PROTEIN COMPOSITION OF NUCLEAR MATRIX PREPARATIONS FROM HeLa CELLS: AN IMMUNOCHEMICAL ... · 2005-08-25 · immunochemical techniques. In this paper we use the terminology of Kaufmann

J. Cell Sri. 80, 103-122 (1986) 103Printed in Great Britain © The Company of Biologists Limited 1986

PROTEIN COMPOSITION OF NUCLEAR MATRIX

PREPARATIONS FROM HeLa CELLS: AN

IMMUNOCHEMICAL APPROACH

RON VERHEIJEN1"*, HELMA KUIJPERS1, PETER VOOIJS1,WALTHER VAN VENROOIJ2 AND FRANS RAMAEKERS1

^Department of Pathology, Radboud Hospital, and 1Department of Biochemistry,University of Nijmegen, Geert Grooteplein Zuid 24, 6525 GA Nijmegen, The Netherlands

SUMMARY

Procedures for the isolation of HeLa S3 nuclear matrices were re-examined with specialemphasis on the use of various nucleases and detergents as well as on the ionic strength of the finalsalt extraction.

The protein composition of the resulting nuclear matrix preparations was analysed by one- andtwo-dimensional gel electrophoresis and found to be extremely reproducible. By means of co-electrophoresis several typical cytoskeletal proteins (actin, vimentin and cytokeratins) andheterogeneous nuclear RNA (hnRNA)-associated core proteins (hnRNP) were shown to bepresent in such nuclear matrix preparations. The nature of some other protein components waselucidated using two-dimensional immunoblotting and immunofluorescence. For this purposemouse monoclonal antibodies to cytoskeletal components (vimentin, cytokeratins), small nuclearRNP (70x \tfMT protein of Ui-RNP), hnRNP (Ci/Q.) and the pore-complex lamina (lamins A,B and C) were used next to human autoimmune sera obtained from patients with connective tissuediseases and directed against the residual nucleoli and the internal fibrillar mass. These antibodiesenabled us to identify a number of proteins present specifically in the nuclear matrix and to showthat part of the cytoskeletal proteins are still present in the isolated structures.

INTRODUCTION

When isolated nuclei are depleted of their membranes, soluble molecules andchromatin by means of subsequent treatments with detergents, nucleases and high-salt solutions, a structural framework, mostly referred to as the nuclear matrix,remains (reviewed by Agutter & Richardson, 1980; Kaufmann & Shaper, 1984).

The isolated nuclear matrix consists of three morphologically distinguishablestructural elements: (1) a peripheral layer, which represents the remainder of thenuclear envelope and contains pore-complexes in association with a lamina; (2)residual nucleoli; and (3) internal fibrillar structures.

The peripheral pore-complex lamina has been isolated separately and itspolypeptide composition has been determined (Franke, Scheer, Krohne & Jarasch,1981). In higher eukaryotes three distinct polypeptides, lamins A, B and C (60 to

• Author for correspondence.

Key words: nuclear matrix, cytoskeleton, hnRNP, monoclonal antibodies, autoantibodies.

104 R. Verheijen and others

74xlO3Mr), can be discerned. Studies dealing with the chromatin-depletednucleolar residue indicate that it contains a unique subset of proteins within nuclearmatrix preparations (Peters & Comings, 1980; Comings & Peters, 1981; Krohneetal. 1982).

Little is known about the structural polypeptides forming the intranuclear fibrillarnetwork, but the few studies that have been done indicate that it is very complex(Peters & Comings, 1980; Capco, Wan & Penman, 1982; Fischer, Berrios & Blobel,1982). Experimental evidence showing which polypeptides form the structuralbackbone is lacking, although some reports have shown that actin, apparently in anon-filamentous form, is the main component of the nuclear matrix (Clark &Rosenbaum, 1979; Krohne & Franke, 1980; Capco et al. 1982; Nakayasu & Ueda,1983; Staufenbiel & Deppert, 1984).

There is good evidence that this internal matrix represents a structure that alsoexists in the intact cell (van Eekelen & van Venrooij, 1981; Kaufmann, Coffey &Shaper, 1981; Brasch, 1982; Capco et al. 1982; van Eekelen et al. 1982; Fischeret al. 1982). The internal matrix structure of HeLa cells, for example, seems not todepend on RNA or DNA for its structural integrity. Even so, disulphide bridgeformation is not likely to be responsible for its formation (van Eekelen et al. 1982).Furthermore, specific proteins can be found in the isolated matrix (Fey, Wan &Penman, 1984).

Studies in which the three-dimensional structural organization of isolated matriceswas viewed using electron microscopy on whole mount preparations instead of thinsections corroborate the hypothesis of the existence of an internal nuclear proteinstructure. These studies also showed a distinct interaction between the cytoskeletonand the nuclear matrix (Capco et al. 1982; Capco, Krochmalnic & Penman, 1984;Veyetal. 1984).

Additional indications of the existence of an intranuclear structural framework canbe deduced from studies of its functional aspects in, for example, DNA replication(McCready et al. 1980; Pardoll, Vogelstein & Coffey, 1980; Vogelstein, Pardoll &Coffey, 1980; Berezney & Coffey, 1985), hormone binding (Barrack & Coffey,1980), association with viral tumour antigens (Staufenbiel & Deppert, 1983), andprocessing and transport of RNA (Herman, Weymouth & Penman, 1978; Miller,Huang & Pogo, 1978; Maundrell, Maxwell, Puvion & Scherrer, 1981; Jackson,Caton, McCready & Cook, 1982; Mariman et al. 1982a; Mariman, Hagebols & vanVenrooij, 19826; Ben-Ze'ev & Aloni, 1983; Mariman, van Beek-Reinders & vanVenrooij, 1983).

The primary transcript of DNA in eukaryotic cells is heterogeneous nuclear RNA(hnRNA). This hnRNA is present in the cell nucleus as fibrillar ribonucleoprotein(RNP) particles and granules (Holoubek, 1984; Wilket al. 1985). The major proteincomponents of hnRNP complexes are the core proteins of about 30 to 41XlO3Mr.The nomenclature proposed for these proteins by Beyer, Christensen, Walker &LeStourgeon (1977) has recently been extended by Wilk et al. (1985) as a result oftheir two-dimensional gel analyses of the core proteins from isolated 35-40 S hnRNPcomplexes.

Immunochemical characterization of nuclear matrix proteins 105

In our approach to identify the nature of the protein components that participate

in the nuclease- and high-salt-resistant nuclear structure we have examined the

effects of several methods of preparation on the polypeptide pattern of this matrix,

including different types of nuclease treatments and high-salt extractions. After the

establishment of such a routine procedure, and verification of its reproducibility,

several of the polypeptides present in such preparations were identified using

immunochemical techniques.

In this paper we use the terminology of Kaufmann & Shaper (1984) and define the

nuclear matrix as the detergent-, nuclease-, and salt-resistant entity composed of

components of the nucleolus, a non-histone intranuclear meshwork, and a peripheral

layer composed of the lamina with its pore-complexes.

MATERIALS AND METHODS

Cell culture and labelling

Tissue culture media and calf sera were purchased from Flow Laboratories Ltd, Irvine,Scotland. HeLa S3 cells (human cervix carcinoma) were grown in suspension at 37°C at densitiesranging from 0-5X106 to 106cells ml"1 on Suspension Minimal Essential Medium supplementedwith 10% newborn calf serum and 1-Sgl"1 lactalbumin hydrolysate (van Eekelen et al. 1982).Cellular protein was labelled by incubating the cells for 16h with S-lO/iCiml"1 [35S]methionine(Amersham U.K. ± 1000 Ci mmol"1) at densities of 106 to 2X106cells ml"1. For the first 2-3 h thecells were incubated in tissue culture medium that contained only labelled methionine, then0-1 vol. of complete medium was added.

Cell fractionation and purification of nuclear matrices

All chemicals were of analytical grade. Buffers were boiled in the presence of 0-02% diethyl-pyrocarbonate and then autoclaved. Cell fractionations were carried out in the presence of 0-5 mM-phenylmethylsulphonyl chloride (PMSC) and 5 mM-./v'-ethylmaleimide (MalNEt) to reduceproteblytic degradation and disulphide bridge formation, respectively. These agents were addedfrom,freshly prepared stocks. Ribonuclease A (RNase A) (Sigma Chemical Co., Mflnchen) waspre-incubated for 15 min at 100°C to reduce possible protease activity. Centrifugation steps werecarried out for 5 min at 800 # and 2°C.

The procedure that we have established for the isolation of nuclear matrices, carried out at0—4°C, is as follows: cells were harvested on frozen NKM buffer (130mM-NaCl, 5mM-KCl,1-5 mM-MgCl2), pelleted by centrifugation, washed twice with isotonic NKM solution and pelletedagain. Each of the following steps in the procedure was preceded by washing the pellet twice withreticulocyte suspension buffer (RSB) (lOmM-NaCl, lOmM-Tris-acetate, pH7-4, l-5mM-MgCl2).Subsequently, the cell pellet was suspended in hypertonic buffer (RSB with 0-3 M-sucrose) andafter addition of 0-05 vol. 10 % Triton X-100 in RSB the suspension (4x 107 cells ml"1) was gentlyswirled in ice for about 1 min and centrifuged to sediment the cytoskeletons. After washing thesewere resuspended in RSB (4X107 cells ml"1) and, after addition of 0-1 vol. of a freshly preparedsolution of 5 % sodium deoxycholate (DOC)/10 % Tween-40 in RSB, homogenized by 10 strokesof a motor-driven Teflon pestle in a Potter tissue homogenizer (Kontes Co., Vineland, N.J.). Thenuclei were pelleted, washed and resuspended in HRSB (HOmM-NaCl, lOmM-Tris-acetate,pH7-4, l-5mM-MgCl2) at a density of 1X108 nuclei ml"1 and incubated with 800/igmP1 deoxy-ribonuclease I (DNase I) (Sigma) and 25/igml"1 RNase A (Sigma) for 15 min at 20°C. Duringthis digestion step MalNEt was omitted, but immediately after the incubation it was added again toa final concentration of 5 mM.

The DNA-depleted nuclei were spun down, washed and gently resuspended in 0 - 4 M -(NH^SO*, 50mM-Tris-acetate, pH 7-4, 1*5 mM-MgCl2. The matrices were pelleted, washed andresuspended in RSB.


Gel electrophoresisSamples were prepared for gel electrophoresis as described by van Eekelen & van Venrooij

(1981). After pelleting, the nuclear matrices were immediately dissolved in sodium dodecylsulphate (SDS) sample buffer. SDS/polyacrylamide gel electrophoresis (SDS/PAGE) wasperformed using the Laemmli (1970) buffer system.

For two-dimensional gel electrophoresis under non-equilibrium isoelectric focusing conditionsin the first dimension, the procedure of O'Farrell was used (O'Farrell, Goodman & O'Farrell,1977), and electrophoresis was performed for 1800 Vh. For the second dimension 10% SDS/polyacrylamide gels were used.

For the identification of non-muscle actin and vimentin on two-dimensional gels a cytoskeletalpreparation from bovine lens cortical fibres was comigrated with [35S]methionine-labelled HeLaproteins. For the localization of cytokeratin 9pots (cytokeratins 7, 8, 18 and 19, according to thenomenclature of Moll et al. 1982) a cytoskeletal preparation of the human bladder carcinoma cellline T24 was used.

The hnRNA-associated core proteins were identified by comigration with unlabelled coreproteins of 35-40 S hnRNP complexes (Wilk et al. 1985).

Blotting and detection of proteinsTransfer of proteins from 10 % polyacrylamide gels onto nitrocellulose sheets was performed as

described by Habets et al. (1983). After transfer the blots were dried and stored at roomtemperature. Detection of the antigens on the blots was essentially performed as described (Habetset al. 1985). For detection of labelled proteins on the gels the procedure of Bonner & Laskey (1974)was used.

Immunofluorescence microscopyHeLa cell nuclear matrix preparations were immunolabelled in suspension essentially as follows:

about 5X106 matrices were centrifuged (5min, 800 # 4°C), the pellet washed twice with 200^1phosphate-buffered saline (PBS) containing 5 % foetal calf serum (FCSy and pelleted again.

The matrices were resuspended in 50 (A of the primary antibodies in the appropriate dilutionsand incubated for 45 min at 4°C, with occasional stirring. Table 1 summarizes the antibodypreparations used for immunoblotting and immunofluorescence studies. Subsequently the

Table 1. Characteristics of the antibodies used for immunoblotting and immuno-fluorescence studies

Antibody

RGE53/CK18-pKerRV201pVim41CC4LN43T100J26T5Z3L154F42-73

Antigen

•2 M Cytokeratin 18R Human callus keratinsM Bovine lens vimentinR Bovine lens vimentinM Lamins A,B,CM Lamin BH Nucleolar proteinsH Nucleolar proteinsH Nucleolar proteinsH 86xlO3A/r

H 56, 70(Xl03Jtfr)M C1/C2 core proteinsM 70xl03Mr/Ul-RNP

Reference

Ramaekers et al. (19836)Ramaekers et al. (1983a)UnpublishedRamaekers et al. (1983a)Burke et al. (1983)UnpublishedUnpublishedUnpublishedUnpublishedvan Venrooij et al. (1985)UnpublishedChoi &Dreyfuss (1984)Billings «* a/. (1982)

Code inFig. 9

18

v,v#,v*»L.,LC

UNb,Ne

N.,NC

Nd

I.Id.Ic

Ib

M, mouse monoclonal antibody; R, polyclonal rabbit antiserum; H, human autoimmune serum;L, lamins; N, ilucleolar proteins; I, proteins localized in the internal fibrillar structure.


I1a

100-

80-

40-

20-

NaCI

-o(NH,)2SO4

0-5 1-0 1-5/ (salt solution)

20

Fig. 1. Estimation of the percentages of high-salt extracted [35S]methionine-labelledproteins from HeLa nuclei as a function of ionic strength of the extraction buffer. Thevalues given are the average of four experiments showing deviations of ± 2 % . The ionicstrength / of a solution is given by the relation / = l/2£,-C,Z,-2, where C,- is theconcentration of an ion of type i and Z,- the number of charges carried by this ion.

matrices were pelleted, washed twice with 200 (A PBS/5 % FCS and incubated for 45 min at roomtemperature in 50/il of the appropriate fluorescein isothiocyanate(FITC)-conjugated secondantibodies (Nordic, Tilburg, The Netherlands). These included: FITC-conjugated rabbit anti-mouse immunoglobulin G (IgG) (heavy and light chains) for detection of the monoclonal primaryantibodies, FITC-conjugated goat-anti-rabbit IgG (heavy and light chains) for the detection of thepolyclonal rabbit primary antibodies and FITC-conjugated goat-anti-human Ig (heavy and lightchains) for the human autoimmune antibodies.

After this incubation the matrices were washed as described above and suspended in 50/ilPBS/glycerol (1:1, v/v). The fluorescent matrix samples were diluted 1:25 in PBS containing10% normal goat serum and spun down onto coverslips using a Cytospin centrifuge. Thesamples were dried overnight at room temperature, mounted in Gelvatol (Monsanto, St Louis,Missouri, U.S.A.) containing lOOmgml"' l,4-diazobicyclo-[2,2,2]-octane (DABCO; JanssenPharmaceutica, Beerse, Belgium) and viewed with a Leitz Dialux 20 EB microscope equipped withepifluorescent illumination using appropriate filters for fluorescein fluorescence. Pictures weretaken on Tri-X film (Kodak) with an automatic camera using an ASA setting of 400.

RESULTS

Effects of high-salt extraction

In studying the effects of salt solutions on the protein patterns of the nuclearmatrices, DNA-depleted nuclei were extracted with salt solutions of various ionicstrengths in the presence of 50 mM-Tris-acetate (pH 7-4) and 1 5 mM-MgC^ (Fig. 1).An increase in ionic strength leads to an increase of the amount of extractableprotein, with the maximal amount of extractable proteins being about 55 % in thecase of (NH4)2SO4 and about 60 % when NaCl is used. The results of extraction withKC1 were similar to those of NaCl (data not shown).


The various high-salt extracts were analysed on one-dimensional SDS/poly-acrylamide gels (Fig. 2) showing that although some variation in the relative amountsof extracted proteins can occur, there is no striking qualitative difference between thepatterns of extracted proteins or between the protein patterns of the remainingnuclear matrices. Only minor differences were observed between the protein patternsof the matrix obtained after treatment with (NH4)2SO4 or NaCl. In particular,somewhat more of a protein that is probably identical to actin could be extractedusing (NH4)2SO4 (arrows in Fig. 2).

Histones were almost completely removed from DNA-depleted nuclei by ionicstrengths of 0-9 and higher. From these data we have chosen a salt concentration inour extraction procedure of 0 -4M-(NH 4 ) 2 SO 4 .

Effects of nucleases

The effects of different nucleases were studied by incubating isolated nuclei eitherwith DNase I (RNase-free) (Fig. 3A), DNase i/RNase A (Fig. 3B) or with micro-coccal nuclease/RNase A mixtures (Fig. 3c). No striking differences between theprotein patterns of the three types of nuclear matrix preparations could be found,except that RNase treatment seemed to reduce the amount of hnRNA-associatedproteins in the nuclear matrix (compare Fig. 3A with B and c) and micrococcalnuclease treatment seemed to remove more of some unidentified polypeptides, asindicated by the open arrowheads in Fig. 3B.

Two-dimensional gel electrophoretic analysis of nuclear matrix proteins

The routine procedure that we use for the isolation of nuclear matrices on the basisof the foregoing experiments does not differ essentially from methods describedearlier by van Eekelen et al. (1982) and Fey et al. (1984). In the procedure of vanEekelen et al. the Triton X-100 step was omitted while the digested nucleic acids andtheir associated proteins were extracted in two steps. Fey et al. (1984) used 0-25 M-(NH4)2SO4 in the high-salt extraction instead of the 0-4 M-(NH4)2SO4 as used by us.

Fig. 4 shows the one-dimensional SDS/polyacrylamide gel patterns and Fig. 5 thetwo-dimensional patterns of the different fractions obtained after the subsequentextraction steps. One of the major protein components that occurs only in the nuclearmatrix fraction consists of a number of protein spots in the 65-72xlO3Mr region,with isoelectric points ranging between 8 and 8-5. This group of apparently closelyrelated polypeptides is shown in detail in Fig. 6. The identity of this cluster, whichconsists of about 15-17 polypeptide spots, is unknown. Fig. 7 shows the electron-microscopic appearance of a nuclear matrix isolated by the routine procedure.

Cytoskeletal proteins

Comparing all the protein patterns shown in Fig. 5, it is obvious that a 43 X 103Mr

polypeptide, comigrating in both dimensions with actin from bovine lens (indicatedas a) occurs in all fractions in relatively high amounts. Polypeptide spots comigrating


with the intermediate filament proteins vimentin (indicated by v in Fig. 5) and thefour HeLa cytokeratins (i.e. nos 7, 8, 18 and 19, indicated as such in the gels) wereobserved in all fractions except in the soluble fraction and in the DNase/RNaseincubation supernatant. In the DNase/RNase extract, however, a small amount of apolypeptide comigrating with cytokeratin 8 can be detected. The polypeptidemigrating in the vicinity of vimentin (asterix in Fig. 5A) may represent either atubulin subunit or a vimentin breakdown product, which has a molecular weightslightly lower than vimentin and behaves in a slightly more acidic way on NEpHGEgels.

hnRNP

hnRNA-associated proteins (hnRNP) can be subdivided into three classes. Themain components are represented by the Ai, A2, Bia, Blb, Bl c, B2, C1; C2, C3 andC3x polypeptides (Wilkef al. 1985), which are indicated as such in Fig. 5c,D,E.

As a result of DNase/RNase treatment, proteins A b A2, Bia and Blb are partlyreleased. After the subsequent high-salt treatment part of the Ci, B2 and probablyBic are released next to a remainder of Aj, A2, Bia and Blb. By using comigration wewere unable to indicate the positions of proteins C2, C3 and C3x in these fractions.However, as will be shown below, using immunoblotting we could show theirpresence in the nuclear matrix fraction.

Identification of nuclear matrix proteins by the immunoblotting method

Two-dimensional gels of [35S]methionine-labelled nuclear matrix preparationswere blotted onto nitrocellulose sheets and used for the immunochemical detectionand characterization of several nuclear proteins. All prominent protein spots, presenton the autoradiographs of the gels, also occurred on the autoradiographs of suchprotein blots, indicating an optimal transfer of polypeptides in all regions ofmolecular weight and isoelectric point (not shown). Using several monoclonalantibodies as well as human autoantibodies directed to specific components of thecytoskeleton and the nucleus (see Table 1), on these [35S]methionine-labelledprotein blots we were able to identify a number of proteins present specifically in thenuclear matrix (Fig. 8).

Monoclonal antibodies to the intermediate filament proteins vimentin and cyto-keratin 18 clearly recognized these proteins in the blots (Fig. 8A). The immuno-fluorescence studies with these monoclonal antibodies and the rabbit antiseradirected against keratin and vimentin stain a fibrillar network apparently surround-ing the nuclear matrix (Fig. 8B,c).

The monoclonal antibody 41CC4 to rat liver lamins A, B and C (Burke, Tooze &Warren, 1983) detected, on two-dimensional blots, only lamins A and C of HeLacells (Fig. 8D). For the detection of lamin B we used the monoclonal antibody LN43(Fig. 8E). Immunofluorescence studies with these two lamin antibodies revealed adiffuse staining of the whole nuclear matrix structure with a higher fluorescenceintensity at the matrix periphery (Fig. 8F).


1 2 3 4 5 1* 2* 3* 4* 5*

1C 3

- 6 9

- 4 6

| M 1 M - 3 0

ff

2A

1 2 3 4 5 1* 2* 3* 4* 5*

• 6 9

•46

- 3 0

B


The core proteins Ci and C2 of the 40 S hnRNP particle (according to thenomenclature proposed by Beyer et al. 1977) were detected by monoclonal antibody4F4 (Choi & Dreyfuss, 1984). From Fig. 8G it can be seen that both proteins arepresent over a relatively broad range of isoelectric points in our blots, probably due tonucleic acid remainders still in tight interaction with these hnRNP core poly-peptides. It is likely that the Ci (39X 103Mr) and C2 (41X 103Mr) correspond to theC3 and C3X core proteins, respectively, described by Wilk et al. (1985). In thefluorescence pictures it can be seen that this antibody stains the nuclear matrix ratherdiffusely except for the nucleoli (Fig. 8H).

The monoclonal antibody 2-73 directed against the 7OXlO3Mr protein ofUl—RNP (Billings, Allen, Jensen & Hoch, 1982) reacts with three discrete proteinspots (two major spots and one weaker spot) in the two-dimensional immunoblots.The three polypeptides migrate together within a small range of molecular weightsand pH values (Fig. 81). These proteins have been described to have a very slow rateof [3sS]methionine incorporation and can be detected only in Coomassie-Blue-stained gels (Billings & Hoch, 1984). Immunofluorescence shows a dot-likedistribution of the 70X 103Mr antigens in the nuclear matrix (Fig. 8J) .

In addition to the mouse monoclonal antibodies, human autoimmune sera frompatients suffering from connective tissue diseases were used for immunoblotting andimmunofluorescence studies. The carefully selected sera were either directed againstthe internal fibrillar mass of the nuclear matrix (Fig. 8L) or against the residualnucleoli (Fig. 8N) and showed, on one-dimensional Western blots, a specific reactionwith only one or two nuclear proteins.

The human autoimmune serum Z3, which was shown in one-dimensional blots toreact with an antigen with an apparent molecular weight of 86X103 that has beensuggested to occur specifically in the nuclear matrix (van Venrooij et al. 1985), reactson a two-dimensional blot with an antigen having an isoelectric point of about 8-4(Fig. 8K). Immunofluorescence studies with this serum on isolated nuclear matrixpreparations revealed a diffuse staining reaction, with exclusion of the nucleolarremainder (Fig. 8L).

In the immunoblots the three nucleolar antibodies T5, J26 and T100 each reactedwith different proteins, apparently specific for nucleoli (see, e.g., the reaction of T5in Fig. 8M,N).

Fig. 9 summarizes all our immunoblotting data. The schematic drawing of theautoradiograph shows the typical nuclear matrix protein pattern and theidentification of some components by immunoblotting. The indications used for thedifferent protein spots correspond to the code used in Table 1 and permits a directcorrelation between antiserum and proteins recognized on the immunoblots.

Fig. 2. Analysis of [35S]methionine-labelled proteins in the high-salt extracts of HeLanuclei and the corresponding remaining nuclear matrices. The proteins were extractedwith: A, (NH^SCV, or B, NaCl and analysed on a 13 % SDS/polyacrylamide gel. Lanes1 to S show the proteins extracted with salt solutions with ionic strengths of 0-3, 0-6, 0-9,1-2 and 1-8, respectively. Lanes 1* to 5* show the corresponding proteins in theremaining structures after these salt treatments. The arrow indicates the position of actin.

Mrx10"3

hnRNPproteins

B

18

- 86

- 74

- 57

- 43

i i i

9-3 8-4 7-1Fig. 3

5-3

- 32

Immunochemical characterization of nuclear matrix proteins

M A B C D M

10-3

- 9 3

4 - 6 9

« - 4 6

- - - - 3 0

Fig. 4. One-dimensional gel electrophoretic analysis of protein fractions of the extractionsteps obtained during the isolation of nuclear matrices. The gel is a 13% SDS/polyacrylamide get. Lanes A, Triton-soluble fraction of HeLa cells; B, DOC/Tween-soluble fraction; C, proteins released after DNase i/RNase A incubation; D, high-saltextractable fraction after nuclease treatment; and E, nuclear matrix preparation; M,molecular weight markers. [14C]-methylated marker proteins (Amersham) used(XlO~2Mt) were: lysozyme (14-3), carbonic anhydrase (30), ovalbumin (46), bovineserum albumin (69) and phosphorylase b (93).

Fig. 3. Two-dimensional gel analysis of nuclear matrix preparations treated withdifferent nucleases. The following treatments were tested:

A. Digestion with DNase I only, under RNase-free conditions. For these experimentsRNase-free DNase I was used (DPFF quality; Worthington Biochemical Corp.). Thenuclei ( lx io ' /ml ) were incubated for 15min at 20°C in HRSB containing 800^gml~'DNase I and 0-5 mM-PMSC.

B. Digestion with DNase i/RNase A. The nuclei (lXlO^/ml) were incubated for15min at 20°C in HRSB containing 800/igmr1 DNase I (Sigma), 25/igml"1 RNase A(Sigma) and 0-5 mM-PMSC.

C. Digestion with micrococcal nuclease/RNase A. A. The nuclei ( lx l t ^ /ml ) wereincubated for ISmin at 10°C in RSB containing 200 U ml"1 micrococcal nuclease (P-LBiochemicals, Inc., Milwaukee, Wis.), 25 ̂ gml"1 RNase A (Sigma), 0-5 mM-PMSC and1 mM-Ca2+. v, vimentin; a, actin; 7, 8 and 18, the different HeLa cytokeratin subunits.

R. Verheijen and others

B

IF •

>

.- &

r10"3

-86-74

-57

-43

-32

9-3 8-4 7-1Fig. 5

5-3


A m>

Fig. 6. Detail of a two-dimensional gel electrophoretic separation of a nuclear matrixpreparation showing the main protein cluster composed of about 15—17 basicpolypeptidea in the 65-72 (X103) Mr region. L, and Lc indicate the lamins A and C.

DISCUSSION

In this study we have tried to identify proteins of the detergent-, nuclease-, andsalt-resistant fraction of HeLa cells, usually referred to as the nuclear matrix(reviewed by Agutter & Richardson, 1980; Kaufmann & Shaper, 1984). The methodthat we now use routinely for the preparation of nuclear matrices has been developedfrom studies in which several conditions of nuclease and high-salt treatments weretested. The final procedure, however, is comparable to those described earlier by vanEekelen et al. (1982) and Fey et al. (1984).

Identification of nuclear matrix proteins in this study has been achieved mainly bya combination of two-dimensional gel ele'ctrophoresis and immunoblotting studiesusing mouse monoclonal antibodies and human autoimmune sera directed againstnuclear and cytoskeletal components (Table 1). In this way several proteins presentin nuclear matrix preparations could be identified.

Over the past years actin has been identified in many studies as a major protein inisolated nuclear fractions, but in most cases the possibility that actin was present as acytoplasmic contamination could not be excluded (Comings & Harris, 1976;LeStourgeon, 1978). Yet, it has been demonstrated that manually isolated andcleaned nuclei of amphibian oocytes contain large amounts of actin (Clark &Rosenbaum, 1979; Krohne & Franke, 1980) and, recently, Scheer, Hinssen, Franke& Jockusch (1984) have shown that nuclear actin of amphibian oocytes might beinvolved in the transcription of lampbrush chromosomes.

Fig. 5. Two-dimensional gel electrophoretic analysis of fractions A to E from Fig. 4.A. Triton-soluble fraction of HeLa cells; B, DOC/Tween-soluble fraction; C, proteinsreleased by DNase i/RNase A treatment; D, high-salt extractable fraction after nucleasetreatment; and E, nuclear matrix preparation. Ai, A2, Bi,, Bib, Bic and Ci indicate thehnRNA-associated core proteins according to the nomenclature of WMaet al. (1985).


Fig. 7. Electron-microscopic appearance of a HeLa nuclear matrix preparation. Electronmicroscopy was performed as described by van Eekelen & van Venrooij (1981). Bar,1-3/ffn.

In nuclear matrix preparations from HeLa cells we find a major component of43xlO3Afr, which comigrates with bovine actin on two-dimensional gels. Ourpreliminary results with rhodamine-conjugated phalloidin applied to nuclear matrixpreparations indicate that filamentous actin may be present in these preparations.However, the exact localization of the staining reaction cannot be determined on thebasis of these light-microscopic studies.

Protein components, which make up the intermediate filament cytoskeleton inHeLa cells (vimentin and cytokeratins), could also be identified as major spots innuclear matrix preparations in the two-dimensional gels and by immunoblotting.However, the immunofluorescence patterns indicate that these proteins occur as anetwork structure around the nucleus, which supports the idea that theseintermediate filament proteins are firmly attached to nuclear matrix components aswas suggested earlier (Woodcock, 1980; Granger & Lazarides, 1982; Peters, Okada& Comings, 1982; Capco et al. 1984; Fey et al. 1984). They are possibly involved in

Fig. 8. Immunochemical identification of proteins present in the nuclear matrix bymeans of two-dimensional immunoblotting (A.D.E.G.I.K.M) and immunofluorescence(B.C.F.H.J.L.N), using the antibodies described in Table 1. A. Vimentin and its breakdownproducts (V, V*, V") and cytokeratin 18 detected on the same immunoblot usingantibodies RV201 and RGE53, respectively. B,C. Filamentous staining patterns seen innuclear matrices when incubated with the monoclonal antibody to cytokeratin 18 (B) orvimentin (c). D. Lamins A and C detected in a blot with antibody 41CC4. E. Lamin B asdetected by the antibody LN43. F. Immunofluorescence pattern of antibody 41CC4 onnuclear matrices. G. C\ and C2 hnRNA-associated core proteins detected by antibody4F4. H. Staining pattern of antibody 4F4 on a nuclear matrix preparation. I,J. Immuno-blotting and immunofluorescence reaction of antibody 2-73. K,L. Immuno(histo)-chemical reactions of the human autoantibody Z3. M,N. Immuno(histo)chemicalreactions of the anti-nucleolar antibody T5.

Immunochemical characterization of nuclear matrix proteins

the positioning of the nucleus (Virtanen, Kurkinen & Letho, 1979; Virtanen, Vartio& Letho, 1982). The major protein components occurring exclusively in the nuclearmatrix fraction are represented by a cluster of basic polypeptides migrating in the65-72XlO3Mr region. Also, Kaufmann & Shaper (1984) have found that theintranuclear material of their nuclear matrix preparations of rat liver cells contain a

18

8A

M

- i

- 3 3

8'-4 5-3Fig. 9. Autoradiograph and schematic representation of a two-dimensional separation ofnuclear matrix proteins containing the immunoblotting data. Next to the componentssummarized in Table 1 actin (a) and cytokeratins 7 and 8 are also indicated. Note that thespot indicated as V* is only partly composed of vimentin breakdown product (cf.Fig. 8A).


series of basic (pi > 8-0) 60-70X 103Mr polypeptides, which are not recognized byanti-lamin antisera. It is likely that these proteins correspond to the cluster ofpolypeptides seen in our two-dimensional gels (Fig. 7). This unidentified set ofproteins do not react with any of the antisera used- in this study. The typical andextremely reproducible two-dimensional gel pattern of this protein cluster showingabout 15-17 discrete spots suggests that some of these proteins are the result of post-translational modifications.

In this study we find that some of the hnRNA-associated core proteins are stillfound to be present in nuclear matrix preparations (Fig. 3). Dreyfuss, Choi & Adam(1984) showed that the 39X103 and 41XlO3Mr proteins (C proteins) are removedquantitatively from the nuclear matrix at 0-5 M-NaCl after digestion with RNase.Using the same sequence of extraction steps we were, however, unable to removethese C proteins completely from the nuclear matrix.

Small nuclear RNAs (snRNAs) have been described to be integral components ofhnRNP particles (Busch, Reddy, Rothblum & Choi, 1982). One of these snRNAs,i.e. Ul-RNA, is known to be involved in pre-mRNA splicing (Kramer, Keller,Appel & Liihrmann, 1984) and its associated proteins are found in the nuclear matrixfraction. It has recently been suggested that the 70xl03Mr snRNP, recognized bythe monoclonal antibody 2-73 used in this study, might be involved in binding of Ul-RNP to the nuclear matrix, since it is not released by incubation with RNase orDNase of nuclei or nuclear matrices, as are the other Ul-RNA-associated proteins(Mariman & van Venrooij, 1985). This could indicate that the 70xl03Mr proteininteracts directly with components of the nuclear matrix or is an integral part of it.Our immunofluorescence and immunoblotting data support this assumption. Themonoclonal anti-70xl03Mr serum shows a dot-like distribution of the 70xl03A/r

antigens in the nuclear matrix. Also, in immunoblots of the matrix preparations wecould demonstrate the presence of this 70x 103Mr polypeptide, which, however, wasnot found in the autoradiographs. This can be explained by the finding of Billings &Hoch (1984) that this polypeptide has a very slow rate of incorporation of [3SS]-methionine, and can be detected only in Coomassie-Blue-stained gels.

In summary, we point out that a combination of two-dimensional gelelectrophoretic and immunoblotting techniques with well-defined antisera permitsthe characterization of nuclear matrix components. These protein constituents of theintranuclear mass may be difficult to study by other techniques because of theirhighly insoluble character. Future studies using immunoelectron microscopy, incombination with antisera such as described here, may provide valuable informationabout the localization and interrelationship of nuclear matrix proteins.

This study was supported by the Netherlands Cancer Foundation (Queen Wilhelmina Fund)grant no. NUKC 1984-11. The authors thank Dr G. Warren (Heidelberg) for his gift of the hybridcell line 41CC4, Dr S. Hoch (La Jolla, Ca) for her gift of the hybrid cell line 2-73, Dr G. Dreyfuss(Evanston) for his gift of the ascites fluid 4F4, Dr K. Schafer (Bochum) for providing the coreproteins of 35 S hnRNP complexes, Dr E. B. Lane (London) for the monoclonal antibody LN43and Dr R. Humbel (Luxembourg) for the human serum T100. Furthermore, we thank


A. Groeneveld for culturing the cells, Dr P. Sillekens for preparing the sample for electronmicroscopy, and G. Schaart and A. Huysmans for their excellent help in testing the intermediatefilament antisera. Mrs Y. Stammes is thanked for her excellent secretarial help in preparing themanuscript.

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{Received 21 June 1985-Accepted 4 July 1985)

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