown).Evidently, the 5E10 domains are related neither tocentromeres, nor to snRNP clusters.

Recently a "novel nuclear domain" was described byAscoli and Maul (1991) with the aid of mAbs andhuman autoantibodies recognizing structures that ap-pear as "nuclear dots" distributed throughout thenucleoplasm. With one of these autoantibodies, desig-nated 1745 (G. G. Maul, personal communication), weobtained the same immunofluorescence pattern as with5E10 (Fig. 7C). This colocalization indicates thatnuclear dots (Ascoli and Maul, 1991) and the 5E10domains represent the same nuclear structures.

Another type of speckled nuclear pattern is observedwith autoimmune sera from patients suffering fromprimary biliary cirrhosis. Immunofluorescent labelingof HeLa cells with such a serum (SUN-3) resulted in adot-like pattern (Szostecki et al., 1987). Doublelabeling of SUN-3 and mAb 5E10 revealed that the twopatterns overlap almost completely (Fig. 7D). Theprincipal antigen recognized by the SUN-3 antiserum isa 100 kDa protein, which differed significantly inmolecular mass from the 126 kDa 5E10 antigen asdetermined by immunoblotting (data not shown). Sincethe SUN-3 antibody is thought to recognize nuclearbodies (Fusconi et al., 1991), this colocalization sup-ports our conclusion from electron micrographs (Fig. 5)that the domains recognized by mAb 5E10 are nuclearbodies.

Discussion

We have isolated a monoclonal antibody (5E10) thatrecognizes discrete domains in interphase nuclei ofhuman and rat cells. These spherical structures have adiameter of about 0.3 jan. The number of domainsvaries considerably from cell to cell. The average is 21± 10 (n=132) in the human bladder carcinoma cell lineT24. The domains were found throughout the nucleusand were predominantly located in areas containing lessdensely packed chromatin. The principal antigen hadan apparent molecular mass of 126 kDa and occurred inseveral forms with a pi around 5.5, suggesting that theantigen is subjected to post-translational modifications.The antigen and the domains were tightly associated


with the nuclear matrix. Since the domains retainedtheir original morphology after removal of most of thechromatin and RNA, their basic structural componentsare probably proteins. Remarkably, 5E10-labeledstructures remained detectable during mitosis. Thestructures did not interact with metaphase chromo-somes.

Previously, we defined a set of nuclear matrixproteins (the so-called minimal matrix) that are presentin matrix preparations from different mammalian celllines and tissues, and may have conserved, possiblystructural functions (Stuurman et al., 1990). Theepitope detected with mAb 5E10 was not found inmouse tissues and cell lines, and it was absent in at leastsome types of rat cells. We do not exclude thepossibility that homologous proteins may be present inmouse cells and in the rat cells that do not react withmAb 5E10. However, in a strict sense, the 5E10 antigendoes not belong to the class of minimal matrix proteinsdefined by Stuurman et al. (1990). We show, however,that the 5E10 antigen is present in most rat and humancell lines and tissues. Therefore, it is likely that theantigen has a general function in these mammaliancells.

Nuclear bodies are discrete electron-dense structuresof diverse morphology, identified exclusively by trans-mission electron microscopy. The most common classof nuclear bodies (so-called 'simple' nuclear bodies)consists of structures with a diameter of 0.3 to 0.5 /an(see Bouteille et al., 1974, for a review). We haveidentified the structures labeled by the 5E10 antibody assuch "simple" nuclear bodies (Bouteille et al., 1974;Padykula and Clark, 1981; Chaly et al., 1983a;Ghadially, 1988), because (i) mAb 5E10 labeleddiscrete electron-dense structures; (ii) the size andshape of these structures are the same as that of simplenuclear bodies; (iii) these structures, like nuclearbodies, are surrounded by electron-translucent ma-terial. We found colocalization of the 5E10 antigen witha 100 kDa protein bound by immunoglobulins presentin serum (SUN-3) from a patient suffering from primarybiliary cirrhosis (Szostecki et al., 1987). Other sera frompatients with the same autoimmune disease gave asimilar speckled nuclear labeling and have been shownto detect proteins located in nuclear bodies. One ofthese proteins is thought to be the same as detected bythe SUN-3 serum (Fusconi et al., 1991). These findingstogether support our conclusion that the 5E10 antigen isconcentrated in nuclear bodies. Several other obser-vations fit the notion that the mAb 5E10 detects anuclear body protein: (i) Chaly et al. (1983b) haveshown that, like the 5E10 domains, nuclear bodies areassociated with the nuclear matrix; (ii) structures thatresemble nuclear bodies have been found in thecytoplasm (Rupee, 1969; Yasuzumi et al., 1981), as weobserve for 5E10 domains; (iii) nuclear bodies aresometimes associated with thread-like nuclear struc-tures (Lane, 1969; MasurOvsky et al., 1970; Bouteille etal., 1974) reminiscent of threads labeled with 5E10 insome T24 cells. It is generally assumed that nuclearbodies are associated with heightened levels of cell

activity (Bouteille et al., 1974; Chaly et al., 1983a).They have, among others, been implicated in steroidhormone action (Padykula and Clark, 1981; Brasch etal., 1989) and transport of rRNA from nucleolus tocytoplasm (Vagner-Capodano et al., 1982).

An important issue is the function of the nucleardomain that is recognized by mAb 5E10. A variety ofnuclear functions and components have been shown tooccupy well-defined domains inside the interphasenucleus, much like the 5E10-labeled domains. More-over, many of these nuclear components and functionsare associated with the nuclear matrix, like the 5E10antigen. We were able to show that the 5E10 antigen isnot associated with centromeres or snRNP clusters,which are involved in RNA processing. DNA repli-cation (Mills et al., 1989; Mazzotti et al., 1990), DNaseI-sensitive sites (Hutchinson and Weintraub, 1985; DeGraaf et al., 1990), and specific mRNAs (Lawrence etal., 1989; Xing and Lawrence, 1991) have also beenshown to be highly localized in the nucleus. In contrastto what is known about these nuclear components, the5E10 antigen clusters are also found in the cytoplasmand remain detectable in metaphase, not being associ-ated with metaphase chromosomes. Therefore, wethink it less likely that the 5E10 antigen is associatedwith any of these aforementioned nuclear elements.The consistent observation of 5E10 domains in thecytoplasm is compatible with a role in nucleo-cytoplas-mic transport. Also the observation that 5E10 antigenclusters are preferentially located in interchromatindomains in interphase nuclei may support this notion.To resolve the enigma of the function of the 5E10-labeled domains we are at present isolating thesestructures. Analyzing their composition and structuremay give clues to their function.

We have shown by double-label immunofiuorescencemicroscopy that antigens present in nuclear domainscalled 'nuclear dots' (Ascoh' and Maul, 1991) colocalizewith those recognized by 5E10. Many other antibodieshave been shown to label the nucleus in a dot-likefashion quite like that described in this paper for mAb5E10 (e.g. see Izant et al., 1982; Turner et al., 1985;Bonifacino et al., 1985; Lehner et al., 1986; Barque etal., 1990; Haaf and Schmid, 1990; Ohta et al., 1990).The function of none of these nuclear compartments isknown. It will be important to compare carefully theantigens and nuclear structures detected by theseantibodies and by mAb 5E10 and look for the functionsof these domains. The answer may shed important newlight on the infrastructure and functional organizationof the cell nucleus.

We thank Drs W. J. van Venrooij, F. A. Bautz and G. G.Maul for their generous gifts of autoimmune sera, and Mrs M.Hendrix and G.-J. de Fluiter for help in preparing monoclonalantibodies.

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{Received 3 October 1991 - Accepted 20 December 1991)

a monoclonal antibody recognizing nuclear matrix ... · chicken hepatoma). t24, hela, caco, mcf7,...

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