A combined ultrastructural approach to the study of nuclear matrix thermal stabilization

Summary Using mouse erythroleukaemia cells and different ultrastructural techniques, the morphology was investigated of the nuclear matrix obtained after incubation at 37° C of isolated nuclei. If purified nuclei were heated for 45 min at 37° C, the final matrix exhibited well-recognizable nucleolar remnants, an inner network and a peripheral lamina. Without such incubation only the peripheral lamina was seen surrounding homogeneous, finely granular material. Similar results were obtained with both araldite-embedded and freeze-fractured nuclear matrices, although in the latter case the loose granular material was not evident. Observations of araldite-embedded, heat-treated nuclei revealed clumping of heterochromatin in small, very electron-dense masses with large interchromatin spaces. These ultrastructural aspects were even more striking in freeze-fractured nuclei. Cytochemical matrix analysis by osmium-ammine staining for nucleic acids and DNase-gold labelling for DNA localization demonstrated that also matrix residual nucleic acids, mostly RNA, are stabilized by heat exposure of isolated nuclei. The results demonstrate that the morphology of heat-stabilized nuclear matrix is not artefactually affected during the preparation for conventional electron microscopy and suggest a possible involvement of nucleic acids in the heat-induced stabilization of the nuclear matrix.     

The effect of sodium tetrathionate stabilization on the distribution of three nuclear matrix proteins in human K562 erythroleukemia cells

Using both conventional fluorescence and confocal laser scanning microscopy we have investigated wheter or not stabilization of isolated human erythroleukemic nuclei with sodium tetrathionate can maintain in the nuclear matrix the same spatial distribution of three polypeptides (M_r 160 kDa and 125 kDa, previously shown to be components of the internal nuclear matrix plus the 180-kDa nucleolar isoform of DNA topoisomerase II) as seen in permeabilized cells. The incubation of isolated nuclei in the presence of 2 mM sodium tetrathionate was performed at 0° C or 37° C. The matrix fraction retained 20–40% of nuclear protein, depending on the temperature at which the chemical stabilization was executed. Western blot analysis revealed that the proteins studied were completely retained in the high-salt resistant matrix. Indirect immunofluorescence experiments showed that the distribution of the three antigens in the final matrix closely resembled that detected in permeabilized cells, particularly when the stabilization was performed at 37° C. This conclusion was also strengthened by analysis of cells, isolated nuclei and the nuclear matrix by means of confocal laser scanning microscopy. We conclude that sodium tetrathionate stabilization of isolated nuclei does not alter the spatial distribution of some nuclear matrix proteins.     

A combined ultrastructural approach to the study of nuclear matrix thermal stabilization.

Using mouse erythroleukaemia cells and different ultrastructural techniques, the morphology was investigated of the nuclear matrix obtained after incubation at 37 degrees C of isolated nuclei. If purified nuclei were heated for 45 min at 37 degrees C, the final matrix exhibited well-recognizable nucleolar remnants, an inner network and a peripheral lamina. Without such incubation only the peripheral lamina was seen surrounding homogeneous, finely granular material. Similar results were obtained with both araldite-embedded and freeze-fractured nuclear matrices, although in the latter case the loose granular material was not evident. Observations of araldite-embedded, heat-treated nuclei revealed clumping of heterochromatin in small, very electron-dense masses with large interchromatin spaces. These ultrastructural aspects were even more striking in freeze-fractured nuclei. Cytochemical matrix analysis by osmium-amine staining for nucleic acids and DNase-gold labelling for DNA localization demonstrated that also matrix residual nucleic acids, mostly RNA, are stabilized by heat exposure of isolated nuclei. The results demonstrate that the morphology of heat-stabilized nuclear matrix is not artefactually affected during the preparation for conventional electron microscopy and suggest a possible involvement of nucleic acids in the heat-induced stabilization of the nuclear matrix.     

Heat-induced stabilization of the nuclear matrix: A morphological and biochemical analysis in murine erythroleukemia cells

Using mouse erythroleukemia cells we performed a comprehensive morphological and biochemical study of the nuclear matrix obtained after exposure of isolated nuclei to 37 degrees C or from cells heat shocked in vivo at 43 or 45 degrees C. At the ultrastructural level it was possible to see that in the absence of a 37 degrees C incubation of purified nuclei, the final matrix lacked well-defined nucleolar remnants but a peripheral lamina was clearly visible, as well as a sparse fibrogranular network which was located at the periphery of the structures. On the contrary, after a 37 degrees C nuclear incubation, very electron-dense nucleolar remnants were observed along with an abundant meshwork dispersed throughout the interior of the structures. When intact cells were heat shocked in vivo, electron-dense residual nucleoli were present only when isolated nuclei had been exposed to 37 degrees C in vitro, whereas without such an incubation, they were not as easily distinguishable and appeared less electron-dense. In the latter case the inner network was more evenly distributed. After purified nuclei were incubated at 37 degrees C for 45 min, the high salt and DNase I resistant fraction retained about 18% of the nuclear protein whereas if the heating was omitted protein recovery dropped to 6%. An increase in the recovery of intact structures in the matrix fraction was the main reason for the higher protein recovery. Heating nuclei in vitro further increased the amount of nuclear protein present in the matrix fraction even if intact cells had been heat shocked in vivo. No major qualitative differences were seen when the polypeptide pattern of the various types of nuclear matrices was analyzed on one-dimensional polyacrylamide gels and this finding was further supported by Western blot analysis with a monoclonal antibody to lamins A and C. These results show that heating mainly stabilizes the nucleolar remnants of the matrix and to a lesser extent the inner network, but the morphology of the final structures is different depending on whether the stabilization is performed in vivo or in vitro.     

The protein composition of Friend cell nuclear matrix stabilized by various treatments. Different recovery of nucleolar proteins B23 and C23 and nuclear lamins

Summary— Using two-dimensional polyacrylamide gels stained with Coomassie blue we have studied the protein composition of the nuclear matrix obtained from mouse erythroleukemic nuclei kept at O°C throughout the isolation procedure to prepare the high ionic strength resistant fraction (control matrix) or stabilized in vitro or in vivo by different procedures prior to subfractionation (ie 37°C incubation of isolated nuclei; sodium tetrathionate exposure of purified nuclei; heat shock of intact cells). When the matrix obtained from 37°C incubated nuclei was compared with the control matrix, striking differences in the polypeptide pattern were seen if the protein was obtained in both cases from an equivalent number of nuclei. On the other hand, if the same amount of protein for both the samples was applied to the gels the differences were less evident. Sodium tetrathionate stabilization of isolated nuclei and heat shock of intact cells produced a matrix protein pattern that was very similar and differed from that of the in vitro heat-exposed matrix. Using specific polyclonal antisera, we demonstrate that nucleolar proteins B23/numatrin and C23/nucleolin were very abundant in the matrix obtained from chemically-treated nuclei or in vivo heat-stabilized nuclei but were recovered in very small amounts (B23) or completely absent (C23) in the matrix prepared from nuclei heated to 37°C in vitro. Differences were seen also in the recovery of nuclear lamins, and especially lamin B, that was poorly represented in the sodium tetrathionate-stabilized matrix. The results demonstrate that in mouse erythroleukemia cells the increased recovery of nuclear matrix protein that is seen after in vitro heating of isolated nuclei is predominantly due to an additional recovery of the same types of polypeptides that are detected also in the absence of such a treatment. The data also indicate that in vivo heat shock of intact cells produces a nuclear matrix protein pattern that is more similar to the pattern seen after stabilization of purified nuclei with sodium tetrathionate and differs significantly from that obtained by exposing nuclei to 37°C in vitro, unlike to that what previous reports have indicated.     

Biochemical and morphological changes in the nuclear matrix prepared from apoptotic HL‐60 cells: Effect of different stabilizing procedures

Apoptotic cell death is characterized by deep morphological changes that take place in the nucleus. It is unclear whether modifications also occur in the nuclear matrix, a mainly proteinaceous structure that conceivably acts as a nuclear framework. We have investigated whether biochemical and morphological alterations of the nuclear matrix prepared from apoptotic HL‐60 cells were dependent on the manipulations to which isolated nuclei were subjected before DNase I digestion and 2 M NaCl extraction. Our results showed that the stabilizing procedures employed to preserve the inner fibrogranular network and nucleolar remnants of the matrix (i.e., a 37°C incubation; exposure to sodium tetrathionate at 4°C; exposure to sodium tetrathionate at 37°C) had no effect on the protein recovery of apoptotic nuclear matrices, which was always approximately two‐ to fivefold less than in control matrices. Moreover, one‐ and two‐dimensional gel analysis of nuclear matrix proteins showed that, in apoptotic samples, striking quantitative changes were present, as compared with controls. Once again, these changes were seen irrespective of the stabilizing procedures employed. Also, transmission electron microscope analysis showed similar morphological alterations in all types of apoptotic nuclear matrices. By contrast, the immunofluorescent distribution of the 240‐kDa NuMA protein seen in apoptotic samples was more sensitive to the stabilizing treatments. Our results indicate that the biochemical and morphological changes of the apoptotic nuclear matrix are largely independent of the isolation protocols and strengthen the contention that destruction of the nuclear matrix network is one of the key events leading to apoptotic nuclear destruction. J. Cell. Biochem. 74:99–110, 1999. © 1999 Wiley‐Liss, Inc.     

The effect of in vitro heat exposure on the recovery of nuclear matrix-bound DNA polymerase alpha activity during the different phases of the cell cycle in synchronized HeLa S3 cells.

HeLa S3 cells were synchronized by a double thymidine block or aphidicolin treatment and the levels of nuclear matrix-bound DNA polymerase alpha activity were then measured using activated calf thymus DNA as template. The nuclear matrix was obtained by 2 M NaCl extraction and DNase I digestion of isolated nuclei incubated at 37 degrees C for 45 min prior to subfractionation. In all phases of the cell cycle 25-30% of nuclear DNA polymerase alpha activity remained matrix-bound, even when cells were in the G1 phase. No dynamic association of DNA polymerase alpha activity with the matrix was seen, at variance with previous results obtained in regenerating rat liver. The variations measured in matrix-bound activity closely followed those detected in isolated nuclei throughout the cell cycle. If nuclei were not heat-stabilized very low levels of DNA polymerase alpha activity were measured in the matrix (1-2% of total nuclear activity). Heat incubation of nuclei failed to produce any enrichment in matrix-associated newly replicated DNA, whereas the sulfhydryl cross-linking chemical sodium tetrathionate did. Therefore the results obtained after the heat stabilization procedure do not completely fit with the model that envisions the nuclear matrix as the active site where eucaryotic DNA replication takes place.     

Further considerations on the thermal stabilization of the nuclear matrix in mouse erythroleukemia cells.

The morphology and the polypeptide composition of the nuclear matrix obtained from 37 degrees C incubated nuclei has been studied in mouse erythroleukemia cells. From a structural point of view, in the absence of heat treatment, the matrix lacked identifiable nucleolar remnants and the internal fibrogranular meshwork whereas a peripheral lamina was seen. On the contrary, the matrix obtained from heat exposed nuclei displayed very electrondense nucleolar remnants and an abundant inner network. These results were obtained irrespective of the type of extracting agent (2M NaCl or 0.2 M (NH4)2SO4) used to remove histones and other soluble proteins. The heat stabilization of the matrix could not be prevented by sulfhydryl blocking chemicals such as iodoacetamide and n-ethylmaleimide, thus suggesting that heat does not stabilize the matrix by inducing the formation of disulfide bonds. Only limited differences in the polypeptide pattern of matrix isolated under different conditions were seen using one-dimensional pore gradient polyacrylamide gels stained with both Coomassie Brilliant Blue and silver despite the fact that the matrix fraction from heat treated nuclei retained about three fold more protein in comparison with controls. The same results were obtained also by means of two-dimensional non-equilibrium gel electrophoresis.     

Subnuclear localization of S/MAR-binding proteins is differently affected by in vitro stabilization with heat or Cu2+

The nuclear matrix, a proteinaceous entity thought to be a scaffolding structure that determines the higher order organization of eukaryotic chromatin, is usually prepared from intact nuclei by a series of extraction steps. In most cell types investigated, the nuclear matrix does not spontaneously resist these extractions, but must rather be stabilized before the application of extracting agents such as high salt solutions or lithium diiodosalicylate. We have examined the effect of two widely used stabilization procedures on the localization of nuclear matrix proteins. Four individual polypeptides were studied, all of which are scaffold or matrix-associated region (S/MAR)-binding proteins: SATB1, SAF-A/hnRNP-U, NuMA , and topoisomerase II α. Nuclei were isolated from K562 human erythroleukemia cells in a buffer containing spermine, spermidine, KCl and EDTA, and the nuclear matrix or scaffold was obtained by extraction with lithium diiodosalicylate after stabilization by heat treatment (37° or 42°C) or incubation with Cu²⁺ ions. When the localization of individual proteins was determined by immunofluorescent staining and confocal scanning laser microscopy, markedly different consequences of the two stabilization strategies became evident, ranging from a total maintenance of the localization (NuMA and topoisomerase II α) to a marked redistribution (SATB1 and SAF-A/hnRNP-U). Our results seem to indicate that a reevaluation of stabilization protocols employed for the preparation of the nuclear matrix is desirable, especially by performing morphological controls. Received: 22 January 1997; in revised form: 17 February 1997 / Accepted: 21 February 1997     

Isolation and characterization of stable nuclear matrix preparations and associated DNA from avian erythroblasts

A novel procedure for isolation of nuclear matrices from chicken erythroblast cells was elaborated. The influence of variations in the isolation procedure on structural integrity and morphology of nuclear matrices as well as on properties of the nuclear matrix-associated DNA fractions was investigated. The incubation of isolated nuclei in the presence of Cu2+ ions provided significant stabilization of the nuclear matrix. Copper treatment of nuclei did not affect the properties of the nuclear skeleton-associated DNA fraction. In both copper-stabilized as well as unstabilized nuclei, nuclear matrix-attached DNA was digested to the same extent with nucleolytic enzymes, and could be totally removed from nuclear matrices by 2 M NaCl-2 M urea treatment. The fine morphology of the nuclear matrix did not change after extraction of nuclear skeleton-associated DNA fragments. In the presence or absence of copper ions, matrix DNA was found to be qualitatively different compared with total DNA, in particular with respect to the representation of specific repetitive sequences of the chicken beta globin gene domain.     

Protein disulfide crosslinking stabilizes a polyoma large T antigen-host protein complex on the nuclear matrix

We have investigated the effects of intermolecular disulfide crosslinking and temperature-dependent insolubilization of nuclear proteins in vitro on the association of the polyoma large T antigen with the nuclear matrix in polyomavirus-infected mouse 3T6 cells. Nuclear matrices, prepared from polyomavirus-infected 3T6 cells by sequential extraction of isolated nuclei with 1% Triton X-100 (Triton wash), DNase I, and 2 M NaCl (high salt extract) at 4 degrees C, represented 18% of total nuclear protein. Incubation of nuclei with 1 mM sodium tetrathionate (NaTT) to induce disulfide crosslinks or at 37 degrees C to induce temperature-dependent insolubilization prior to extraction, transferred an additional 9-18% of the nuclear protein from the high salt extract to the nuclear matrix. This additional protein represented primarily an increased recovery of the same nuclear protein subset present in nuclear matrices prepared from untreated nuclei. Major constituents of chromatin including histones, hnRNP core proteins, and 98% of nuclear DNA were removed in the high salt extract following either incubation. Polyoma large T antigen was quantified in subcellular fractions by immunoblotting with rat anti-T ascites. Approximately 60-70% of the T antigen was retained in nuclei isolated in isotonic sucrose buffer at pH 7.2. Most (greater than 95%) of the T antigen retained in untreated nuclei was extracted by DNase-high salt treatment. Incubation at 37 degrees C or with NaTT transferred most (greater than 95%) of the T antigen to the nuclear matrix. T antigen solubilized from NaTT-treated matrices with 1% SDS sedimented on sucrose gradients as a large (50-S) complex. These complexes, isolated by immunoprecipitation with anti-T sera, were dissociated by reduction with 2-mercaptoethanol, and SDS-PAGE analysis revealed that T antigen was crosslinked in stoichiometric amounts to several host proteins: 150, 129, 72, and 70 kDa. These host proteins were not present in anti-T immunoprecipitates of solubilized nuclear matrices prepared from iodoacetamide-treated cells. Our results suggest that the majority of polyomavirus large T antigen in infected cells is localized to a specific subnuclear domain which is distinct from the bulk chromatin and is closely associated with the nuclear matrix.     

Androgen-dependent nuclear proteins in rat ventral prostate are glycoproteins associated with the nuclear matrix

The major rat ventral prostate androgen-dependent nuclear proteins were studied using isolated nuclei, nuclear matrix and nuclear envelope fractions. Nuclear and subnuclear fractions obtained were characterized by electron microscopy and SDS-polyacrylamide gel electrophoresis. A group of approximately 20 kDa peptides is demonstrated to be present in nuclei, nuclear matrices and nuclear envelopes from normal prostate. Time course experiments indicate that the 20 kDa peptides become drastically reduced after 7 or 10 days following castration and are incompletely restored after 3 daily testosterone injections. Lectin binding studies demonstrate that the 20 kDa peptides bind both to Concanavalin A and Wheat Germ Agglutinin. These peptides represent the major nuclear Concanavalin A binding glycoproteins from normal prostate nuclei and nuclear matrices.     

Influence of different metal ions on the ultrastructure, biochemical properties, and protein localization of the K562 cell nuclear matrix.

The higher order of chromatin organization is thought to be determined by the nuclear matrix, a mainly proteinaceous structure that would act as a nucleoskeleton. The matrix is obtained from isolated nuclei by a series of extraction steps involving the use of high salt and nonspecific nucleases, which remove chromatin and other loosely bound components. It is currently under debate whether these structures, isolated in vitro by unphysiological extraction buffers, correspond to a nucleoskeleton existing in vivo. In most cell types investigated, the nuclear matrix does not spontaneously resist these extractions steps; rather, it must be stabilized before the application of extracting agents. In this study nuclei, isolated from K562 human erythroleukemia cells, were stabilized by incubation with different metal ions (Ca2+, Cu2+, Zn2+, Cd2+), and the matrix was obtained by extraction with 2 M NaCl. By means of ultrastructural analysis of the resulting structures, we determined that, except for Ca2+, all the other metals induced a stabilization of the matrix, which retained the inner fibrogranular network and residual nucleoli. The biochemical composition, analyzed by two-dimensional gel electrophoresis separation, exhibited a distinct matrix polypeptide pattern, characteristic of each type of stabilizing ion employed. We also investigated to what extent metal ions could maintain in the final structures the original distribution of three inner matrix components, i.e. NuMA, topoisomerase IIalpha, and RNP. Confocal microscopy analysis showed that only NuMa, and, to a lesser extent, topoisomerase IIalpha, were unaffected by stabilization with divalent ions. On the contrary, the fluorescent RNP patterns detected in the resulting matrices were always disarranged, irrespective of the stabilization procedure. These results indicate that several metal ions are powerful stabilizing agents of the nuclear matrix prepared from K562 erythroleukemia cells and also strengthen the concept that NuMA and topoisomerase IIalpha may act as structural components of the nuclear matrix.     

Study of the structural and structure-functional subdomains in the interphase nucleus by photostabilization

The role of the nuclear matrix components in the organization of structural and functional domains of interphase nuclei was studied using irradiation with blue light in the presence of a photosensibilized agent (Ethidium bromide). Nuclear domain resistance to extractive solution (2 M NaCl) treatment served as a criterion of irradiation-induced stabilization of different nuclear domains. The following results have been obtained: 1) the structural organization of the complexes of chromatin and clusters of replication does not depend on the state of the nuclear matrix in isolated nuclei; 2) chemical stabilization of the nuclear matrix by Cu(2+)-ions is not sufficient for the organization of chromatin domains; 3) irradiation in the presence of Ethidium bromide stabilizes domains of the nuclei, but does not lead to stabilization of the nuclear matrix internal network. Hence, the irradiation prevented extraction from the nuclear domains of nonhistone proteins which were not standard matrix proteins. Based on the results obtained, a hypothesis was proposed about a coexistence of two groups of nonhistone proteins in the cell nucleus. The first group includes proteins of the nuclear matrix involved in immobilization of scafford attachment regions and active genes. The second group includes some hypothetical structural proteins participating only in compaction of DNA of condensed chromatin.     

Small heat shock protein p26 associates with nuclear lamins and HSP70 in nuclei and nuclear matrix fractions from stressed cells

The small heat shock/alpha-crystallin protein p26 undergoes nuclear translocation in response to stress in encysted embryos of the brine shrimp Artemia franciscana. About 50% of total p26 translocates to nuclei in embryos treated with heat shock or anoxia, and in embryo homogenates incubated at low pH. Nuclear fractionation shows that the majority of nuclear p26 and a nuclear lamin are associated with the nuclear matrix fraction. To further explore the roles of p26 and other HSPs in stabilizing nuclear matrix proteins (NMPs), nuclear matrices from control, and heat-shocked embryos were disassembled in urea and evaluated by one and two-dimensional (2-D) gel electrophoresis and Western immunoblotting after reassembling. Nuclear lamins were present only in reassembled fractions and, in the case of heat shock, p26 and HSP70 were also present. HSP90 was not detected in any nuclear fraction. Confocal microscopy on isolated nuclei and nuclear matrix preparations from control and heat-shocked embryos showed that the majority of p26 and a nuclear lamin share similar nuclear distributions. The combination of microscopy and fractionation results suggests that p26 and HSP70 play a role in the protection of nuclear lamins within the nuclear matrix.     

The nuclear matrix is a thermolabile cellular structure

Heat shock sensitizes cells to ionizing radiation, cells heated in S phase have increased chromosomal aberrations, and both Hsp27 and Hsp70 translocate to the nucleus following heat shock, suggesting that the nucleus is a site of thermal damage. We show that the nuclear matrix is the most thermolabile nuclear component. The thermal denaturation profile of the nuclear matrix of Chinese hamster lung V79 cells, determined by differential scanning calorimetry (DSC), has at least 2 transitions at Tm = 48 degrees C and 55 degrees C with an onset temperature of approximately 40 degrees C. The heat absorbed during these transitions is 1.5 cal/g protein, which is in the range of enthalpies for protein denaturation. There is a sharp increase in 1-anilinonapthalene-8-sulfonic acid (ANS) fluorescence with Tm = 48 degrees C, indicating increased exposure of hydrophobic residues at this transition. The Tm = 48 degrees C transition has a similar Tm to those predicted for the critical targets for heat-induced clonogenic killing (Tm = 46 degrees C) and thermal radiosensitization (Tm = 47 degrees C), suggesting that denaturation of nuclear matrix proteins with Tm = 48 degrees C contribute to these forms of nuclear damage. Following heating at 43 degrees C for 2 hours, Hsc70 binds to isolated nuclear matrices and isolated nuclei, probably because of the increased exposure of hydrophobic domains. In addition, approximately 25% of exogenous citrate synthase also binds, indicating a general increase in aggregation of proteins onto the nuclear matrix. We propose that this is the mechanism for increased association of nuclear proteins with the nuclear matrix observed in nuclei Isolated from heat-shocked cells and is a form of indirect thermal damage.     

The development of branched silk gland nuclei

Nuclei in the giant polyploid silk gland cells of Calpodes ethlius grow by endomitosis and can develop hundreds of branches during larval life. The shape of the these nuclei is characteristic for each region of the gland. We have found shape to be correlated with arrangement of the nuclear matrix. Scanning electron microscopy showed nuclear matrices with shapes similar to those of feulgen stained nuclei. Profiles of isolated matrices seen by transmission electron microscopy had filaments aligned parallel to the long axis of nuclear branches. DNA stained by Hoechst had a similar parallel alignment within the branches. Nuclear shape may be maintained by a small number of components, since electrophoretic analysis showed only a few abundant polypeptides in the matrix fraction. Silk gland nuclei have some of the same nuclear matrix antigens found in smaller, more regularly shaped, eukaryote nuclei.     

Residual structure and constituent proteins of the peripheral framework of the cell nucleus in somatic embryos from Daucus carota L.

Nuclei were isolated from somatic embryos of carrot (Daucus carota L.) using a buffer system containing non-ionic detergent. To prepare nuclear matrices, the purified membrane-depleted nuclei were digested with DNase I in combination with RNase A, followed by extraction with 1 M NaCl. The DNA residue in the final insoluble fraction was less than 4% of that in isolated nuclei, and most of the residual nuclei retained their sphericity. Electron microscopy revealed that the nuclear matrix was composed of a distinct peripheral layer, an internal matrix structure and some fibrils; residual nucleoli were observed when exogeneous RNase was not incorporated. The proteins extracted from the nuclei and nuclear subfractions were compared by gel electrophoresis, which showed that the residual fraction contained many minor proteins. To identify proteins showing specific localization at the nuclear periphery, we prepared monoclonal antibodies (MAbs) against an ion-exchange chromatography fraction extracted from carrot nuclear matrices. Immunofluorescence microscopy with one of the MAbs, CML-1, showed exclusive staining of the nuclear periphery. The MAb recognized several spots showing microheterogeneity, with a narrow range of pI and molecular mass upon immunoblotting. A complete set of these spots was shown to be conserved in nuclear matrices. On the other hand, MAb CML-13 appeared to react with the nuclear interior as well as the periphery, recognizing a 96-kDa polypeptide of the nuclear matrix. These proteins were thus demonstrated to lie at the nuclear periphery, and to constitute the nuclear matrices in carrot. The 96-kDa polypeptide is suggested to be similar to the 92-kDa nuclear protein reported by Beven et al. in carrot (Beven et al., 1991, J. Cell Sci. 98, 293–302).Abbreviations DEAE diethylaminoethyl - MAb monoclonal antibody - NEPHGE nonequilibrium pH gradient electrophoresis We wish to thank Ms. Akiko Itoh for excellent technical assistance. This work was supported by a Grant-in-Aid (05640738) from the Ministry of Education of Japan.