REVIEW The nuclear matrix: structure and composition

RON VERHEIJEN1'*, WALTHER VAN VENROOIJ2 and FRANS RAMAEKERS1

'Department of Pathology and department of Biochemistry, University of Sijmegen, The Xetherlands

•Author for correspondence at: Department of Pathology, University Hospital of Nijmcgcn, Geert Grootcplein Zuid 24, 6525 GA Nijmcgcn, The Netherlands

Summary

Introduction Enzymes involved in DNA and RNA The pore complex-lamina metabolism The nuclear pore complexes Virus-specific proteins The nuclear lamina Associated transcripts The nucleolar residue The nuclear matrix and RNA transport The internal matrix Behaviour of nuclear matrix components during Morphological and biochemical aspects mitosis Heterogeneous nuclear RNP particles Small nuclear RNP particles Key words: nuclear matrix, pore complex-lamina, Nuclear actin nucleolar matrix, internal matrix, chromosomal scaffold.

Introduction However, it has been shown not to be an obligatory nuclear component (Lafond & Woodcock, 1983). The term nuclear matrix was first introduced by In general, nuclear matrix preparations from differ- Berezney & Coffey (1974) to denote a highly structured ent cells and tissues possess some common structural residual framework obtained from rat liver nuclei by entities: (1) the residual elements of the nuclear sequential salt extractions, detergent and nuclease envelope, also designated the pore complex-lamina; treatments. The isolated three-dimensional structure (2) the residual nucleoli; (3) a granular and fibrous consisted almost entirely of protein. Subsequent internal matrix structure that extends throughout the studies showed that when protease inhibitors were interior of the nucleus. included in all isolation steps and ribonuclease (RNase) In recent years various studies have implicated the was omitted, the isolated nuclear matrix contained nuclear matrix as being involved in nuclear activities RNA as the second most abundant component (Her- such as DNA metabolism (reviewed by Berezney, man et al. 1978; Miller et. al. 1978a; Shaper

Nucleolus Nuclear lamina Fig. 1. Schematic view of a cell nucleus. The nuclear envelope is composed of Outer inner and outer nuclear nuclear membrane membranes, which fuse at the regions where nuclear pores Inner are situated. The nuclear nuclear membrane lamina is localized on the Rough endoplasmic nucleoplasmic site of the inner reticulum membrane. molecules in nuclear matrix preparations, and thus ribosomes on its outermost surface. The inner mem- comprises only circumstantial evidence. This aspect brane is smooth. The nuclear pore complexes are has to be borne in mind in discussing the relation of situated in those regions where the two membranes particular nuclear functions with the nuclear matrix. fuse (Franked al. 19816; Gerace, 1986). Although in this review we want to focus mainly on The nuclear lamina is a fibrillar meshwork of pro- recently characterized nuclear matrix proteins, increas- teinaceous material, which is intercalated between the ing evidence indicates that heterogeneous nuclear RNA chromatin and the inner membrane of the nuclear (hnRNA or pre-mRNA) plays a structural role in the envelope (see Fig. 1). organization of the internal nuclear matrix (Miller et al. In preparing nuclear matrices, the nuclear envelope 1978*7; Berezney, 1980; Brasch, 1982; Gallinaro et al. is exposed to buffers containing non-ionic detergents, 1983; Long &"Schrier, 1983; Fey et al. 1986a,b). nucleases and high-molarity salt buffers. Morphologi- Therefore, part of this review will be dealing with cally, only the pore complexes and the nuclear lamina RNA processing and transport in the nucleus as well. seem to be resistant to such treatments. This residual The main thesis proposed in this respect is that RNA framework is, therefore, mostly referred to as the polymerase II transcripts, soon after the initiation of nuclear pore complex-lamina, and is considered to be a their synthesis, bind to proteins to set up a structural part of the nuclear matrix. It should be noted, how- backbone. Among these proteins are the so-called core ever, that much of the data about the pore complex- proteins. Subsequently, other functional molecules lamina have not originated from studies of nuclear that are involved in RNA and DNA metabolism may matrix preparations but from investigations on isolated be tethered to this hnRNP structure. During process- nuclear envelopes. ing and transport the RNA remains involved in the maintenance of the integrity of the internal matrix and The nuclear pore complexes is not released until after the last maturation step, that The nuclear pore complexes are large organelles that is after changing its set of core proteins for proteins form channels for nucleocytoplasmic transport through known to be associated with cytoplasmic mRNA. the nuclear envelope (reviewed by Newport & Forbes, 1987). They have also been postulated to serve as gene- The pore complex-lamina gating organelles capable of interacting specifically with expanded (transcribable) portions of the genome The major structural components of the nuclear envel- (Blobel, 1985). ope are the inner and outer nuclear membranes enclos- The pore complexes are non-randomly distributed ing a lumen or perinuclear space, as well as the nuclear on the nuclear surface. Their three-dimensional struc- lamina and pore complexes. The outer membrane on ture has been determined by electron microscopy using the cytoplasmic side appears to be continuous with the nuclear envelopes from Xenopus oocytes (Unwin & rough endoplasmic reticulum and is covered with Milligan, 1982). These authors found the pore complex

12 R. Verheijen et al. to be a symmetrical structure (outer diameter ± protein seems to be modified by addition of A'-acetyl- 120 nm) framed by two widely separated, coaxial rings. glucosamine residues. Each ring is composed of eight globular subunits, and gpl90 as well as the 62K protein remain associated attaches to the nuclear membranes. Connected to these with the pore complex-lamina after Triton X-100 rings and extending radially inwards from them along a extraction at low-ionic strength. However, the interac- central plane are elongated structures called spokes. tion of both proteins with the pore complex-lamina These spokes appear to contact a large central spherical fractions was found to be destabilized in the presence of particle, the plug. elevated salt concentrations. Until 1982 none of the pore complex polypeptide Recently, Gerace and co-workers reported on the constituents had been defined and characterized. In identification of eight structurally distinct pore com- recent years, however, the nuclear envelope of the plex proteins with common epitopes, isolated from rat Xenopus laevis oocyte (see Fig. 2) has been shown to liver cells (Snow et al. 1987). These polypeptides with contain one principal polypeptide of 68K (K = 10 Mr) apparent molecular weights of 210, 180, 145, 100, 63, that appeared to be a major component present in both 58, 54 and 45 (Xl0J) copurified with the pore com- lamina and pore complex preparations (Stick & plexes under various conditions of ionic strength and Krohne, 1982; Benavente et al. 1984a). Gerace et al. non-ionic detergent, and were characterized using (1982) identified a prominent 190K nuclear pore com- monoclonal antibodies. All members of this group of plex glycoprotein (gpl90) in rat liver nuclear envel- proteins contained multiple O-linked A:-acetylglucos- opes. On the basis of its biochemical characteristics, amine residues (see also Holt et al. 1987). Using these authors have suggested that the protein is in- immunoelectron microscopic techniques it was found volved in anchoring the pore complex to the nuclear that the proteins recognized by the monoclonal anti- envelope membranes. bodies were situated on the cytoplasmic as well as on Davis & Blobel (1986) have identified and character- the nucleoplasmic surfaces of the nuclear membranes, ized a 62K protein of the nuclear pore complex from rat but were absent from the lumen. Because of some liver. This protein was shown to be synthesized as a similarities between the biochemical properties of the soluble cytoplasmic precursor of 61K. After incorpor- 63K protein and the 62K protein described by Davis & ation of the protein into the nuclear fraction the Blobel (1986), it was concluded by Snow et al. (1987)

Fig. 2. Electron micrograph showing the nuclear envelope manually isolated from oocytes of A', laevis (for details see Franke & Scheer, 1970; Scheer, 1972). In the upper picture numerous pore complexes are denoted by vertical arrows, brackets denote some of intranuclear tangles of the fibrils associated with the pore complexes. In the lower picture the arrows denote the individual annular granules on either side of the pore margin, the arrowhead points to the central granule. N, nucleoplasmic side; C, cytoplasmic side; o, outer side; i, inner side of the nuclear envelope. Note that in these cells the nuclear lamina at the inner aspect of the nuclear envelope is very thin and barely detectable. In whole-mount preparations it appears as a single layer of a loose filamentous meshwork (Scheer et al. 1976; Aebi et al. 1986). Bars, 0-1 /im. (Courtesy of Professor Dr W. W. Franke.)

The nuclear matrix 13 that these two pore complex constituents are probably The nuclear lamina identical. The nuclear lamina consists of a proteinaceous layer Berrios et al. (1983) have identified a glycoprotein situated subjacent to the inner nuclear membrane associated with nuclear matrix pore complex-lamina (reviewed by Franke et al. 19816; Gerace & Blobel, preparations obtained from Drosophila melanogaster 1982; Gerace, 1986; Krohne & Benavente, 1986; embryos. The molecular weight of this protein was Newport & Forbes, 1987). The lamina is usually co- initially estimated to be 17X103, but more recent purified together with the pore elements. The predomi- studies have shown that it was only 2x 10 smaller than nant polypeptides in such preparations are the lamins, gpl90 described by Gerace et al. (1982) in rat liver. proteins in the molecular weight range of 60-80 This Drosophila glycoprotein is therefore designated (XlO3), which are immunologically related. In mam- here as gp 188. It seems to be the homologue of the rat mals and avian species three main lamin proteins, i.e. liver gpl90, since the two polypeptides have some lamins A, B and C, have been characterized, while at biochemical properties in common (Filson et al. 1985). least five different lamins have been described in Antisera prepared against gpl90 were found not to amphibia and one or two in certain invertebrates cross-react with gpl88 (Filson et al. 1985). In contrast, (reviewed by Krohne & Benavente, 1986). Immuno- one of the two antibodies raised against gpl88 cross- histochemical studies using specific antibodies to the reacted weakly with glycoproteins of similar molecular lamin proteins have confirmed the localization of these weight in isolated nuclear fractions from Xenopus proteins at the rim of the nucleus (see, e.g., Fig. 3). oocytes, as well as chicken, opossum and rat liver. Several authors have detected lamin precursors There was no detectable release of gpl88 from the (Gerace et. al. 1984; DagenaiseZ al. 1985; Lehnere/a/. nuclear fraction after treatment with Triton X-100 or 1986), while additional minor components that display DNase I and RNase A. Extraction of the residual the biochemical properties characteristic of the lamins nuclear material with 1 M-NaCl resulted in the apparent have been described for rat and chicken by Lehner et solubilization of approximately 10-20% of the glyco- al. (1986). This indicates that the composition of the protein, but the majority of this component resisted nuclear lamina in these species is probably more salt extraction (and even 5 M-urea) and was found complex than previously assumed. associated with the nuclear matrix pore complex- Recently, McKeon et al. (1986) and Fisher et al. lamina. (1986) have characterized the cDNA clones for human Berrios et al. (1983) have also reported on the lamins A and C. From the protein sequences deduced existence of a 188K protein in the nuclear matrix pore from these cDNAs it became apparent that lamin A has complex-lamina fraction of Drosophila embryos, an additional region of approximately 9K at its car- which is distinct from gpl88. This protein was ident- boxyl terminus as compared to lamin C (Fisher et al. ified as an ATPase/dATPase and appeared not to be 1986). Both lamin sequences show a marked homology glycosylated, as it was completely resistant to digestion with the intermediate filament proteins (reviewed by by endoglycosidase H. It remains to be determined if Franke, 1987). In vitro translation studies performed the ATPase/dATPase is a real constituent of the pore by Laliberte et al. (1984) showed that lamins A and C complex in vivo. are encoded for by different mRNAs, with lamin A Nuclear matrix preparations described by many being a precursor approximately 2K larger than mature authors differ considerably with respect to the high- lamin A. ionic strength conditions under which they are isolated. Using a mouse monoclonal antibody (IFA) raised It is not known to what extent the pore complex against a common domain of all intermediate filament proteins described above remain associated with such proteins, Lebel & Raymond (1987), as well as Osborn different preparations. When nuclear envelopes are & Weber (1987), have shown that lamin B also shares exposed to rigorous extraction treatments involving some sequence homology with the intermediate fila- solutions of high-ionic strength or non-denaturing ment proteins. This homology of the lamins to the detergents, the basic structural elements of the pore intermediate filament proteins may account for the complexes are still identifiable (Franke et al. 19816). fibrillar nature of the lamina as seen, for example, in An interesting postulation in this respect is that only Fig. 4. the components on the cytoplasmic surface would be Lamin B is thought to fulfil a role in anchoring the sensitive to salt extraction (Davis & Blobel, 1986), lamina to the inner membrane of the lipid bilayer, since whereas the components on the nuclear surface would this protein is more resistant to chemical extractions remain attached to the lamina by a link that is resistant from nuclear membranes compared with lamins A and to extraction with high-salt solution. Whether or not its C (Gerace & Blobel, 1982; Lebel & Raymond, 1984). It link to the lamina makes the nuclear part of the pore also remains selectively associated with membrane complex resistant to high-salt extraction remains to be vesicles after nuclear envelope disassembly during examined. mitosis (Burke & Gerace, 1986).

14 R. Verheijen et al. Fig. 3. Peripheral localization of the nuclear lamina in mouse P19 cells as detected by an antiserum to lamin B. The set of pictures was obtained by the use of a confocal scanning laser microscope (Brakenhoff et at. 1985), which scanned through the nucleus from bottom (1) to top (9) at 2-j.im intervals. Bar, 10/tm. (Courtesy of Dr G. J, Brakenhoff & Dr R. van Driel.)

Recent studies by Georgatos & Blobel (\987a,b) A direct association between the cytoskeletal frame- have demonstrated that lamin B also constitutes an work and the nuclear lamina had been described by intermediate filament attachment site at the nuclear Capco et al. (1982) by using whole-mount microscopy envelope. Their approach consisted of in vitro binding to visualize nuclear matrices prepared from mouse 3T3 studies with isolated bovine lens vimentin and avian fibroblasts and HeLa cells. Their electron micrographs erythrocyte nuclear membranes. Removal of lamin B showed that cytoskeletal filaments were attached to the from the nuclear envelope by urea extraction or nuclear lamina. Two-dimensional gel electrophoresis blocking with anti-lamin B antibodies were found to revealed vimentin to be present in their nuclear matrix reduce the binding of vimentin to these membranes. fractions. Several other investigators have demon- Other techniques, such as immunoprecipitation, rate strated the presence of vimentin and cytokeratins in zonal sedimentation and affinity chromatography, nuclear matrix preparations from cells grown in sus- pointed to a specific vimentin—lamin B association pension culture (see, for example, Verheijen et al. under;/? vitro conditions. The 6-6K carboxyl terminus 1986a). of vimentin was found to be involved in this inter- Another indication of the attachment of intermediate action, whereas the binding was positively influenced filaments to the nuclear envelope has been reported by when lamin A was present. From these data Georgatos Staufenbiel & Deppert (1982), who showed that after & Blobel (19876) concluded that intermediate fila- isolation of nuclei from cells grown in suspension ments may be anchored directly to the nuclear lamina. culture the majority of the cytokeratin and vimentin These anchorage places are suggested to be restricted filaments had collapsed onto the nuclear surface but to certain distinct foci along the lamina, coinciding still constituted a filamentous system. These collapsed with nuclear pores, and not to be uniformly distributed filaments could be partially unfolded again by centrifu- over the nuclear surface (see also Goldman etal. 1985). gation through an isotonic buffer.

The nuclear matrix 15 Fig. 4. Rotary-shadowed platinum/carbon replica of an isolated and cntical-point-dned BHK—nuclear matrix preparation showing the lamina as a meshwork of anastomosing 8-10nm filaments. Cell extraction was essentially performed as described by Simard el al. (1986) (0-5% Triton X-100, 5 fig ml"1 DNase I, 2lM-NaCl). Bar, 1-OjUm. (Courtesy of Dr V. Bibor-I lardy.)

16 R. Yerlieijen el al. A remarkable observation in the field of intermediate al. 1982). Small nuclear RNPs (U3 and U8 in particu- filament-nuclear matrix interactions has recently been lar), which may function in rRNA processing, are also made by Carmo-Fonseca et al. (1987). These investi- accumulated in the nucleolus (Epstein et al. 1984; gators isolated nuclear matrix-intermediate filament Reddy et al. 1985). Many authors have dealt with the scaffolds from cultured rat ventral prostate cells and morphological and biochemical aspects of the nucleolus isolated rat uterine epithelial cells. Subsequently, the (for reviews see Jordan & Cullis, 1982; Goessens, 1984; scaffolds were critical-point dried, platinum-carbon Hadjiolov, 1985; Sommerville, 1986). replicated and examined by electron microscopy. In At the Eighth European Nucleolar Workshop in 1983 such preparations the intermediate filaments were not an attempt was made to standardize nucleolar no- seen to abut on the nuclear lamina, but rather to be menclature. Thus, the nucleolar matrix was defined as looped and to follow the nuclear surface. Short, direct the residual structure left after extraction procedures to connections of a cross-bridge type (5-7 nm in diam- reveal the nuclear matrix (Jordan, 1984). Such nu- eter) extended laterally from the intermediate filaments cleolar matrices retain the size and shape of the original and fused with the nuclear pore complexes (see Fig. 5). nucleoli. However, as nucleolar residues present in These cross-bridges appeared to be about 75-100 nm nuclear matrix preparations may have been subjected long in uterine epithelial cells and were shown to be to treatment with DNase I only or with a combination associated with cytokeratin filaments, while in fibro- of DNase I and RNase A, it is necessary to extend the blasts they were considerably shorter (approximately definition given by Jordan (1984). The term nucleolar 14 nm long) and probably associated with vimentin matrix is used here to indicate the residual nucleolus in filaments. The cross-bridges were not recognized by nuclear matrix preparations in which only DNase I has anti-cytokeratin antibodies. Considering the very short been used in the nuclease step. length of these linking structures, the authors con- Little is known about the composition of the nu- cluded that their finding does not contradict the cleolar remainders in nuclear matrix preparations. It is observations of Georgatos 8c Blobel (1987a,b). Also difficult to detect any morphological resemblance be- Fey et al. (1984a) have clearly shown interactions of tween the nucleolar residues found in nuclear matrices intermediate filament structures with the nuclear per- and structural components of the intact nucleolus. iphery (see Fig. 6). Aggregation and condensation of nucleolar structures, Apart from investigations on cytoskeleton-nuclear probably due to the presence of divalent cations, make lamina interactions, other studies have established that interpretation of electron-microscopic images very dif- during interphase the lamina is in intimate contact with ficult. Procedures using EDTA, however, can yield the peripheral chromatin (Boulikas, 1986; reviewed by fairly decondensed nucleolar matrices. This is illus- Hancock & Boulikas, 1982; Hubert & Bourgeois, 1986; trated by the studies of Long & Ochs (1983), who Hancock & Dessev, 1987). Such interactions are prob- prepared chromatin-depleted nuclei from Friend ably important for stabilizing or maintaining certain erythroleukaemia cells under conditions that avoid the aspects of higher-order chromosome architecture (Leb- use of high salt concentrations. These authors partially kowski & Laemmli, 19826). digested the DNA, and then washed the cells twice in In conclusion, the lamina not only determines the 2mM-EDTA. Compared with the amounts in whole nuclear shape and the spatial organization of the pore nuclei, the amounts retained in the resulting structures complexes, but seems also to be directly involved in were approximately 1 % of the DNA, 65 % of the total anchoring both the intermediate filaments and the RNA, 70% of the hnRNA, 74% of the snRNA, 29% chromosomes at the nuclear periphery during inter- of all protein and 2% of the histories. Although in phase. electron micrographs of nuclei treated in this way no morphological evidence was found for residual nu- cleoli, immunofluorescence studies showed that pro- The nucleolar residue tein C23 was located in distinct centrally localized regions. Exposing these EDTA-prepared chromatin- The nucleolus is the site of synthesis and processing of depleted nuclei to 2mM-MgCl2 resulted both in the pre-ribosomal RNA, and of assembly of the ribosomal reformation of morphologically distinct residual nu- proteins and ribosomal RNA into pre-ribosomal par- cleoli and in aggregation of matrix fibrils. Similar ticles. Only a relatively small number of the many effects have been observed in rat liver nuclei by nuclear proteins are confined exclusively to the Galcheva-Gargova et al. (1982) and also by Hubert et nucleolus and presumably play specific roles in its al. (1981), Bouvier et al. (1980) and Aaronson & Woo structure and function. One such major nucleolar- (1981). specific protein is nucleolin or C23. This phosphoryl- For some time it was not clear whether the nucleolar ated protein (HOK/pI 5-5) is probably involved in pre- matrix structures and the intranuclear matrix network rRNA transcription and ribosome assembly (Bugler et were composed of distinct or identical constituents.

The nuclear matrix 17 Fig. 5. A. Rotary-shadowed platinum/carbon replica of an isolated, extracted and critical-point-dried rat uterine epithelial cell. Cell extraction was performed according to Fey et al. (1984a) in order to obtain nuclear matrix-intermediate filament scaffolds. Nuclear pore complexes are seen attached to intermediate filaments through short filamentous cross-bridges approximately 5 nm in diameter (arrows). B. Lateral view of the thin filaments, extending from the intermediate filaments to the nuclear matrix in a similar preparation as shown in A. The thin filaments are seen to abut on the nuclear lamina (arrows) and pore complexes (open arrow). Filaments with identical diameters link adjacent intermediate (cytokeratin) filaments (arrowheads). Bars, 0'2,i(m. (Courtesy of Dr M. Carmo-Fonseca & Dr A. Cidadao; see also reference Carmo- Fonseca*?/ al. (1988).)

18 R. Verheijen et al. The first experiments that provided evidence for differ- 16% of the RNA and less than 4% of its original ent protein compositions of these two structures were protein content, being enriched for proteins of 34K, performed in rat liver by Berezney & Coffey (1977). 36K, 43K, 57K, lamins A and C (70 and 62K). Also Later, Todorov & Hadjiolov (1979) found five distinct higher molecular weight proteins, including a lOOK/pI protein bands with apparent molecular weights of 30, 6-8 and a phosphorylated 160K/pI5-5 protein were 40, 56, 70 and 82 (XlO3) enriched in the nueleolar found in these preparations. A portion of ribosomal matrix fraction. Distinct nueleolar matrix proteins spacer DNA remained tightly bound after treatment from rat liver were found also upon two-dimensional with2M-NaCl (see also Bollaef al. 1985). Shiomi et al. gel electrophoresis by Comings & Peters (1981). They (1986) found the C23 protein to be quantitatively found many basic proteins specific for the nucleolus, released from the nueleolar matrix by 2M-NaCl, which the most prominent one being a 33K protein. is in contrast to the results of Olson et al. (1986). This The elegant studies of Franke et al. (1981a) led to was verified with a specific antibody test indicating that the isolation of a nueleolar matrix from Xenopus oocyte the 110K protein was distinct from protein C23. The nuclei comprising filaments of about 4nm diameter, observations of several investigators that the nucleolus which were densely coiled into higher-order fibrils of is often in close contact with the nuclear envelope (Rae 30-40 nm diameter. This matrix was resistant to treat- & Franke, 1972; Goessens, 1979; Bouteillee/ al. 1982; ments with low-salt and high-salt buffers, DNases and Bourgeois et al. 1981, 1984) may explain the presence RNases, sulphydryl agents and non-ionic detergents, of lamins A and C in the nueleolar matrix preparations and contained a single 145K/pI 6-2 protein (see also of Shiomi et al. (1986). Evidence for a nucleolus- Benavente et al. 1984a,b). Furthermore, Olson & nuclear envelope junction in the form of a 'pedicle' or Thompson (1983) showed that a 160K polypeptide was 'stalk' has been presented by Rae & Franke (1972) in predominantly found in the nueleolar matrix fraction mouse hepatocyte nuclei and more recently by Hubert from Novikoff hepatoma ascites cells prepared by et al. (1984), who isolated nucleoli containing nuclear digestion with DNase I and extraction with high-salt. shells from membrane-depleted rat liver nuclei (see Recent studies of Olson et al. (1986), also performed also Bouviere/ al. 19856). Hubert e? al. (1984) showed on Novikoff hepatoma cells, revealed a nueleolar the nucleoli to be anchored to the peripheral lamina by matrix fraction that was enriched in polypeptides with a pedicle that was continuous with an intranucleolar molecular weights of 28, 37-5, 40, 70, 72, 110 and network. The pedicle and the network that supports 160X103. The 110K protein was recognized by an the nueleolar DNA were composed mainly of non- antibody directed against protein C23 (Olson et al. histone proteins insoluble in 2M-NaCl. 1981). About 25 % of the protein, 50 % of the RNA and In conclusion, the nueleolar residues in nuclear less than 4% of the DNA of untreated nucleoli were matrix preparations appear to be complex structures recovered in such nueleolar matrix preparations. Olson comprising several different elements, including many et al. (1986) also showed that pre-ribosomal RNP proteins not yet adequately characterized. particles are major constituents of nueleolar matrix preparations extracted with DNase I and high-salt solutions. RNase A treatment during the DNase I digestion stage, together with the inclusion of 1 % The internal matrix /3-mercaptoethanol in the high-molarity salt washes, reduced the protein content to 15 % and the RNA Morphological and biochemical aspects content to about 2-5% of that of untreated nuclei. Several authors have provided evidence of the protcin- Under such conditions protein C23 and the 160K aceous nature of the internal matrix, based on the fact protein were also removed. The remaining polypep- that a fibrillar structure is observed in the nucleus when tides were largely represented in the 30-50K range, both DNA and RNA are removed by nucleases (Capco and electron microscopy revealed only amorphous et al. 1982). Other authors (Galcheva-Gargova et al. material instead of the granular elements usually found 1982; Kaufmann & Shaper, 1984; Kaufmann et al. in nueleolar matrix preparations (Olson et al. 1986). 1986) have observed a complete reorganization of the In preparing nueleolar matrices from mouse L-cells, internal matrix structure, depending on the isolation Shiomi et al. (1986) used 50/xgmr1 DNase I, a conditions used. In this regard there is concern about concentration five times higher than that used by Olson aggregation or precipitation of otherwise soluble nu- et al. (1986), and reproducibly obtained core nucleoli clear components, due to the high-salt concentrations (the nueleolar fraction that remains after extensive required to extract chromatin during matrix prep- DNase I action, without a high-salt extraction) with a aration, or about cross-links formed by oxidation of minimum protein content. The nueleolar matrix frac- sulphhydryl groups. tions prepared by Shiomi et al. (1986) contained about Other studies indicated that the structural integrity 5 % of the amount of DNA present in isolated nucleoli, of the internal network depends on the maintenance of

The nuclear matrix 19 . ••• /

20 /?. Verheijen et al. certain metalloprotein interactions during matrix iso- been studied sufficiently. Some of these technical lation, e.g. with Ca2+ and Cu2+ (Lebkowski & problems have been tackled in recent papers but Laemmli, 1982a,b) or with Mg2+ (Bouvier et al. answers are still incomplete (Long & Ochs, 1983; 1985a). These latter authors found the intranuclear Kaufmann & Shaper, 1984; Staufenbiel & Deppert, structures in HeLa nuclear matrix preparations to be 1984; Fey et al. I984a,b, I986a,b; Mirkovitch et al. composed of residual (DNase- and salt-resistant) RNP 1984; Verheijen et al. 1986a). complexes of both nucleolar and non-nucleolar origin. Electron-microscopic studies performed by Pouche- These intranuclear structures comprised two distinct let et al. (1986) have shown the existence of a well- but superimposed networks, which appeared as thin defined network in nuclei of resting mouse lympho- fibrillar elements (2-3 nm) and as thick fibrogranular cytes in situ. These authors prepared nuclear matrices elements of varying size. Both networks disappeared as from formaldehyde-fixed cells. The nuclease step in a result of RNase digestion in the presence of low-ionic such isolations was performed either with DNase only strength EDTA before extraction of nuclei in 2M-NaCl. or with DNase in combination with RNase. In both However, the thick fibrillar elements were preserved types of preparations three well-defined networks were from being eluted in 2 M-NaCl when RNase was used in observed: the lamina, an intra-chromatin network and 2+ buffers containing Mg . This remaining network was an inter-chromatin network. This latter structure could enriched in two proteins of 49K and 70K. From these be superimposed on the internal network of isolated results Bouvier et al. (1985c/) concluded that in the nuclear matrices. Its sensitivity to pepsin digestion was + presence of Mg interactions between certain RNP increased tenfold when the digestion was preceded by complexes are established, which then become able to treatment with RNase. This latter finding indicates form a salt-resistant intranuclear network. that RNA appears to be essential for the maintenance Herman et al. (1978) showed that after removal of of this structure. Also Fey et al. (1986a) have shown 99 % of the chromatin in a two-step extraction pro- that the morphology of the internal matrix changed cedure, both steady-state and newly synthesized drastically when RNases were used in the extraction hnRNA were associated with the remaining nuclear buffers (see Fig. 7). structure. Their suggestion that the integrity of the In conclusion, the relevance of an internal matrix nuclear matrix is dependent on the RNA was in structure in vivo is still a matter of controversy and at contrast with the conclusion of Miller et al. (1978a) present time many questions are still unanswered. It is that RNase treatment of the nuclear matrix does not beyond the scope of this review to compare all the alter the morphology of this network. different isolation procedures for the preparation of Considering the results obtained by the many nuclear matrices or to discuss all the indications that workers on nuclear matrix structure and composition, support or deny the existence of an extranucleolar it will be obvious that the presence or absence of the network in vivo. We merely conclude that considerable internal network in nuclear matrix preparations evidence indicates that when an internal matrix struc- depends on the experimental protocol used. The effects ture is obtained without prior treatment with RNase, of divalent cations, the molarity of extraction buffers, hnRNA is found to be an integral constituent of this the effect of high-salt treatment, the extent of disul- structure (Miller et al. 1978«; Berezney, 1980; Brasch, phide cross-linking during preparation of the matrices, 1982; Gallinaroe/ al. 1983; Long & Schrier, 1983; Fey the order in which the various preparation steps are et al. 1986a,b). applied, the use of (NH^SC^ instead of NaCl for the A relevant question is whether the presence of an extraction itself, the presence of endolytic enzymes internal matrix is a general feature of cells. Using other than proteases inhibited by phenylmethylsul- procedures similar to those published by Merman et al. phonyl fluoride (PMSF) or phenylmethylsulphonyl (1978) and Miller et al. (1978a) a relatively stable chloride (PMSC), and many other factors that are used RNA—matrix association can be found in various types for the preparation of the matrix structure have not yet of cells (Berezney, 1980; van Eekelen & van Venrooij, 1981; Brasch, 1982; Gallinaro et al. 1983; Long et al. Fig. 6. A. Whole-mount transmission electron 1979; Fey et al. \986a,b). The adult chicken erythro- micrographs of the nuclear matrix-intermediate filement cyte nucleus, however, in which virtually no DNA or (NM-IF) scaffold from a breast carcinoma cell line. The RNA synthesis takes place, was found to lack an chromatin-depleted nuclear matrix (NM) is apparently in internal nuclear matrix. Also, mild extraction pro- association with intermediate filaments (IF) largely cedures resulted in empty shells of pore complex- consisting of cytokeratins. Note the nuclear pores (NP) present in the nuclear lamina. Bar, 0-5jum. B. Immunogold lamina together with loose aggregates of core histones staining of intermediate filaments (IF) as described by Fey (Lafond & Woodcock, 1983). In contrast, rat liver et al. (1984«) in a similar preparation as in A, using anti- nuclei showed a typical intranuclear salt-resistant skel- cytokeratin antibodies. Bar, 01 /*m. (Courtesy of Dr E. G. eton after the same treatments. These results indicate Fey.) that an internal matrix is not an obligatory nuclear

The nuclear matrix 21 Fig. 7. Transmission electron micrographs of HeLa nuclear matrix preparations in unembedded resin-free sections (0-2fim thick) as described by Fey et al. (1986c;). A shows an RNP-containing nuclear matrix preparation, and B an RNP-depleted nuclear matrix. The RNP-containing nuclear matrix reveals fibres (F) that extends throughout the nucleus, forming continuous associations between nucleoli (Nu) and the nuclear lamina (L). Cytoplasmic filaments (Cy) are observed in association with the lamina. The RNP-depleted nuclear matrix displays a distortion of nuclear shape. The interior of the nuclear matrix is composed of condensed and fragmented filament aggregates (FA). The distortion of the interior by digestion with RNase suggests that RNA is an important structural component of the nucleus. Bars, l-Of/m. (Courtesy of Dr E. G. Fey.) component and that in erythrocytes it is apparently not nuclear volume increased up to sixfold, together with required for the spatial organization of chromatin. In an extensive synthesis of stable interchromatinous contrast, much more active 5-day-old embryonic eryth- matrix proteins. All these results suggest a correlation rocytes did contain an interchromatinous nuclear between the presence of nuclear matrix structures and matrix (Lafond & Woodcock, 1983). Subsequent ob- nuclear 'activity'. servations from the same group showed that an internal In summary, the composition and organization of nuclear matrix is generated during the reactivation of hnRNA as part of the granular and fibrous internal chick crythrocyte nuclei in mouse L-cell cytoplasts. nuclear matrix structure still require more precise The nuclei enlarge and chromatin decondenses, ac- characterization. The results obtained to date permit companied by an influx of proteins from the host the conclusion that when nuclear matrices are isolated cytoplasm and the onset of RNA synthesis (Lafond & in the absence of RNase, hnRNA can be isolated Woodcock, 1983). Recently, Woodcock & Woodcock almost quantitatively as an intimate part of it. Con- (1986) have used the same experimental system to sidering the complicated composition of the nuclear identify 15 major polypeptides that, after a 16-h matrix it will be obvious that it is not a static structure, reactivation period, had migrated into the nucleus. but must display considerable dynamic activity. Five of the identified proteins in the 30-70K molecular weight range appeared to be nuclear matrix proteins; Heterogeneous nuclear RNP (hnRNP) particles two of these had their counterparts in L-cell nuclei. Shortly after hnRNA has been synthesized it associates During the concanavalin-A-induced stimulation of with proteins to form fibrillar ribonucleoprotein lymphocytes Setterfield et al. (1983) observed that the (RNP) particles and granules resembling 'beads on a

22 R. Verheijen et al. string' that extend away from the DNA-protein axis. hand, releases RNP particles by the shearing forces These fibres (7 nm thick) and granules (20-25 nm employed. Faiferman & Pogo (1975) have shown that diameter) are mostly referred to as the nuclear RNP the yields of particles from disrupted nuclei are pro- network. hnRNP structures can be isolated from cell portional to the shearing forces applied. It is evident nuclei in a wide range of sedimentation values that this procedure also destroys the delicate nuclear (30-250 S), depending on the isolation procedure ap- infrastructure. In studying the release of RNA from plied (reviewed by LeStourgeon et al. 1981; Holoubek, the nuclear matrix several authors have found that 1984). The monomeric forms of these structures are hnRNA is bound tenaciously to other matrix com- hnRNP particles of 30-40 S. Next to an RNA fragment ponents and that it can be separated from the attaching in the range of 125-800 nucleotides, these 40 S hnRNP structure only after disruption of the nuclear integrity particles contain a set of proteins that comprise (Longed al. 1979; van Eekelen & van Venrooij, 1981). 75-90% of its mass (Holoubek, 1984; Dreyfuss, 1986). In summary, it can be concluded that isolation of Although non-specific binding of proteins to the RNA hnRNP particles necessarily implies the fragmentation during the isolation of the particles has never been of the internal nuclear matrix. As a consequence, excluded, it is generally accepted that a distinct set of depending on the procedure used, varying quantities of so-called core proteins is present in 40 S hnRNP large hnRNP complexes and RNP core particles must particles. According to the nomenclature of Beyer et al. have been present in nuclear matrix preparations (1977), HeLa cells contain the following core proteins: isolated by various workers during the last decade. The A1(34K), A2(36K), B1(37K), B2(38K), C1(41K) and complexity of the nuclear matrix and salt-resistant C2(43K) (reviewed by LeStourgeon et al. 1981; hnRNP structures has been compared by Gallinaro et Holoubek, 1984; Dreyfuss, 1986). Proteins Cl and C2 al. (1983). Apart from small nuclear RNAs about 40 appear to play a role in hnRNA processing, as a proteins in the 25-120K molecular weight range were monoclonal antibody to these proteins inhibits in vitro characterized as common constituents of the nuclear splicing of an mRNA precursor, while depletion of matrix and the hnRNP particles. In addition, the pre- these proteins from the splicing extract abolishes its mRNAs and maturation products present in both capacity to splice pre-mRNA (Choi et al. 1986). Two- structures were compared. The results confirmed the dimensional gel separations of the core proteins have similarity of the structures, strongly suggesting that made it necessary to extend the nomenclature of Beyer pre-mRNA in the nuclear matrix and in the salt- et al. (1977), as has been done by Wilknucleoplasm. lengthy low-salt extraction procedure or by sonication The association of U RNAs with the nuclear matrix of isolated nuclei (reviewed by Holoubek, 1984). The was first reported by Zieve & Penman (1976) and Miller extraction of hnRNP particles from intact nuclei is et al. (19786), who demonstrated that, like hnRNA, dependent on the action of nuclear RNases and on the small nuclear RNAs remained in the nuclear matrix slightly alkaline pH that is required for the release of after removal of nuclear membranes and chromatin. the particles from nuclei. Since the nuclear matrix has Similar observations were made by Herlan et al. been shown to be extremely susceptible to proteolytic (1979), Maundrell et al. (1981) and Ross et al. (1982) activities (Miller et al. 1978a), it is likely that this for Tetrahymena, duck erythroblast and chicken eryth- procedure may also release RNP particles from the roblast nuclear matrices, respectively. nucleus by proteolytic degradation of the nuclear Miller et al. (19786) and Gallinaro et al. (1983) structure. The sonication procedure, on the other found a quantitative association of all snRN As with the

The nuclear matrix 23 nuclear matrix. Zieve & Penman (1976), however, the matrix structure via RNA. When a human autoim- characterized U2, U3, U4 and U6 RNA as being mune serum containing anti-La antibodies was used as nuclear matrix-associated, whereas Ul RNA was a control, immunodecoration of the matrices did not mainly lost upon chromatin extraction. In contrast, occur. This means that the La RNP particles (contain- Ciejek el al. (1982) have shown that only some of the U ing polymerase III transcripts such as pre-tRNAs and snRNAs were tightly associated with chicken oviduct precursor forms of 7 S RNA and 5 S rRNA; Rinke & matrices. They did not find a quantitative association Steitz, 1982) were lost during matrix preparation. nor a specific enrichment of one or more of the Similar experiments to immunodecorate isolated snRNAs. matrix structures with antibodies directed against U snRNPs can be released efficiently from isolated RNP antigens have been performed by van Eekelen el nuclei or from nuclear matrices by disintegration of the al. (1982), Spector

24 R. \ erheijen et al. Fakan et al. (1986) have applied immunoelectron Additional evidence for a possible role of nuclear microscopic techniques to mouse and Drosophila tis- actin in RNA metabolism has been documented in sue-culture cells, using monoclonal antibodies directed several studies by Nakayasu & Ueda (1984, 1985, against hnRNP core proteins or against U RNP pro- 1986). These authors have shown an interaction be- teins. Their studies provided direct evidence for an tween pre-mRNAs and actin filaments in the nuclear association of Ul RNP and possibly also of other U matrix of mouse L-cells (Nakayasu & Ueda, 1985). In a RNP species with extranucleolar RNA during early previous study it had been shown that actin filaments transcription elongation. In addition the results of are closely associated with small nuclear RNPs these authors confirmed the presence of hnRNP pro- (Nakayasu & Ueda, 1984). Recently, the existence of teins within the growing RNP chains in the transcrip- two additional acidic species of actin in the nuclei of tion complexes. mouse L-cells were reported next to the two common In summary, the limited number of studies per- /3-actins and y-actins (Nakayasu & Ueda, 1986). The formed on the interaction between snRNA or snRNA most acidic actin (pi 5-1) was localized predominantly and nuclear matrix structures indicates a specific in the nuclear matrix. Other authors have also found interaction between these two complexes. It will be of actin as a major protein in nuclear matrix preparations great interest to elucidate the nature of such an (Capco et al. 1982; Staufenbiel & Deppert, 1984; interaction and to establish its functional significance. Verheijen et al. 1986a; Nakayasu & Ueda, 1986). Although the possibility of contamination with cyto- plasmic actin cannot be totally excluded, the obser- Nuclear actin vations described above justify the conclusion that Considerable evidence has accumulated over the past actin may be a major and well-defined nuclear matrix decade to show that actin is a constituent not only of the protein that might have a defined function in RNA cytoplasm but also of interphase nuclei in a wide synthesis in vivo. variety of cells (for a review, see Scheer et al. 1984). Actin has also been demonstrated as a major protein in Enzymes involved in DNA and RNA metabolism manually isolated and cleaned nuclei of Amoeba and Enzymes involved in DNA and RNA metabolism that amphibian oocytes, in which concentrations of have been found in nuclear matrix fractions are numer- 3-4mgml~' can be found (Krohne & Franke, 1980; ous. These include DNA alpha and beta polymerases Gounon & Karsenti, 1981). (Nishizawa et al. 1984; Smith et al. 1984; Foster & Experiments by Manley et al. (1980) have indicated Collins, 1985), topoisomerase I (Nishizawa et al. that at least part of the nuclear actin may be involved in 1984), topoisomerase II (Halligan et al. 1984; Berrios RNA transcription. The transcription of protein- et al. 1985), RNA polymerase II (Lewis et al. 1984), coding genes in eukaryotic systems is performed by poly(A) polymerase (Schroder et al. 1984), DNA RNA polymerase II, which in vitro requires sup- methylase (Burdon et al. 1985) and DNA primase plementation with crude cellular extracts to initiate (Wood & Collins, 1986; Tubo & Berezney, 1987a,b). accurate transcription. Such cellular extracts contain multiple factors, some of them recognizing specific Vims-specific proteins promotor elements such as the TATA box (Breathnach It has been shown that the nuclear matrix is an & Chambon, 1981). One of these factors has been important site of viral interaction (reviewed by Berez- purified and characterized as a protein that is strikingly ney, 1984; Simard et al. 1986). Viral DNA (see, e.g., similar to actin (Egly et al. 1984). Smith et al. 1985) and virus-specific proteins (see, e.g., In a completely different approach, Scheer et al. Jones &Su, 1982; Bibor-Hardy et al. 1985; Khittoo et (1984) observed that injection of antibodies against al. 1986) have been found enriched in nuclear matrix actin into oocytes resulted in the cessation of transcrip- preparations. tion by RNA polymerase II, loop retraction and The bulk of large tumour antigen (large T) in simian chromosome condensation. Moreover, even stronger virus 40 (SV40)-infected cells is present in three inhibition was observed after injection of actin-binding subnuclear locations: in the nucleoplasm, associated proteins from different sources, such as fragmin from with the cellular chromatin and tightly bound to the Physarum polycephalum and an actin modulator pro- nuclear matrix. Schirmbeck & Deppert (1987) have tein from mammalian smooth muscle. The idea that analysed the distribution of large T in lytically infected actin is involved in some way in RNA transcription is monkey cells and found that the amounts of large T also supported by the finding that this protein is tightly associated with both chromatin and nuclear matrix associated with purified RNA polymerase II (Smith et increased markedly after transition from early to late al. 1979) and possibly involved in the regulation of phase of viral infection. The amount of nuclcoplasmic poly(A) metabolism mediated by poly(A) polymerase large T increased only slightly. During the course of (Schroder et al. 1982). infection large T accumulated in the chromatin and in

The nuclear matrix 25 the nuclear matrix fraction, in parallel with the increase (or mRNP) component induced by ATP or its deriva- of viral DNA synthesis. Recent studies by the same tives without cleavage of any high-energy bond. The group have indicated that the association of SV40 large hnRNA remained completely bound to the matrix in T with the chromatin and the nuclear matrix is the presence of ATP. Furthermore, the release of mediated by protein-protein interactions, rather than mature mRNA by ATP could be strongly inhibited by by sequence-specific DNA binding (Hinzpeter & Dep- various inhibitors of topoisomerase II, by a mechanism pert, 1987). not yet understood. Other remarkable results were that both mature and pre-mRNA were released from the matrix structure in the presence of poly(A), ethidium Associated transcripts bromide or the copper chelator 1,10-phenanthroline Newly synthesized SV40 RNA appears to be quantitat- (Schroder et al. 1987a). The general conclusion ively associated with the nuclear matrix, while its reached by these authors was that nucleoplasmic RNA processing and transport also appear to take place on transport is apparently regulated not only during this structure (Ben Ze'ev & Aloni, 1983; Abulafia et al. passage through the pore complex but also at the level 1984). Similarly, influenza viral RNA sequences (Jack- of RNA release from the nuclear matrix. son et al. 1982) and the primary transcripts as well as Although the possibility that the non-matrix-bound the spliced RNA intermediates of adenovirus-specific mRNAs in the studies of Ciejek et al. (1982) and genes (Mariman et al. 1982a; van Venrooij et al. 19826, Schroder et al. (1987a) are contaminations from cyto- 1985) have been shown to be tightly bound to the plasmic mRNAs cannot be completely ruled out, the matrix structure. Rearrangements in the nuclear data indicate that a substantial part of the processed matrix morphology after infection with adenovirus mRNA in the nucleus is bound differently and not as type 2 have been demonstrated by the electron-micro- tightly to the nuclear matrix as are the mRNA precur- scopic studies of Zhonghe et al. (1987). sors. Since nuclear mRNAs are associated with a Other studies concerning nuclear matrix-associated different set of proteins, as compared to cytoplasmic transcripts have been presented by Ciejek e£ al. (1982). RNA molecules (van Eekelen et al. 19186; van Ven- RNA was isolated from oviduct nuclear matrices and rooij et al. 1982a), one would expect the selection of analysed by hybridization to cloned probes for ovalbu- mature mRNA for nucleocytoplasmic transport to min and ovomucoid mRNA. More than 95% of all of occur by means of exchange of hnRNP proteins the precursors of these mRNAs, including various (Dreyfuss, 1986) with a set of mRNA-binding pro- splicing intermediates, were associated with the teins. Possible candidates for such proteins that could matrix. Less than 50 % of the mature mRNA present in exchange with hnRNP proteins are the transport intact nuclei was recovered in the nuclear matrix. proteins described by Moffet & Webb (1983) or cyto- Schroder et al. (1987a) have also studied the release of plasmic mRNP proteins (Wagenmakers et al. 1980; total mRNA, as well as of specific high-abundance Setyono & Greenberg, 1981; van Eekelen et al. 1981a; ovalbumin mRNA, from hen oviduct nuclear matrices. van Venrooij et al. 1982a). Their results confirmed the earlier findings of Ciejek et All these results support the concept that the nuclear al. (1982), by demonstrating that ovalbumin pre- matrix may be the structural site for RNA processing mRNA was almost quantitatively associated with the within the nucleus of eukaryotic cells. oviduct nuclear matrix, whereas only one-third of the mature ovalbumin mRNA of whole nuclei was re- The nuclear matrix and RNA transport covered in the nuclear matrix fraction. In addition, they showed that the binding of both pre-mRNA and Recently, Schroder et al. (19876) have reviewed nu- matrix-bound mature mRNA displayed no difference cleoplasmic mRNA transport and discussed require- in strength when the matrices were subjected to ments for mRNA release. For this reason we will focus treatments with high-salt (3M-NaCl), urea (4M), deter- only on the fact that different RNA species have gent (2% Triton X-100) or EDTA (SOmM). The different rates of transportation. mature mRNA, however, was released selectively from A general finding is that smaller RNAs are trans- the nuclear matrix by either ATP, AMP plus pyrophos- ported faster to the cytoplasm than the larger mRNAs. phate, ADP or ATP analogues containing non-hydro- Newly synthesized globin mRNA (9 S), for example, is lysable a,/3 or j6,y bonds. Whereas mRNA translocation released into the cytoplasm about 5 min after initiation through the nuclear pores is dependent on hydrolysis of of its synthesis (Bastos & Aviv, 1977; Kinniburgh & ATP or GTP, mRNA release from the nuclear matrix Ross, 1979). This RNA is polyadenylated and spliced. apparently does not require hydrolysis of the ft,y Histone mRNA, which is non-polyadenylated, phosphodiester bond. From these results Schroder et unspliced and similar in size to the globin mRNA, is al. (1987a) suggested that the release of RNA might be released into the cytoplasm within 10 min (Adesnik & caused by a conformational change of a nuclear matrix Darnell, 1972). Adenovirus pIX mRNA (about 9S,

26 R. Verheijen et al. polyadenylated and unspliced) reaches the cytoplasm Thomas (1985) found that the hnRNP core proteins in within 4 min after the start of synthesis (Mariman et al. mitotic HeLa cells were not free but stably associated 19826). In contrast, the bulk of the late adenovirus with high molecular weight RNA in the form of mRNAs reach the cytoplasm only after 16 min (Mari- hnRNP particles that sedimented between 80 and man et al. 19826), a phenomenon that has been found 200 S. Recent studies by these authors (Lahiri & for most cellular mRNAs as well (see, e.g., van Thomas, 1987) showed that during mitosis at least Venrooij et al. 1975). The reason for these divergent 95 % of the total cellular snRNPs was also present in rates of transportation between matrix-bound mRNAs such hnRNP complexes, sedimenting at about 100 S. is not understood. Probably most of the larger pre- Other nuclear proteins, when investigated with im- mRNAs require more complicated processing patterns, munocytochemical techniques, appear to be associated which means that the rate of transportation of mRNA with the condensed chromosomes in mitosis (Chaly et depends mainly on the rate of maturation. Another al. 1984). Such behaviour has been specifically de- possibility is that mRNAs emerging rapidly into the scribed for some nucleolar proteins (see, e.g., Pfeifle et cytoplasm are not assembled into the usual hnRNP al. 1986), topoisomerase I (see, e.g., Verheijen et al. structures, as has been suggested by Pederson for 19866) and topoisomerase II (Earnshaw et al. 1985). transcripts lacking introns (Pederson, 1983). In this The question arises as to how this group of nuclear respect it should be mentioned that the final maturation (matrix) proteins is associated with the chromatin. The step of mRNA, that is the binding of the typical models for chromosome architecture that are in vogue cytoplasmic mRNA-associated proteins (Wagenmakers have been reviewed by Earnshaw (1986). In one of et al. 1980), is accompanied by the release of hnRNA- these models a non-histone scaffold supposedly main- associated proteins (van Eekelen et al. 19816; van tains the higher-order topological organization of DNA Venrooij et al. 1982a). It has also been established that mature mRNAs in the nucleus cannot be considered as in mitotic chromosomes. Such scaffolds have been integral components of the nuclear matrix, as are visualized in the electron microscope by Paulson & precursor RNAs (Ciejek et al. 1982; Schroder et al. Laemmli (1977) after treating isolated mitotic HeLa 1987a). chromosomes with dextran sulphate and heparin in order to remove the histones. Their preparations consisted of a subset of non-histone proteins attached Behaviour of nuclear matrix components to intact chromosomal DNA. The proteinaceous com- during mitosis ponent or chromosome scaffold has been isolated from such structures after nuclease digestion and extraction The onset of mitosis is accompanied by an extensive of chromosomal proteins (Adolph et al. 1977; Lewis & rearrangement of nuclear components (see Fig. 8). As Laemmli, 1982) and was initially proposed to be a rigid the cell approaches mitosis, the nucleolus first de- linear axial backbone in each chromatid, responsible creases in size and then disappears as the chromosomes for the morphology of metaphase chromosomes (Paul- condense and all RNA synthesis ceases. At prophase son & Laemmli, 1977). Among the scaffold proteins, the lamins become highly phosphorylated (Ottaviano & Lewis & Laemmli (1982) found two prominent high- Gerace, 1985), followed by dissassembling of the molecular weight polypeptides, Sc-1 and Sc-2, of about nuclear envelope. In immunofluorescence localization 170 and 135K, respectively. Using a polyclonal anti- studies it has been shown that during prophase many body that recognized chicken scaffold protein Sc-1, nuclear (matrix) proteins shift from their distinct nuclear locations to a diffusely cytoplasmic distri- Earnshaw et al. (1985) have shown this polypeptide to bution, excluding the condensed chromosomes (Chaly be topoisomerase II (see also Gassere/ al. 1986). Other et al. 1984). At telophase this process is reversed. Such components that have been found in isolated chromo- behaviour has been documented for several pore com- some scaffolds are centromeric proteins (Earnshaw et plex proteins (see, e.g., Davis & Blobel, 1986; Snow e( al. 1984). al. 1987), the lamins (see, e.g., Gerace, 1986; Ver- Chromosome scaffolds respond dramatically to heijen et al. 19866), snRNP proteins (see, e.g., Reuter changes in the ionic environment. Scaffolds isolated in et al. 1985; Spector & Smith, 1986; Verheijen et al. the presence of 2 M-NaCl differ completely in appear- 19866) and hnRNP proteins (see, e.g., Martin & ance from those isolated at low ionic strength in the Okamura, 1981). For some of these proteins the presence of dextran sulphate and heparin (Earnshaw & molecular state of association in mitotic cells has been Laemmli, 1983), even though the protein composition investigated. Lamin B in Chinese hamster ovary of the two forms appears to be identical (Lewis & (CHO) cells, for example, has been found to remain Laemmli, 1982). Exposure of isolated scaffolds to associated with phospholipid vesicles during mitosis, millimolar concentrations of Mg2+ also causes a similar while lamins A and C are converted into a soluble form dramatic alteration in scaffold appearance (see Fig. 9; (Burke & Gerace, 1986). In a previous study, Lahiri & and references, Earnshaw & Laemmli, 1983).

The nuclear matrix 27 Fig. 8. Immunofluorescent localization of various nuclear antigens in MR65 cells (human lung carcinoma) in interphase (Al-Dl) and metaphase (A2-D2) with monoclonal antibodies directed against: A, the lamins (antibody 41CC4); B, the Ul RNP-specific 70K protein (antibody 2.73); C, the hnRNP-associated C proteins (antibody 4F4); D, a nucleolus- associated cell proliferation marker (antibody Ki-67). X1150.

''••«•- •

Fig. 9. Electron micrographs of chromosome scaffolds prepared from highly purified HeLa mitotic chromosomes. A. Scaffold prepared at low ionic strength using a dextran sulphate/heparin lysis mix. B. Scaffold prepared at low ionic strength using a dextran sulphate/heparin lysis mix, followed by exposure to S mM- MgC^. Centromere region, indicated by arrows. Bar, 1-0 jUm. (Courtesy of Dr W. C. Earnshaw; see also Earnshaw & Laemmh (1983).)

28 R. Verlieijen et al. Hancock & Dessev (1987) have isolated chromo- dimensions of the skeletal element determined by the somes by lysis with Nonidet P40 in low ionic strength DNA associated with it. buffer without divalent cations or EDTA. These Using the same polyclonal antibody to chicken chromosomes slowly stretched to up to several times topoisomerase II as described above, Earnshaw & Heck their original length while conserving an identifiable (1985) have examined the distribution of this enzyme chromosome morphology (see Fig. 10). This finding is in intact, swollen but unextracted, chromosomes from incompatible with a rigid nature of a supposed shape- MSB-1 chicken lymphoblastoid cells. Under the con- determining skeletal element. These authors further ditions used in their experiments, topoisomerase 11 was observed that each chromatid was not linear, but localized in numerous separate spots that appeared to consisted of a spiraled fibre, many times the chroma- be 120-200 nm across when covered with bound anti- tid's length, which was unwound upon incubation of polyamine-stabilized chromosomes with deoxvcholate body. It was therefore concluded that these data did or diiodosalicylate (see Fig. 11). The winding of this not provide evidence for the existence of a rigid core- fibre was suggested by Hancock & Dessev (1987) as like scaffold structure. being determined by protein-protein interactions be- In summary, despite the controversial experimental tween neighbouring gyres. These authors also con- data, we think it is justifiable to conclude that, if cluded that if a skeletal element does exist in mitotic present /// vivo, the chromosomal scaffold is not a rigid chromosomes it does not itself dictate the dimensions structure, but rather a component of the mitotic of the chromosome, but rather are the form and chromosome with dvnamic features.

Fig. 10. Mitotic chromosomes from CHO cells, prepared by lysis with 0-25% Nonidet P40 in 0-15 M- sucrose/0-2M-phosphate, pH7-5. The lysate was stained with Hoechst 33258 and examined in suspension by fluorescence microscopy. The chromosomes showed a dramatically extended appearance (B,C) as compared to their initial shape (A). Magnifications in A, B and C are the same. Bar, 10;/m. (Courtesy of Dr R. Hancock; see also Hancock & Dessev (1988).)

The nuclear matrix 29 Fig. 11. Mitotic chromosomes from CIIO cells prepared in a polyamine-containing medium by lysis with Nonidet P40. Lithium diioclosalicylate was added to a final concentration of 25 mM and the chromosomes were immediately examined by fluorescent staining with Hoechst 33258, showing that chromatids are wound in helical gyres. Bar, lOjUm. (Courtesy of Dr R. Hancock; see also Hancock & Dessev (1988).)

This study was supported by the Netherlands Cancer ALEXANDER, R. B., GREENE, G. L. & BARRACK, E. R. Foundation, grant no. NUKC 1984-11. The authors thank (1987). Estrogen receptors in the nuclear matrix: direct Professor Dr W. W. Franke (Heidelberg, FRG) for provid- demonstration using monoclonal antireceptor antibody. ing Fig. 2, Drs G. Brakenhoff and Dr R. van Driel Endocrinology 120, 1851-1857. (Amsterdam, The Netherlands) for Fig. 3, Dr V. Bibor- BARRACK, E. R. & COFFEY, D. S. (1982). Biological Hardy (Sherbrooke/Montreal, Canada) for Fig. 4, Drs A. properties of the nuclear matrix: steroid hormone Cidadao and M. Carmo-Fonseca (Oeiras, Portugal) for Fig. binding. Recent Prog. Honn. Res. 38, 133-195. 5, Drs E. G. Fey and S. Penman (Cambridge, USA) for Figs BASTOS, R. N. & Aviv, II. (1977). Globin RNA precursor 6 and 7, Dr W. C. Earnshaw (Baltimore, MD, USA) for Fig. molecules: biosynthesis and processing in crythroid cells. 9, and Dr R. Hancock (Quebec, Canada) for Figs 10 and 11. Cell 11, 641-650. We also acknowledge the kind gifts of the monoclonal BENAVENTE, R., KROHNE, G., SCHMIDT-ZACHMANN, M. S., antibodies 41CC4 (from Dr G. Warren; Heidelberg, FRG), HOGLE, B. & FRANKE, W. W. (19846). Karyoskeletal 2.73 (from Dr S. I loch; La Jolla, USA) and 4F4 (from Dr G. proteins and the organization of the amphibian oocyte Dreyfuss; Evanston, USA). nucleus. J. Cell Sci. Suppl. 1, 161-186. BENAVENTE, R., KROHNE, G., STICK, R. & FRANKE, W. W. References (1984a). Electron microscopic immunolocalization of a karyoskeletal protein of molecular weight 145,000 in AARONSON, R. P. & Woo, E. (1981). Organization in the nucleoli and perinucleolar bodies of Xenopus laeris. Expl cell nucleus: divalent cations modulate the distribution Cell Res. 151, 224-235. of condensed and diffuse chromatin. J. Cell Biol. 90, BEN-ZE'EV, A. & ALONI, Y. (1983). Processing of SV40 181-186. RNA is associated with the nuclear matrix and is not ABULAFIA, R., BEN-ZE'EV, A., HAY, N. & ALONI, Y. followed by the accumulation of low-molecular-wcight (1984). Control of late SV40 transcription by the RNA products. Virology 125, 475-479. attenuation mechanism and transcriptionally active BEREZNEY, R. (1980). Fractionation of the nuclear matrix. ternary complexes are associated with the nuclear matrix. J. moiec. Biol. 172, 467-487. I. Partial separation into matrix protein fibrils and a ADESNIK, M. & DARNELL, J. E. (1972). Biogenesis and residual ribonucleoprotein fraction. J. Cell Biol. 85, characterization of histone mRNA in HeLa cells. 641-650. J. molec. Biol. 67, 397-406. BEREZNEY, R. (1984). Organization and functions of the ADOLPH, K. W., CHENG, S. M. & LAEMMLI, U. K. (1977). nuclear matrix. In Chmmosomal Nonhistone Proteins- Role of nonhistone proteins in metaphase chromosomes. Structural Associations, vol. IV (eel. L. S. Hnilica), pp. Cell 12, 805-816. 119-180. Boca Raton, Florida: CRC Press. AEBI, U., COHN, J., BUHLE, L. & GERACE, L. (1986). The BEREZNEY, R. & COFFEY, D. S. (1974). Identification of a nuclear lamina is a meshwork of intermediate-type nuclear protein matrix. Bioclieni. biophys. Res. Coniinun. filaments. Nature, Land. 323, 560-564. 60, 1410-1417.

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36 R. Verheijen el a\.