Histol Histopathol (1996) 1 1: 1035-1048 Histology and Histopathology
Invited Re vie W
The nuclear matrix: a critical appraisal
A.M. ~artellil,L. Cocco2, B.M. ~iederer~and L.M. Neri4~5 Department of Human Morphology, Universita di Trieste, Trieste, %stitute of Anatomy, University of Bologna, Bologna, Italy, 31nstitute of Anatomy, Universite de Lausanne, Lausanne, Switzerland, 41nstitute of Anatomy, University of Ferrara, Ferrara, Italy, 5C.N.R. Institute of Normal and Pathological Cytomorphology, c10 I.O.R., Bologna, ltaly
Summary. It is becoming increasingly clear that the cell compartments (Hernandez-Verdun, 1991) and hetero- nucleus is a highly structurized organelle. Because of its chromatin, the nucleus also contains a heterogeneous tight compartmentalization, it is generally believed that a group of inclusions, such as interchromatin granules framework must exist, responsible for maintaining such (Clevenger and Epstein, 1984; Thiry, 1993, 1995; a spatial organization. Ferreira et al., 1994), nuclear bodies (Brasch and Ochs, Over the last twenty years many investigations have 1992) and coiled bodies (Thiry, 1994), that have been been devoted to identifying the nuclear framework. known since a long time. Structures isolated by different techniques have been Thanks to the outstanding advances made by obtained in vitro and are variously referred to as nuclear molecular biology techniques, our knowledge about the matrix, nucleoskeleton or nuclear scaffold. Many genetic machinery and its regulation or how DNA is different functions, such as DNA replication and repair, replicated is continuously growing. Now genes can be mRNA transcription, processing and transport have been identified, purified, sequenced, changed at our will, described to occur in close association with these reintroduced into cells and expressed there as proteins. structures. However, there is still much debate as to However, an overall view about how functions are whether or not any of these preparations corresponds to structurally integrated within the nucleus is still lacking a nuclear framework that exists in vivo. In this article we (Manfredi Romanini and Fraschini, 1996). summarize the most commonly-used methods for During the last few years, morpho-functional studies obtaining preparations of nuclear frameworks and we employing antibodies against molecules involved in also stress the possible artifacts that can be created in DNA replication and transcription, DNA repair, mRNA vitro during the isolation procedures. processing and transport, steroid hormone binding sites, Emphasis is placed also on the protein composition have revealed that these phenomena are highly of the frameworks as well as on some possible signalling compartmentalized in the nucleus, because they take functions that have been recently' described to occur in place in well-distinct domains, of which some tight association with the nuclear matrix. correspond to the aforementioned inclusions (e.g. Lawerence et al., 1989; Nakayasu and Berezney, 1989; Key words: Nuclear matrix, Isolation techniques, Mazzotti et al., 1990; Carter et al., 1991; Neri et al., Stabilization, Protein composition, Functions 1992; Hassan and Cook, 1993; Wansink et al., 1993; Blencowe et al., 1994; Jackson et al., 1994; Zhang et al., 1994; Bisotto et al., 1995; van Steensel et al., 1995). Introduction Thus, these morpho-functional investigations have demonstrated that a division in compartments is indeed It has commonly been thought that the cell nucleus present in the nucleus, though it is not clearly evident at is relatively unstructured, especially when compared first glance. A belief common to many investigators is with the tight compartmentalization found within the that, if the nucleus is indeed strictly compartmentalized, cytoplasm. An obvious reason for such a view is that a structure must exist, responsible for maintaining such a there are no membrane structures inside the nucleus. rigid subdivision. Nevertheless, apart from very prominent domains such Investigations carried out over the last twenty years as the nuclear envelope with the pore complexes (Pant6 have demonstrated that when the nucleus is stripped of and Aebi, 1995), the nucleolus with its sub- most DNA, RNA and soluble proteins (mainly histones) a structure remains that has been variously referred to as Offprint requests to: Dr. Alberto M. Martelli, Dipartimento di Morfologia the nuclear matrix, or scaffold or the nucleoskeleton Urnana Normale, Universita di Trieste, via Manzoni 16, 1-34138 Trieste, (Berezney, 1984, 1991; Verheijen et al., 1988; de Jong et ltaly al., 1990; van Driel et al., 1991; Jack and Eggert, 1992; An overview of nuclear matrix
Cook, 1995). Such a structure, acting as a framework, well-distinct domains: a peripheral lamina containing would provide attachment sites to the structural domains residues of the pore complex; an ill-defined inner (or DNA loops) in which the eukaryotic genome is fibrogranular network; and residual nucleoli (Maraldi et subdivided during interphase (e.g. Gerdes et al., 1994). al., 1986). Using the EDTA regressive staining and thin Indeed, it is bewildering to realize that in a typical sectioning, a similarity was seen between the matrix diploid mammalian cell about 2 m of double-stranded fibrogranular network and a framework that can be DNA are packed within a nucleus with a diameter of evidenced by transmission electron microscopy in the approximately 10 pm. To achieve this, the DNA double nucleus of intact cells (Berezney, 1984). Such an helix is first coiled into nucleosomes (11 nm diameter), observation strengthened the belief that a matrix would then nucleosomes into solenoids (30 nm diameter) which also exist in vivo. The original protocol devised by are thought to be folded into loops of tens of Berezney and Coffey (1974) called for the use of 2M kilobasepairs (Cook, 1995). It is clear that only a highly NaCl to extract histones and soluble or loosely-bound structured organelle may handle the topological nuclear proteins, but subsequently it has been claimed problems arising for example during duplication of that use of lower salt concentrations (for example 0.25M DNA. (NH4)2S04) may lead to a better preservation of the However, there is always uncertainity as to whether morphology without significantly affecting the or not the nuclear matrix, isolated in vitro, could really efficiency of protein extraction (e.g. Belgrader et al., represent a framework of the nucleus in vivo. In fact, the 1991a). It is very important to note that many variations only nuclear framework of which the existence is from the original protocol have been described, universally accepted is the nuclear lamina, a continuous depending on the cell type being used. Indeed, the thin fibrous layer that underlies the inner nuclear original matrix isolation technique was described for rat membrane and is mainly composed from three related liver nuclei, that are extremely resistant to repeated proteins named lamin A, B and C (Moir and Goldman, centrifugations. 1993; Dessev, 1994). Moreover, during nuclear isolation, in rat liver there A problem that has plagued this field of is a spontaneous formation of disulfide bonds, with a investigation is the use of different names to denote very consequent <
The LIS extraction technique allowed the importantly, these structures synthesize DNA and RNA identification of matrix-associated regions (MARs), also at a rate found in vivo (e.g. Jackson et al., 1992). It called scaffold-associated regions (SARs), that is AT- should also be noted that these nucleoskeletons do not rich stretches of 300-1000 bp, highly conserved during require heat ccstabilization~in vitro (see later, and the evolution and believed to anchor DNA loops to a Jackson et al., 1990a). In any case, this isolation protocol nuclear framework (Mirkovitch et al., 1984; Izuarralde has not been frequently used, perhaps because the et al., 1988, 1989). It is worthwhile recalling that MARs method is quite complex and the preparations are not sequences bind to heterologous nuclear matrices well-suited to biochemical studies. Nevertheless, such a (Izuarralde et al., 1988, 1989). MARs are common at the method has permitted to size in 86 kb the average length boundaries of transcription units and are often found of DNA loops in HeLa cells (Jackson et al., 1990a,b). near enhancer-like regulatory sequences (see Wang et Moreover, electron microscope immunolabelling has al., 1995) but have been implicated also in DNA shown in this type of framework the existence of replication and chromosome segregation, at least in ccreplication or transcription factories,, (Hughes et al., yeasts (Amati and Gasser, 1988). However, subsequent 1995), that is large structures containing, beside newly- studies have shown that MAR binding sites are present synthesized DNA or RNA, several factors involved in also in matrices prepared by 2M NaCl extraction DNA duplication or transcription, such as DNA (Izuarralde et al., 1988) but further evidence suggests polymerase a, proliferating cell nuclear antigen (or that the major part of the binding sites are generated in PCNA, i.e. the 36 kDa accessory protein to DNA vitro during stabilization of nuclei by heat (Jackson et polymerase a), RP-A, RNA polymerase 11, etc (Hozak et al., 1990a,b). Moreover, in nuclei not exposed to al., 1993; Hughes et al., 1995). It is of interest that at nonionic detergent, an ectopic SAR sequence does not least some of these factories correspond to a sub-set of bind to the nuclear scaffold, whereas it does in nuclear bodies observed in the past in thin sections permeabilized nuclei (Eggert and Jack, 1991). The (Brasch and Ochs, 1992; Hozak et al., 1994). Cook and classic biochemical studies by Laemmli and coworkers associates have elaborated a model of matrix-associated hinted at the fact that proteins binding MAR sequences DNA transcription and replication in which it is should be components of the inner matrix network envisaged that both of these events take place as (Izuarralde et al., 1988). So far, a few nuclear proteins template slides through fixed sites (i.e. the factories) that can bind SARs in vitro have been identified (see located around a nucleoskeleton (but see also Pardoll et later). al., 1980; Vogelstein et al., 1980; Tubo et al., 1987; Tubo and Berezney, 1987b). Overall, many of the results The agarose-embedded matrix obtained with this type of preparation are in agreement with data gained from nuclear frameworks obtained in a Since chromatin tends to aggregate under isotonic more conventional way. Lastly, it should be recalled that conditions, it is necessary to isolate nuclei using Hozak et al. (1995) have demonstrated, by immuno- unphysiological (highly hypotonic) salt concentrations. labelling at both the light and electron microscope level, The aggregation can also be prevented by using Mg++, the presence, in the interior network, of nucleoskeletal but this has an adverse effect on chromatin structure and structures, of nodes containing lamin A. Such an activates nucleases (Cook, 1988). For this reason, Cook observation, which follows a number of previous reports and coworkers have developed a procedure in which dealing with the intranuclear presence of lamins (e.g. HeLa cells, growing embedded in agarose microbeads Moir et al., 1994), suggests that the inner framework (50-150 pm in diameter), are permeabilized with a mild could be somewhat related to the cytoskeleton, as nonionic detergent (0.1% Triton X-100) in a nuclear lamins share much sequence homology with ccphysiological,, buffer (e.g. Jackson et al., 1988). Then intermediate filaments (e.g. McKeon et al., 1986). DNA is removed by restriction enzymes and Therefore, the authors envisioned the existence of a subsequently electroeluted. However, it should be common cell skeleton, spanning from the plasma emphasized that, so far, nobody really knows the exact membrane to the nuclear interior (Hozak et al., 1995), in ionic composition of the nuclear interior, thus the term agreement with other suggestions (Pienta et al., 1989; ccphysiological,, must be interpreted with caution. Getzenberg et al., 199la,b;Pienta and Coffey, 1992) and Moreover, even if this technique has been claimed to be their own previous observation about lumps of very mild, it should be noticed that evidence has chromatin attached to an intermediate-filament-like demonstrated that when cultured cells are permeabilized skeleton (Jackson and Cook, 1988). with 0.1% Triton X-100 there is a redistribution of cytoplasmic proteins to the cell nucleus (Melan and The in situ matrix Sluder, 1992). Nevertheless, these kind of preparations (referred to Capco et al. (1982) and Staufenbiel and Deppert as nucleoskeletons) offer several advantages over the (1984) first described a method in which adherent cells others, because the gross structural morphology is are extracted in situ with nonionic detergents and salt preserved, there is DNA integrity (i.e. DNA is solutions and digested with nucleases. These structures supercoiled after removing histones) and, most are referred to as in situ nuclear matrix or nuclear An overview of nuclear matrix
matrix-intermediate filament complexes. The obvious and maturation, while Frey and Matera (1995) have advantage of this type of isolation technique is that the demonstrated that they also contain snRNP U7 and time required for obtaining the final structures is much associate with specific DNA sequences. Another striking shorter than in conventional preparations, because there example is the doughnut-shaped nuclear body containing are no time-consuming centrifugations. Furthermore, the 126-kDa matrix protein recognized by monoclonal there is probably a better preservation of the morphology antibody 5E10 (Stuurman et al., 1992a) as well as the because of the absence of centrifugations at high speed. PML protein, that is part of a fusion product with the The major disadvantage is that these preparations also retinoic acid receptor a (RARa), resulting from the retain cytoskeletal components, so that biochemical t(15;17) chromosomal translocation associated with studies (for example electrophoretic analysis of acute promyelocytic leukemia (De ThC et al., 1991). It is polypeptides) must deal with this fact, even though Fey extremely interesting to note that the PML-RARa hybrid and Penman (1988) subsequently claimed the discovery displays an altered nuclear localization when compared of a method allowing the separation of the nuclear to that of its normal PML and RARa counterparts (Weis matrix from cytoskeletal components. Penman and et al., 1994). In leukemic cells the major part of PML coworkers have also emphasized the necessity to use nuclear bodies are disorganized into several aberrant 0.25M (NH4)+04 as a milder extracting agent and they microstructures containing both PML and PML-RARa. have stressed the role played by RNA in maintaining Importantly, treatment of the neoplastic cells with the structural integrity of the inner network by retinoic acid restores both the normal morphology and demonstrating that the RNase inhibitor, vanadyl the number of the bodies (Koken et al., 1994). Moreover, rybonucleoside complex, is necessary for isolating intact infection with adenovirus causes both a redistribution structures. Conversely, the use of RNase A completely and morphological changes of the nuclear bodies destroys the inner matrix (Nickerson et al., 1989; He et (Puvion-Dutilleul et al., 1995). Several viral proteins al., 1990). If the nuclear matrix-intermediate filament (E4-ORF3 E1A and SV40 large T antigen) localize to or complex (obtained after treatment with (NH4)2S04) is accumulate in close proximity to these bodies suggesting further extracted with 2M NaCl, a network of 9 and 13 that they might be a preferential target for small DNA nm cccore filaments,, becomes visible, which can be tumor virus oncoproteins (Carvalho et al., 1995). These studied by whole mount and resinless thick section data are in agreement with the results by Smith et al. electron microscopy (He et al., 1990). The filaments can (1985) and Pombo et al. (1994) concerning nuclear be completely disrupted by treatment with RNase A. By matrix and adenovirus DNA replication. This nuclear means of a monoclonal antibody, Nickerson et al. (1992) domain is the same as described by Ascoli and Maul have identified a high molecular weight (240-kDa) (1991), identified by monoclonal antibodies and human protein (HlB2) that is a component of the core filament. auto antibodies. It is also indicated as NDlO and Interestingly enough, the antigen is masked in the contains protein SP100, and autoantigen in primary interphase nucleus while it is uncovered as chromatin biliary cirrhosis, as well as 55- and 52-kDa polypeptides condensation takes place during mitosis. It should be (see Korioth et al., 1995). It should be recalled, however, pointed out that protein composition of core filaments, that other nuclear domains have been identified that are though quite complex, is not very different from the not part of the nuclear matrix, because they do not resist polypeptide profile of the material extracted by 2 M treatment with DNase I. A well-known example is the NaCl (He et al., 1990). region called PIKA (polymorphic interphase karyosomal The in situ matrix is well suited for morpho- association) described by Saunders et al. (1991) functional studies having as a goal the identification of containing three to four structurally-related proteins (23- the distribution of nuclear matrix proteins and their 25-kDa in size) that are recognized by a human relationship with phenomena like DNA replication and antiserum. transcription, DNA repair, RNA splicing, mRNA transport etc. (see Introduction for the references). Stabilization of the matrix This technique has demonstrated that some types of nuclear bodies can be considered part of the nuclear In 1981 Kaufmann et al. first demonstrated that matrix, because at least some of their constituents resist when the nuclear matrix is prepared from rat liver nuclei, treatment with salt and nucleases (Brasch and Ochs, formation of S-S bonds spontaneously occurs during 1992). This applies to coiled bodies, a structure first isolation of nuclei. If formation of the bonds is inhibited identified almost one hundred years ago by Ramon-y- by alkylating agents such as n-ehtylmaleimide andlor Cajal (for a review see Bohmannn et al., 1995a), and iodoacetamide the inner matrix and residual nucleoli are containing the small nuclear ribonucleoproteins barely recognizable while the peripheral lamina is still (snRNPs) U1, U2, U4, U5 and U6, the autoantigen p80 detectable (Kaufmann and Shaper, 1984). Treatment coilin, as well as proteins deriving from the nucleolus with RNase A and dithiothreitol results in even more like fibrillarin, Nopp 140, NAP 57 and ribosomal protein empty structures showing only the peripheral lamina. S6 (Bohmann et al., 1995a). Very recently Bohmann et For this reason, formation of disulfide bonds, acted by al. (1995b) have shown that coiled bodies might interact the cross-linking agent sodium tetrathionate (NaTT), has with nucleoli in regulating ribosomal RNA transcription been deliberately used to stabilize the inner network An overview of nuclear matrix
(Stuurman et al., 1990; Nakayasu and Berezney, 1991). al., 1995a). Our recent results also indicate that an in It is worth remembering that the stabilization created by vitro incubation at 37 T of isolated nuclei causes Nan had been erroneously interpreted as the result of changes in the distribution of nuclear matrix proteins protease inhibition (Berezney, 1979). Two important and, most interestingly, that changes seen after in vitro issues need to be emphasized about this form of incubation closely resemble those visible after heat stabilization. Firstly, available evidence suggests that shock in vivo (Neri et al., 1994, 1995a). Moreover, proteins of the nuclear matrix are not cross-linked by changes are also seen when nuclei are incubated in a disulfide bonds in vivo (Kaufmann and Shaper, 1991). buffer without Mg++ (Neri et al., manuscript in Secondly, it seems that spontaneous formation of preparation) which instead contains the polyanions, disulfide bonds occurs only when nuclei are isolated spermine and spermidine as well as KC1 (Izuarralde et from normal rat liver or hepatoma cells (Kaufmann and al., 1988). An example of how a thermal exposure can Shaper, 1984, 1991) because treatment with iodo- influence the distribution of nuclear matrix polypeptides acetamide is ineffective when used in HeLa cells or is presented in Fig. 1. While the 240-kDa antigen, mouse erythroleukemia cells (Van Eeekelen et al., 1982; referred to as NuMA (nucleus-mitotic apparatus) was Belgrader et al., 1991a; Martelli et al., 1992a). In these insensitive to a 20 min incubation of isolated nuclei at cases, the inner matrix and nucleolar remnants are 37 T in a buffer containing spermine/spermidine/KCl, a present even if nuclei were exposed to sulfhydryl ribonucleoprotein (RNP) component showed dramatic blocking agents. Interestingly enough, the formation of changes in comparison with intact cells. In the same intranuclear disulfides also takes place when the matrix picture, it can be observed that when isolated nuclei is prepared in situ from hepatoma cells (Kaufmann and were kept at 0 T for 20 min no variations in the Shaper, 1991). immunofluorescent pattern due to a 105-kDa splicing Laemmli and coworkers described the stabilizing component were seen, while they were very evident after effects of divalent cations such as Cu++ or Ca++, a 37 T incubation (data not shown). Although Mg++ especially when nuclei were exposed to these agents at a ions are usually held responsible for creating artifacts temperature of 37 T or above (Lebkowski and Laemmli, during isolation of nuclei (Cook, 1988), our data clearly 1982; Lewis et al., 1984). Mirkovitch et al. (1984) used a show that other molecules can have a similar effect. It 37 T incubation of isolated nuclei from Drosophila cells should also be emphasized that at least two nuclear to stabilize the inner matrix before LIS extraction and to matrix proteins contain hydrophobic domains (see later). preserve interactions between the scaffold and SARs. In Thus, the 37 'T stabilization of isolated nuclei could be 1985, Evan and Hancock unequivocally demonstrated due to protein aggregation andlor precipitation, likewise the stabilizing effect of a 37 V incubation of isolated the stabilization caused by heat shock in vivo. However, nuclei, leading to the insolubilization of a specific subset there must be differences between the two phenomena, of nuclear proteins. It si very important to note that up to because the protein composition of the matrix prepared that time a 37 T incubation has been inadvertently used from nuclei heat-stabilized in vitro is different, when by many investigators, for example during nuclease analyzed by two-dimensional gel separations, from the digestion. Therefore, an unnoticed stabilization of the matrix isolated from nuclei deriving from cells subjected matrix inner network had been obtained. to heat shock in vivo (Martelli et al., 1995). The protein The stabilizing effect of heat is a universal composition of the matrix after a heat shock in vivo phenomenon because it has been observed in nuclei closely resembles that seen after stabilization of isolated isolated from a variety of cell lines and even from yeasts nuclei with Nan (Martelli et al., 1995). For example, (Boyle and Baluda, 1987; Humphrey and Pigiet, 1987; nucleolar proteins B23lnumatrin and C23/nucleolin are McConnell et al., 1987; Berrios and Fisher, 1988; very abundant in the nuclear matrix derived from in vivo Martelli et al., 1990, 1991). So far, the biochemical heated cells or chemically cross-linked nuclei, whereas mechanism(s) underlying this stabilization have escaped they are very scarce (B23) or totally absent (C23) from clarification. However, it should be stressed that an the matrix after exposure of nuclei to 37 T in vitro analogous stabilization occurs when nuclei are (Martelli et al., 1995). immediately prepared from cells exposed in vivo to Nevertheless, our data also suggest that formation of temperatures that produce a heat shock response S-S bonds during in vitro heat stabilization of isolated (Littlewood et al., 1987; Martelli et al., 1991; nuclei is unlikely to occur in agreement with our Wachsberger and Coss, 1993). It is commonly thought previous suggestions (see Martelli et al., 1994a). that during heat shock nuclear proteins become Furthermore, two-dimensional gel analysis has revealed denatured with a subsequent exposure of hidden that the protein composition of the in vitro heat- stretches of hydrophobic aminoacids (Becker and Craig, stabilized matrix is not very different from that of 1994; Hart1 et al., 1994). This in turn leads to structures isolated without this form of treatment aggregation and precipitation ensuing in insolubilization (Martelli et al., 1995), thus suggesting that the increased of nuclear proteins. It is very interesting to note that recovery of protein, measurable in heat-exposed during heat shock a redistribution of nuclear proteins, of structures, is predominantly due to an additional which some belong to the nuclear matrix, takes place recovery of the same types of polypeptides. (McConnell et al., 1987; de Graaf et al., 1992; Neri et Functional studies have evidenced that in vitro heat An overview of nuclear matrix
stabilization can lead to the formation of artifacts and in the localization of matrix polypeptides (Neri et al., these cases the available data do not fit well with the manuscript in preparation). model envisaging the matrix as the active site for DNA As far as stabilization with NaTT is concerned, it replication and transcription (Fisher et al., 1989; Martelli should be stressed that it does not change the distribution et al., 1992b, 1994b). Moreover, the data from Jackson of some nuclear matrix proteins (Neri et al., 1995b). et al. (1990a,b) have clearly established that the ccthermal Stabilization with this chemical has also been used for stabilizationn creates five new (artefactual) DNA loop preparing the in situ matrix (e.g. Wansink et al., 1993). attachment sites in HeLa cell nuclei for every one that However, since NaTT does not act only by inducing pre-existed. Therefore, in our opinion, such an in vitro formation of S-S bonds, but also through other, as yet stabilization should be avoided. Our unpublished data unidentified mechanism(s) (Stuurman et al., 1992b), we have shown that in isolated nuclei, Cu++ also changes feel that caution is necessary when employing this
Fig. 1. lmmunofluorescent staining of intact K562 human erythroleukemia cells (a, c, e) and isolated nuclei (b, d, f) reacted with antibody to NuMA protein (a,b), RNP constituent (c,d) and 105-kDa splicing component (e,f). In b and d nuclei were incubated for 20 min at 37 'C in a buffer containing spermine/spermidine/KCI, whereas in f they were kept in the same buffer for 20 min at 0 TC.Samples were analyzed by a confocal laser scanning microscope. Bar: l pm. An overview of nuclear matrix
chemical, also because enzymic activities cannot be polypeptide acting as a shuttle protein playing a role in recovered after this treatment (Martelli and Cocco, transport from cytoplasm to the nucleus (Fields et al., 1994). 1986; Feuerstein et al., 1988; Hernindez-Verdun, 1991; Martelli et al., 1995). Concerning proteins that bind Protein composition of the nuclear matrix MARs, a few of them have been identified, including topoisomerase 11, a constituent of the matrix isolated Even though the first available reports indicated that from different cell types (e.g. Berrios et al., 1985; the protein composition of the nuclear matrix was quite Adachi et al., 1989; Kaufmann and Shaper, 1991; Zini et simple when compared with that of nuclei (e.g. Berezney al., 1994). Other polypeptides sharing such a property and Coffey, 1977), the use of two-dimensional gel are SATBl (Dickinson et al.. 1992), particularly electrophoresis has evidenced that the polypeptide expressed in thymocytes, and SAF-A, a 120-kDa profile of the matrix is exceedingly complicated. This protein, identical to hnRNP-U (Fackelmayer et al., has been mostly demonstrated in the ccnuclear matrix- 1994), that has also been recognized and studied by intermediate complex,, preparations (e.g. Fey and other groups (e.g. Tsutsui et al., 1993; von Kries et al., Penman, 1988; Dworetzky et al., 1990). These 1994) and the 240-kDa protein referred to as NuMA investigations have shown differences depending on the (LudCrus et al., 1994). Very recently, a nine amino acid cell type and related to neoplastic transformation sequence motif has been identified in SATBl, conferring (Getzenberg et al., 1991b; Keesee et al., 1991). to the protein unique MAR binding activity (Wang et al., Stuurman et al. (1990) have investigated the NaTT- 1995). Using nuclear matrices prepared by a high-salt stabilized matrix protein composition of different cell extraction, Zong and Scheuermann (1995) have types from several mammalian species. Silver staining described a novel 33-kDa DNA binding protein (MAR- combined with two-dimensional separations have BP1) that specifically binds to MARs associated with the revealed several hundreds of polypeptides. The immunoglobulin heavy chain (IgH) enhancer. Results electrophoretic profiles differed depending on the cell have also shown that in cells where the IgH locus is types being studied. The authors referred to these transcriptionally inactive, the nuclear factor-p negative proteins as the ((minimal matrix,,. Nakayasu and regulator binds to IgH enhancer and interferes with Berezney (1991) have identified several abundant MAR-BPlJenhancer interactions, thus preventing proteins that are ubiquitous in matrix preparations attachment to the nuclear matrix. Also, protein obtained from different cells and tissues and have C23/nucleolin binds MARs and it seems that poly- denominated these polypeptides as ccnuclear matrins,,. peptides sharing such a property can be divided into two Polyclonal antibodies, staining an inner nuclear groups: those that bind to double-stranded DNA fibrogranular network, have been raised against nuclear (possibly through recognition of a peculiar tertiary matrins. Some of the matrins have been cloned and structure such as a narrow minor groove) like MAR-BP1 sequenced. Matrin FIG are two proteins with a molecular as well as SATBl; and those preferring single-stranded weight of 75-kDa and 65-kDa, respectively, containing DNA, such as C23 and lamin B1 (LudCrus et al., 1992; approximately 50% hydrophobic amino acids and Zong and Scheurmann, 1995). Our unpublished results exhibiting two putative Cys-Cys zinc finger DNA (Neri et al., manuscript in preparation) have shown that binding motifs (Hakes and Berezney, 1991b). both SATBl and SAF-A are localized in the nuclear Interestingly enough, these two proteins are capable of interior and antibodies raised against them reveal a binding DNA in a 2M NaC1-resistant fashion that, fibrogranular network. In this context it should also be however, is apparent only when incubation is performed recalled that lamins A and C are DNA-binding proteins, at 37 'X (Hakes and Berezney, 1991a). A 225-kDa and bind MAR sequences (Hakes and Berezney, 1991a; protein (matrin 3) contains a highly acidic domain Luderus et al., 1992, 1994). Very recently, Yanagisawa et (Belgrader et al., 1991b), a feature shared by other al. (1996) have identified a 114-kDa MAR-binding nuclear proteins (Earnshaw, 1987). protein that is expressed only in human breast Fields and Shaper (1988) identified a major 62-kDa carcinomas and not in either normal and benign breast matrix polypeptide as a component of metaphase disease tissues or epithelia1 carcinomas. Undoubtedly, chromosomes. This observation underlies the similarity the number of known polypeptides showing MAR- existing between the nuclear matrix and the scaffold binding capability will continue to grow in the future. prepared from metaphase chromosomes (Pieck et al., Then, there is a plethora of other proteins identified 1987), also because topoisomerase I1 is an abundant as minor components of the nuclear matrix: autoantigens component of both types of structures (see Earnshaw and like fibrillarin (Ochs and Smetana, 1991), fibronectin Laemmli, 1983; Earnshaw et al., 1985; Boy de la Tour (Zerlauth et al., 1988), keratin-like proteins (AliguC et and Laemmli, 1988). In this connection, it is worth al., 1990), oncogene products (Evan and Hancock, noting that several nuclear matrix proteins are functional 1985), tumor-suppressor genes (such as the retino- components of the mitotic spindle (e.g. Kallajoki et al., blastoma gene product, Durfee et al., 1994; Mancini et 1991; Wan et al., 1994; Liao et al., 1995). al., 1994), splicing factors (Smith et al., 1989). hetero- A very abundant component of the NaTT-stabilized geneous nuclear ribonucleoprotein (He et al., 1991), matrix is protein B23/numatrin, a 38-kDa nucleolar transcription factors like NMP-l and NMP-2 (Guo et al., An overview of nuclear matrix
1995; Merrimann et al., 1995), trans-acting factors like specific DNA sequences, the results are strictly NF1 (Sun et al., 1994), DNA binding proteins such as dependent on the technique used for preparing the RFP (Isomura et al., 1992), primer recognition proteins framework and a definitive conclusion cannot be drawn (Vishwanatha et al., 1992), or enzymes like DNA yet (Razin and Vassetzky, 1992; Razin and Gromova, polymerase a (Smith and Berezney, 1982, 1983), DNA 1995). primase (Tubo and Berezney, 1987c), RNA polymerase I A function that has been recently linked to the (Dickinson et al., 1990) or I1 (Lewis et al., 1984), poly- nuclear matrix of which awareness is continuously ADP-ribosome polymerase (Kaufmann et al., 1991), increasing, concerns the autonomous inositide cell cycle histone deacetylase (Hendzel et al., 1991), RNase H operating within the nucleus (see Manzoli et al., 1989; (Karwan et al., 1990), phospholipases (Tamiya-Koizumi Cocco et al., 1994). Following the original report by et al., 1989; Bertagnolo et al., 1995), and protein kinases Capitani et al. (1987) regarding protein kinase C (PKC) such as kinase C or casein kinase 2 (Capitani et al., bound to the nuclear matrix, Payrastre et al. (1992) have 1987; Tawfic and Ahmed, 1994), etc. The biological shown the association with the nuclear matrix of several significance of several of these molecules is still to be key enzymes of the inositide cycle such as phospho- ascertained. inositide kinases, diacylglycerol kinase and phospho- It could be hypothesized that, while the most lipase C (PLC). Ultrastructural observations obtained by represented matrix proteins are components of a means of colloidal gold-conjugated secondary antibodies common network (or a few frameworks) spanning the have confirmed these findings based on enzymatic entire nuclear interior, the minor constituents are assays (Zini et al., 1995a,b; Maraldi et al., 1995; localized to specific matrix sub-domains, each of which Mazzotti et al., 1995). In particular, the a isoform of conceivably performs a different task (Stuurman et al., PKC has been identified bound to the matrix after 1992~). stimulation of quiescent Swiss 3T3 mouse fibroblasts Lastly, it should be noted that, by definition, the with insulin-like growth factor I, and PLC 131 has been nuclear matrix is mostly composed from nonhistone recognized as the major PLC isoform present in the proteins (Berezney, 1984). Usually, extraction with high nucleus, in agreement with previous data based on ionic strength solutions (NaC1 or (NH4)2S04) is western blot experiments carried out on highly-purified effective in removing almost all core histones (about nuclear preparations (Martelli et al., 1992d). Such a 95%) from the matrix preparations (histone H1 is system might play a fundamental role in conveying completely removed at a lower ionic strength). signals from the cell periphery to active sites of DNA According to Laemmli and coworkers LIS treatment also replication and transcription within the nucleus. results in scaffolds that are devoid of core histones (Mirkovitch et al., 1984; Izuarralde et al., 1988). Concluding remarks Surprisingly, they have also reported that LIS is less efficient in removing histone HI, that could be extracted An intriguing new concept for the nuclear matrix has and purified from scaffold preparations and it has been been recently formulated by Razin and Gromova (1995) shown that this protein specifically associates with SARs in order to reconcile many of the contradictory data (Izuarralde et al., 1989; Kas et al., 1989). Other available in the literature. These authors envision the investigators, including us, have come to opposite nuclear matrix as a system of channels connecting the conclusions, because LIS extracted H1 but not most core nuclear interior with nuclear pores. The chromosomal histones (Belgrader et al., 1991a; Neri et al., manuscript domains are assembled around this channel system to in preparation). which active DNA sequences (both in terms of replication and transcription) are attached, thus making Functions associated with the matrix the transport of different molecules possible (RNA and DNA precursors, enzymes and regulatory factors) from The functions associated with the nuclear matrix are cytoplasm to the nucleus and, at the same time, endless and the reader is referred to other excellent migration in the opposite direction of mRNA molecules. reviews (e.g. Berezney, 1991) for a more comprehensive Indeed, a recent observation from Panzeter and Ringer treatment of the topic. (1993) corroborates such a model by showing that DNA Rather, we would like to stress that some of the precursors are channeled directly to DNA replication functions that have been studied in more depth (for sites present on the matrix. Such a model could also example DNA replication) have been found in explain why antibodies to nuclear matrix proteins never frameworks prepared by different techniques, that is the decorate a uniformously-sized inner network but rather a high-salt nuclear matrix, the in situ matrix and the fibrogranular framework showing branches and dilations agarose-embedded matrix (e.g. Tubo and Berezney, (Stuurman et al., 1992~).Indeed, the channels would not 1987a; Nakayasu and Berezney, 1989; Hozak et al., have the same size but they would branch and expand, 1993). Thus, the evidence that these functions take place forming caverns around which replication and attached to some sort of nuclear frameworks seems transcription factories are assembled. Certainly, this new compelling. attractive model for the nuclear matrix deserves further For other functions, however, like the attachment of investigations. An overview of nuclear matrix
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