Status of the nuclear matrix in mature and embryonic chick erythrocyte nuclei

Experimental Cell Research 147 (1983) 3 1-39

Copyright @ 1983 by Academic Press, Inc. AII rights of reproduction in any form reserved 0014-4827/83$3.00

Status of the Nuclear Matrix in Mature and Embryonic Chick Erythrocyte Nuclei RICHARD E. LAFOND

and C. L. F. WOODCOCK

Department of Zoology, Universiry of Massachusetts, Amherst, MA 01003, USA

SUMMARY The adult chicken erythrocyte nucleus was found to lack an internal nuclear matrix: even milder extraction procedures resulted in the production of empty shells of pore complexlamina together with loose aggregates of core histone. In contrast, rat liver nuclei showed a typical intranuclear salt-resistant skeleton. These results show that an internal matrix is not an obligatory nuclear component, and is not required for the spatial organization of chromatin. Sday-old embryonic erythrocytes did, however, contain an interchromatinic nuclear matrix, suggesting a correlation between the presence of matrix structures, and nuclear ‘activity’.

A subject of major interest in the study of the nucleus has developed from the isolation by Berezney & Coffey [5, 61of a nuclear framework from rat liver nuclei which remains after almost all of the chromatin, RNA and phospholipids are removed. This residual structure, termed the nuclear matrix (see refs [l-4] for reviews), has been found in nuclei from many cell types [5, 7-141 and has been implicated in a wide variety of nuclear functions, including DNA replication [15-191 and transcription [20-24, 30-311, as well as providing the foundation for nuclear shape [2], and non-random anchoring of specific gene sequences [25-281. In addition, the nuclear matrix contains receptors for both androgens and estrogens [32, 331, has been coisolated with heat shock proteins in Drosophila [34] and has been associated with polyoma T antigen and DNA in lytic infection of 3T6 cells [29]. Although treatments used to produce the nuclear matrix are not all identical, the original method consists of the digestion of isolated nuclei by either endogenous nuclease or DNase I followed by low salt and high salt extractions and treatment with Triton X-100. The residual nuclei may then be digested with DNase I together with RNase [5, 61, although this step is often omitted (see for example [8, 171). In the case of the rat liver nucleus, the resulting structure represents IS15 % of total nuclear protein and typically consists of 98 % protein with approx. 1% or less of nuclear DNA and RNA, together with only a trace of phospholipids [5, 61. The nuclear matrix has three morphologically distinct components: the fibrous pore complex-lamina that surrounds the residual nucleus, the fibrous and granular interchromatinic matrix, and residual nucleolar structures [5, 61. The adult chicken erythrocyte nucleus offers an unusual opportunity to examine the properties of the internal matrix in view of its relative metabolic quiescence and low content of non-histone chromosomal proteins [41-43]. In addition, the chromatin is uniformly condensed and its possible anchorage and dependence on an underlying internal matrix could be better studied in this ‘simple’ system. 3-838334

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We have examined the status of the nuclear matrix in this nucleus both by the standard preparative procedures and by milder isolation regimes; under no conditions have we been able to observe an internal matrix [54]. Similar treatments of mouse liver nuclei, on the other hand, resulted in the production of a typical internal matrix bounded by the pore complex-lamina and showing residual nucleolar structures. These findings suggest that the interchromatinic matrix is not required for nuclear organization per se, but may be correlated with metabolic activity. Further support for this hypothesis was obtained by examining active erythrocyte nuclei: a typical internal nuclear matrix was found in erythrocytes from 5-day embryos. MATERIALS Isolation

AND METHODS

of nuclei

Nonidet P-40 method. Adult chicken erythrocytes were collected from the wing vein of mature roosters and washed in buffer consisting of 10 mM PIPES, pH 8.0 150mM KC1 and the cells disrupted by mixing at room temperature for 15 min in the presence of 0.4% Nonidet P-40. Nuclei were recovered after at least two washings in the same buffer by sedimentation at 375 g. Nitrogen cavitation. Adult nuclei were also isolated by the method of Shelton et al. [36]. Erythrocytes were collected in l/10 vol of 3.8 % (w/v) sodium citrate, washed three times in 0.146 M NaCl and resuspended in the same salt solution to 10-14 times packed cell volume. They were then subjected to 1050 psi of nitrogen for 20 min at ice temperature in a Parr 4635 Cell Disruption Bomb (Parr Instrument Company, Moline, Ill.) and the resulting nuclei purified by sedimentation at 600 g for 20 min through a 20% glycerol barrier containing 0.146 M NaCl and 1.0 mM CaCl*. Finally, the nuclei were washed three times in 50 mM Tris-HCl, pH 7.5, 25 mM KCI, 5 mM MgClz containing 250 mM sucrose. Mouse liver nuclei. Mouse liver nuclei were isolated according to Burgoyne et al. [37]. Each gram of minced liver fragments was homogenized at ice temperature in 7 ml of buffer consisting of 60 mM KCI, 15 mM NaCl, 0.15 mM spermine, 0.5 mM spermidine, 15 mM /?-mercaptoethanol, and 15 mM Tris-HCl, pH 7.0 containing 340 mM sucrose. The resulting homogenate was layered on 0.33 vol of buffer containing 1.37 M sucrose and centrifuged for 15 min at 16000 g. The nuclear pellet was dispersed in the same buffered sucrose and sedimented for 45 min at 75000 g. The final pellet was washed again in the same buffer containing 340 mM sucrose. Embryonic nuclei. Blood was collected from 5-day-old embryos by severing the allantoic vessels and allowing the blood to accumulate in the surrounding cavity. Cells were then taken up in semi-solid agar which formed a block upon cooling. The cells, embedded in the agar, were then treated with 0.1% Triton X-100 containing 0.001% spermine [68] for 30 min at ice temperature. Alternatively, the collagen overlay method (see below) was used.

Table 1 Digestion time (mitt)

NaCl

% DNA remaining

% Protein remaining

15 15

+

88 63

97 51

35 35

+

83 48

95 39

Chick erythrocyte nuclei were digested for indicated times and the amount of protein and DNA remaining was determined.

Exp Cell Res 147(1983)

Nuclear matrix in mature and embryonic chick erythrocyte nuclei

Preparation of nuclear matrix Nuclei were resuspended at a protein concentration of 1 mg/ml in 10 mM Tris-HCl, pH7.4, 5.0 mM MgC12. Digestion was initiated by adding 200 &ml DNase I (Worthington, RNase-free) in the same buffer and allowed to proceed for 1 h at room temperature. Nuclei were collected by centrifugtion at 780 g for 20 min and treated three successive times with 2 M NaCl, 10 mM Tris-HCI, pH 7.4,0.2 mM MgClr for 15 min each at ice temperature. Nuclei were then treated with 1.0% Triton X-100 in 10 mM Tris-HCl, pH 7.4, containing 5 mM MgQ, for 15 min also on ice, followed by two successive washes in the same buffer minus Triton X-100. An additional DNase I digestion was imposed, identical with the first, except that the time was extended to 1.5 h. Also a final 2 M NaCl extraction was carried out and the final pellet washed twice in DNase I digestion buffer. All buffers contained phenyl methyl sulfonyl fluoride (PMSF) and disodium tetrathionate at a concentration of 1.O mM.

Mild nuclear depletion regime Nuclei were resupended to a protein concentration of 1 mg/ml in DNase digestion buffer and DNase I digestion carried out under the same conditions as described above for various times at ice temperature. This step was sometimes followed by a single extraction with buffered 2 M NaCl, also at ice temperature. In the case of the agar block method, cells were treated with the T&on-spennine solution also for 30 min at 0°C and washed three times with DNase digestion buffer. Nuclei were then digested with 200 u&nl of DNase I for 20 min followed by a single 2 M NaCl extraction for 20 min. When the collagen overlay method was used, cells were treated with Triton-spermine solution for 15 min at 0°C. After detergent treatment, the cells were thoroughly washed with DNase digestion buffer, and then digested with 20 ug/ml of DNase I, 200 &ml for 1 h at 0°C. An additional wash with digestion buffer or 10 mM Tris, pH 7.4, 0.2 mM MgC12was then followed by extraction with buffered 2 M NaCl for 1 h at 0°C. Finally the cells were washed once more with low salt buffer and prepared for electron microscopy. All solutions contained PMSF and disodium tetrathionate at a concentration of 1.0 mM.

Collagen overlay method Adult chick erythrocytes were dispersed on the surface of 60 mm tissue culture dishes and overlaid with collagen. A 0.2 % stock solution of collagen (Sigma, type VII) was made up in 0.1% acetic acid. Before use it was neutralized and diluted into Eagle’s minimal essential medium (MEM) to a final concentration of 0.16 % [62]. Approx. 0.4 ml of ice-cold collagen was added to each dish, and the cells were incubated at 37°C for 10-15 min. During this time, collagen monomers polymerize to form a protective film over the cells, which can be treated with aqueous solutions without detachment from the plate. DNA andprotein quantitation. DNA was estimated by a modification of the thiobarbituric acid assay [38] and protein was quantitated by the Bio-Rad Coomassie Brilliant Blue G-250 assay (Bio-Rad Laboratories). Polyacryfumide gel electrophoresis. SDS gel electrophoresis of nuclear proteins followed the method of Komberg & Thomas [39]. Samples were dissolved in 10 mM Tris, pH 6.8, containing 10% glycerol, 0.16% bromphenol blue and 1% sodium dodecyl sulfate (SDS), heated at 55°C for 1 h and electrophoresed at 30 mA for approx. 5 h or until the tracking dye had traversed the 18% acrylamide slab gel. Gels were stained with Coomassie Brilliant Blue R-250. Electron microscopy. Nuclear pellets, matrix preparations, or cells overlaid with collagen were fixed in 4% glutaraldehyde in 0.1 M sodium phosphate buffer, pH 7.4 for 2 h at 4°C and postfixed in 1% osmium tetroxide in the same buffer for 1 h at 4°C. Specimens were dehydrated through a step-wise, ascending series of ethanol and embedded in Epon-Araldite. Ultrathin sections were stained with many1 acetate and lead citrate, and examined in a Siemens Elmiskop 102.

RESULTS Treatment of adult chicken erythrocyte nuclei by the nuclear matrix isolation procedure resulted in the production of membranous fragments rather than the nucleus-shaped structures obtained with other material. On the basis of subsequent work, described below, we interpret these fragments as collapsed pore

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Exp Cell Res 147 (1983)

Figs 1-3. Nitrogen cavitation nuclei. Fig. 1. Untreated adult erythrocyte nucleus showing some plasma membrane contamination. Fig. 2. After 30 min digestion with DNase I alone. Fig. 3. After digestion for 35 min followed by extraction with 2 M NaCl for 15 min. Arrowheads point to retained pore complex-lamina. Bar, 1 pm. Figs 44. Fig. 4. Untreated mouse liver nuclei. Fig. 5. After digestion with DNase 1 alone for 35 min. Fig. 6. Mouse liver nuclear matrix prepared by standard protocol (see Materials and Methods). Bar, 1 w.

Em Cell Res 147 (1983)

Nuclear

matrix in mature and embryonic

chick erythrocyte

nuclei

Fig. 11. SDS gels of nuclear fractions. a, Profile of untreated adult chick nucleus; b, after digestion with DNase I for 30 min; c, after complete matrix isolation procedure, d, bovine serum albu-

min marker, 66 kD.

complex-lamina material such as observed by Hodge et al. [8] in HeLa cells and Shelton et al. [61] in erythrocytes. The same treatment of mouse liver nuclei, on the other hand, resulted in the appearance of a typical nuclear matrix complete with residual interchromatinic and nucleolar structures (fig. 6). Since the erythrocyte pore complex-lamina is known to be especially fragile [5-81, milder treatments were used in an attempt to reveal any high salt-resistant structures. We also employed nuclei which retained the plasma membrane ‘ghost’ as this has been shown to assist in preserving the pore complex-lamina [57-591. When nitrogen cavitation nuclei, which were slightly contaminated with plasma membrane but not surrounded by it (fig. 1) were treated for 30 min with DNase, and then processed for electron microscopy, the partial loss of nuclear contents was observed (fig. 2). At this point, the pore-lamina remained intact. However, when samples were digested to a similar extent with DNase I, but then extracted with 2 M NaCl, the pore-lamina disintegrated (fig. 3). When the same treatments were given to NP-40 nuclei, which retain a more or less complete plasma membrane covering, we were able to preserve the pore complex-lamina completely. At the end of the DNase-2 M NaCl sequence, almost all of the nuclear contents had been removed, leaving a nearly empty shell of pore-lamina (figs 7, 8). This result is seen even more clearly in erythrocytes treated in situ to DNase - 2 M NaCl. In these experiments, washed erythrocytes were allowed to settle on a plastic culture dish, then overlaid with a permeable collgen skin (see 7-8. NP-40 nuclei. Fig. 7. Adult chick erythrocyte nucleus showing surrounding plasma membrane. Fig. 8. After digestion with DNase I for 20 min followed by 2 M NaCl extraction also for 20 min. Bar, 1 pm. Figs 9-10. Collagen overlay technique. Fig. 9. Whole adult erythrocyte. Fig. 10. After matrix preparation (see Materials and Methods). Figs

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Exo Cell Res 147

Figs 12-13. Fig. 12. Five-day-old embryonic erythrocyte nucleus prepared by the agar block method (see Materials and Methods). Fig. 13. Embryonic nucleus after digestion with DNase I for 20 min and followed by 2 M NaCl extraction for 20 min. Arrowheads point to retained pore complex-lamina. Note distinct internal matrix material compared with figs 8 and 10. Bar, 1 urn.

Materials and Methods for details) and treated for 15 min with 0.1% Triton X100, 0.001% spermine [68] before digestion and high salt extraction. Erythrocytes fixed before and after such treatment are shown in figs 9 and 10. Again, the nuclear contents have been completely removed leaving only a shell of pore complex-lamina. As expected, mouse liver nuclei retained their internal structure after the mild DNase - 2 M NaCl extraction (fig. 5). To investigate the loss of erythrocyte nuclear material further, DNA and protein determinations were made during the extraction process, and the protein species present at each stage displayed on gels. As seen from table 1, after 35 min of DNase I digestion of nitrogen cavitation nuclei, followed by 2 M NaCl, 48 % of DNA and 39 % of protein remained. Even so, the residual nuclear structures were largely disintegrated (fig. 3). These results were confirmed by the gel electrophoresis studies; after mild depletion, the core histone content was lowered from that of whole nuclei, but still substantial (fig. 11a, 6). However, after the complete matrix preparation, which yielded only fragments of pore complex-lamina, histones were markedly decreased in amount relative to non-histone proteins (fig. 11c). During digestion and extraction, chromatin forms electron-opaque granules that accumulate at the nuclear boundary (fig. 8); from the gel analyses (fig. 11) we conclude that they are aggregates of core histone. As previously mentioned, the adult erythrocyte nucleus is unusually low in non-histone proteins, and metabolically dormant. To determine whether there was any correlation between the inactive state and the lack of an internal nuclear matrix, embryonic erythrocytes were examined. Nuclei from transcriptionally active 5-day embryonic erythrocytes did indeed show an internal matrix (figs 12, 13) although it was clearly less extensive than that in mouse liver nuclei. By 1l-14 days however, when the adult line of erythrocytes predominates, the internal matrix was no longer present (not shown). Cook & Braze11[5&52] also found a marked difference between 5-day embryonic nuclei and mature (1 l-14 day) nuclei with respect to DNA super-twisting. Whereas DNA from the 5-day nucleus responded to ethidium bromide by changing its degree of supertwisting, the mature erythrocyte DNA did not.

(1983)

Exo Cell Res 147 (1983)

Nuclear matrix‘in mature and embryonic chick erythrocyte nuclei

DISCUSSION The apparent absence of an internal nuclear matrix in mature chicken erythrocyte nuclei was unexpected in view of its generally assumed ubiquity [2]. Even the milder depletion regime utilizing DNase I digestion alone or DNase I followed by a single 2 M NaCl extraction failed to reveal an internal matrix, both treatments resulting in the partial or complete removal of the nuclear contents, leaving nuclear shells consisting of pore complex-lamina. In all cases, the protease inhibitors, PMSF and disodium tetrathionate, which have been shown to increase the yield of complete nuclear matrices [35, 401, were included throughout our preparative procedures. When mouse liver nuclei were treated similarly, however, nuclear integrity was retained. Our inability to detect an internal nuclear matrix in mature erythrocytes could be related to the special properties of this nucleus: it is inactive in DNA synthesis [41], virtually inactive in RNA synthesis [43], contains no detectable RNPs [42], has no active nucleoli [41] and is low in non-histone chromosomal proteins [41]. The possibility that the presence of an internal nuclear matrix was correlated with transcriptional or other metabolic activity led to an examination of erythrocyte precursors. An internal matrix was present in 5-day-old nuclei (fig. 13). There is considerable evidence that in actively transcribing nuclei, ribonucleoprotein particles have a role in either providing the basis for the internal matrix, or affecting its stability. In nuclear depletion experiments in which RNase is included with DNase as an initial treatment before high salt extraction, empty nuclear shells surrounded by pore complex-lamina were observed by Herman et al. 1211and Adolph [45] in HeLa nuclei, and by Barrack & Coffey 1321,Gerace et al. [46] and Kaufmann et al. [40] in rat liver nuclei. In addition, Peters & Comings [47] and Berezney [48] have shown that a significant portion of matrix polypeptides consists of ribonucleoprotein. These results suggest that RNase treatment renders both the interchromatinic and nucleolar components extractable in solutions of high ionic strength. However, the precise role of RNPs and the stability of the internal matrix in different cell types remain unclear, particularly in view of the recent isolation of complete matrix from Drosophila embryonic nuclei by Fisher et al. [49] and from HeLa nuclei by van Eekelen et al. [69] in which the nuclei were treated with DNase I and RNase A prior to high salt and detergent treatment. Also, an internal matrix has been described recently in biosynthetically active duck erythroblasts even though the bulk of proteins associated with 40s RNPs were extracted during matrix preparation [44]. If the internal matrix is operationally defined by its low salt and high salt insolubility after DNase treatment, it is apparently not an obligatory component of nuclear structure and not a requirement for spatial organization of chromatin in the adult erythrocyte nucleus. The absence of an underlying matrix in this nucleus, which contains uniformly condensed chromatin, is consistent with the conclusions of Berezney & Coffey [ 1, 51 that a matrix is not associated with the condensed chromatin of the nuclear periphery and perinucleolar areas; recently, however, Brasch [60] has challenged this interpretation. Our findings of an internal matrix in S-day-old embryonic nuclei strongly

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suggests that its presence is associated with an active nucleus. Since embryonic blood consists of cells of various lineage, it is probable that the matrix we observe is found in cells that are not direct progenitors of the mature erythrocyte. However, the correlation between internal matrix and nuclear activity can be tested more directly. Avian erythrocytes can be ‘reactivated’ [53] if transplanted into mouse L-cell cytoplasts [63, 641. During reactivation, nuclei swell, take up proteins from the host cytoplasm, chromatin decondenses and nucleic acid synthesis resumes [63-67]. We find that within 16 h of fusion, mature erythrocytes gain an internal nuclear matrix [55, 561. This system therefore provides a novel means to assess the composition and development of the internal matrix and its relationship to DNA synthesis, transcription, and post-transcriptional events. This work was supported by NSF PCM 8104079.

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