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Cell Biology International (2004) 28, 835–843 (Printed in Great Britain)
Stabilization of macromolecular chromatin complexes in mitotic chromosomes by light irradiation in the presence of ethidium bromide
Eugene V. Shevalab*, Igor I. Kireeva and Vladimir Yu. Polyakovab
aA. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, GSP-2, Moscow 119992, Russia
bInstitute of Agricultural Biotechnology of the Russian Academy of Agricultural Sciences, Timiryazevskaya Street 42, Moscow 127550, Russia


Abstract

A method was developed for stabilizing mitotic chromosomes. Light irradiation of permeabilized cells in a low concentration of ethidium bromide made chromatin resistant to high salt concentrations and decondensing buffer. This resistance was abolished by proteinase treatment, but not by DNase or RNase treatment. In photostabilized and extracted chromosomes, chromatin appeared as thick fibers with discrete high electron density regions. These stabilized structures might correspond to the higher-level structures (chromonemata) observed in native chromatin. Moreover, the electron density was higher in the centromeric regions than the chromosome arm material. Thus, the method allows chromatin substructures (chromonemata and centromeric heterochromatin) to be stabilized inside mitotic chromosomes.


Keywords: Chromosome, Chromonema, Scaffold, Chromatin photostabilization.

*Corresponding author. Department of Electron Microscopy, A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Leninskie Gory GSP-2, Moscow 119992, Russia. Tel.: +7 095 9395528; fax: +7 095 9393181.


1 Introduction

Chromosome structure can be described as a hierarchy of levels of DNA compaction. Histones are necessary for packaging the DNA into 10-nm nucleosomic fibers and for compacting these into 30-nm fibers. The histones are not only structural proteins; histone H3 phosphorylation is required for cell cycle progression and for the changes in chromatin structure during chromosome condensation (Van Hooser et al., 1998; De la Barre et al., 2000).

Little is known about the higher levels of chromosome organization and several different models have been proposed. According to the radial loop model, the structural non-histone proteins (so-called ‘scaffolding proteins’) form a protein skeleton (chromosome scaffold) organizing the 30-nm fibers into topologically constrained loops during mitosis (Marsden and Laemmli, 1979; Stack and Anderson, 2001; Swedlow and Hirano, 2003). The main proteins of the chromosome scaffold are DNA topoisomerase IIα and components of the 13S condensin complex (De, 2002; Swedlow and Hirano, 2003; Gassman et al., 2004). However, according to published data, the formation of 30-nm fibers is not the final step in DNA folding. Thick fibers (chromonemata) have been described in mitotic chromosomes fixed in situ (Sparvoli et al., 1965; Chentsov et al., 1984; Zatsepina et al., 1983; Belmont et al., 1989; Hao et al., 1990, 1994; Iwano et al., 1997).

Thus, the higher-order organization of mitotic chromosomes still remains unclear. To study chromosome structure and the chemical composition of chromatin complexes, it is necessary to isolate mitotic chromosomes and their substructures. However, the isolation procedure can significantly affect structure (Laughlin et al., 1982; Paulson and Langmore, 1983). The sensitivity of chromosomes to different isolation conditions can be diminished by the preliminary stabilization of native complexes. Previously, we developed a method for stabilizing higher-level chromatin structures in isolated interphase nuclei (Sheval et al., 2002; Prusov et al., 2003). In the current work, we describe for the first time an approach to stabilize macromolecular complexes in mitotic chromosomes: light irradiation in the presence of ethidium bromide makes higher-level chromatin structures resistant to 2M NaCl extraction. The role of non-histone proteins in chromatin complex integrity is discussed in the light of the results.

2 Materials and methods

2.1 Cell culture

Embryonic pig kidney cells (PK cells) were grown in 199 culture medium supplemented with 10% fetal calf serum and antibiotics. Cells were grown as monolayers on coverslips and used during exponential growth phase. In some cases, cells were pretreated with 0.1μg/ml nocodazole (Sigma, USA) for 2h to arrest metaphase.

2.2 Cell permeabilization and extraction

PK cells were briefly washed with cold PBS and immersed in buffer A (5mM triethanolamine–HCl pH 7.6 (TEA–HCl; Sigma), 3mM CaCl2, 1mM MgCl2, 0.1mM PMSF and 0.5% Triton X-100) for 4min. The permeabilized cells were washed with the same buffer without Triton X-100 and extracted with buffer B (2M NaCl, 10mM EDTA, 20mM TEA–HCl) for 6min.

2.3 Stabilization of nuclear structures by irradiation

Coverslips with permeabilized cells were immersed in buffer A without Triton X-100, containing 10μg/ml ethidium bromide (Sigma). For irradiation, a 250W mercury lamp was used. The light was passed through a glass filter with λmax 460nm, λ1/2 90nm. The incident light power density was 60mW/cm2. Cells were exposed for 4min and extracted with buffer B.

2.4 Microscopy

After permeabilization and extraction, the cells were fixed for 2h at 4°C in 1% glutaraldehyde, postfixed with 1% OsO4 for 1.5h and embedded in Epon. Some coverslips were used for electron microscopy: ultrathin sections were cut with a LKB Ultratome-III (LKB, Sweden) and examined in HU-11B and HU-12 electron microscopes (Hitachi, Japan). Others were attached to microscope slides and after Epon polymerization were used for light microscopy. The cells were photographed under phase contrast using an Opton III microscope (Carl Zeiss, Germany) with a Planapo 63/1.40 objective.

In order to obtain cytogenetic preparations, cells were incubated with nocodazole, subjected to hypotonic treatment for 10min in 0.075M KCl, fixed in methanol:acetic acid (3:1) and spread by dropping cell suspension on to slides.

2.5 Enzymatic treatments

After irradiation, PK cells were incubated in buffer containing either 0.1mg/ml DNase I (Boehringer Mannheim, Germany) for 15min at 37°C, 0.1mg/ml RNase A (Sigma) for 30min at 37°C, or 0.1mg/ml proteinase K (Sigma) for 10min at 37°C. During nuclease treatments, but not the proteinase treatment, 1mM PMSF was added to all solutions. For chromosome decondensation, the cells were immersed in 5mM TEA–HCl, 1mM PMSF for 4min, fixed for 15min with 2% paraformaldehyde in appropriate buffer, stained with 4′,6-diamidino-2-phenylindole (DAPI; Sigma) and mounted in Mowiol (Calbiochem, USA). Photographs were taken with the Opton III microscope equipped with a Neofluar 63/1.25 objective.

3 Results

3.1 Light microscopy

Cells permeabilized in buffer containing 3mM CaCl2 and 1mM MgCl2 were visualized under phase contrast (Fig. 1a). The internal space of the interphase nuclei was occupied by dense blocks of condensed chromatin and nucleoli; in metaphase cells there were compact chromosomes, aligned at the cell equator.


Fig. 1

Light irradiation in the presence of ethidium bromide induces structural stabilization of mitotic chromosomes and interphase nuclei; phase contrast microscopy. (a) Permeabilized cells; (b) cells extracted with 2M NaCl; and (c) cells irradiated before extraction. Mc – mitotic cell; mchr – mitotic chromosomes; n – nucleolus; cchr – condensed chromatin; zdchr – zone of decondensed chromosomes; and imn – internal matrix network. Bar=10μm.


The permeabilized cells did not detach from coverslips after treatment with 2M NaCl, so it was possible to study the nuclear matrix morphology in situ during both interphase and metaphase (Fig. 1b). Under phase contrast, interphase nuclei appeared slightly swollen, with residual nucleoli and internal matrix network elements (small globules connected with each other by thin threads). Metaphase chromosomes were destroyed by high salt extraction and the central part of each cell was occupied by a zone of decondensed chromosomes.

Irradiation in the presence of ethidium bromide induced resistance to high salt extraction in interphase chromatin and mitotic chromosomes. The interphase nuclei did not swell, and the blocks of condensed chromatin and dense nucleoli were well preserved (Fig. 1c). In metaphase cells, discrete chromosomes were visible, their diameters being slightly larger than in unextracted permeabilized cells.

3.2 Enzymatic treatments of photostabilized mitotic chromosomes

In order to determine the role of each class of chromatin constituents (DNA, RNA, and proteins) in stabilizing mitotic chromosomes, the irradiated cells were treated with DNase I, RNase A or proteinase K before decondensation. A low ionic strength buffer without bivalent cations was used to effect decondensation.

Control untreated permeabilized chromosomes were compact (Fig. 2a, a'). After decondensation they become invisible under phase contrast (Fig. 2b) and appeared swollen after staining with DAPI (Fig. 2b'). In contrast, photostabilized chromosomes did not disappear under decondensing conditions, although they were slightly increased in diameter (Fig. 2c, c').


Fig. 2

Enzymatic treatment of photostabilized metaphase chromosomes. (a–f) Phase contrast and (a'–f') DAPI. (a, a') Permeabilized metaphase cell; (b, b') cell treated with decondensing buffer after permeabilization; (c, c') cell irradiated before decondensation; (d, d') irradiated cell digested with DNase I before decondensation; (e, e') irradiated cell digested with RNase A before decondensation; and (f, f') irradiated cells (interphase – left and metaphase – right) digested with proteinase K before decondensation. Bars=5μm (a'–e') and 10μm (f').


Mitotic chromosomes were not destroyed by DNase I treatment before decondensation (Fig. 2d, d'), although they were not stained with DAPI, implying that the DNA was mainly digested. After DNase treatment the metaphase chromosomes appeared thinner. The thicknesses of the prophase and anaphase chromosomes also seemed to decrease (data not shown). The partial swelling of the undigested chromosomes after photostabilization was probably due to electrostatic repulsion among charged groups on the DNA, so that when the DNA was removed, the protein remnants of the chromosomes did not swell but retained their initial proportions. After RNase treatment (Fig. 2e, e') the chromosome structure was not significantly different from controls (Fig. 2c, c'). After the photostabilized cells had been incubated with the proteinase, the chromosomes were completely destroyed by decondensation (Fig. 2f), and the DNA appeared as a meshwork of thin fibers distributed throughout the mitotic cell (Fig. 2f').

3.3 Ultrastructural organization of photostabilized chromatin

Electron microscopy revealed that the mitotic chromosome arms were organized in 40–60nm fibers, chromonemata (Fig. 3a, b), which were absent only from the centromeric regions of metaphase chromosomes (Fig. 3b). Splitting of chromosome arms was observed in some metaphase cells, but the centromeric regions were never split. According to published data, chromosome decondensation is induced by 50–100μg/ml ethidium bromide (Hirano and Mitchison, 1993). Ethidium bromide at the concentration used in the current study (10μg/ml) did not influence the chromosome morphology or decondense the chromonemata (data not shown).


Fig. 3

Ultrastructural organization of metaphase chromosomes in permeabilized cells. The chromosome arms consist of 40–60nm fibers, chromonemata (arrowheads). Arrow indicates kinetochore. Bars=1μm (a) and 0.5μm (b).


High salt extraction destroyed mitotic chromosomes and only a few deproteinized fibers were observed in the central regions of extracted metaphase cells. All cytoplasmic organelles were shifted to the cell periphery (Fig. 4a). DAPI staining revealed that the central regions contained only small amounts of homogeneously distributed DNA. Most of the DNA was observed in the peripheral zones, where it was probably tethered during extraction (Fig. 4b, b').


Fig. 4

Organization of extracted metaphase cells. (a) Ultrustructural organization and (b, b') DNA distribution inside metaphase cell. (b) DAPI and (b') phase contrast. Bars=5μm.


Irradiation made the chromosomes resistant to high salt extraction. As shown in Fig. 5a, the photostabilized metaphase cell contained chromosomes with slightly increased diameters compared with permeabilized cells (0.6 and 1.0μm, respectively). Chromosomal morphology remained largely unchanged but there was no splitting of the arms into two chromatids. The internal structure of the chromosome arms was homogeneous; the whole volume appeared to be filled with a dense meshwork of photostabilized material. Inside this meshwork no typical nucleosomic (10nm) or solenoid (30nm) fibers were observed (Fig. 5b, b'). It was so dense that it was hard to reveal any regularity. However, color coding of the pixel intensity made it possible to visualize large-scale fibers within the meshwork (Fig. 5c, c'). Sometimes these fibers were parallel to each other; the distance between the axial regions of neighbouring fibers was 60–120nm. The highest density zones appeared as small globules inside these fibers. Small aggregates of dense material were localized on the chromosome surface, but the origin of this material is unknown. Kinetochores and microtubules of the mitotic spindle, easily observed in permeabilized cells, were not seen and were therefore presumably not stabilized by irradiation.


Fig. 5

Ultrustructural organization of photostabilized metaphase chromosomes after 2M NaCl extraction. (a) General view of metaphase plate and (b) high magnification of selected metaphase chromosome. Arrows indicate centromeric regions. (b') Cropped region. (c) Image from (b) with reduction to 4 levels of gray. Black zones correspond to the chromosomal regions with highest electron density, forming discrete foci within less dense threads (labeled in gray). (c') Cropped region. Bars=2μm (a), 1μm (b) and 0.3μm (b').


The region of primary constriction consisted of denser material than the chromosome arms. Increased intercalation of ethidium bromide into the centromeric heterochromatin (hence, a higher level of crosslinking) might account for this, but in cytogenetic preparations all chromosomes were stained homogeneously with ethidium bromide (Fig. 6a). A similar staining pattern was observed in permeabilized cells (Fig. 6b).


Fig. 6

Mitotic chromosomes homogeneously stained by ethidium bromide. (a) Fragment of metaphase plate in chromosome spread and (b) permeabilized metaphase cell. Bar=10μm.


After removal of most of the DNA from the photostabilized chromatin by DNase treatment, the chromosomes remained resistant to decondensing buffer (Fig. 2d, d') and 2M NaCl (data not shown). We analyzed the ultrastructural organization of these ‘residual’ chromosomes (Fig. 7). Chromosomes treated with DNase I before high salt extraction were paler than untreated ones, due to the absence of DNA, which binds with uranyl acetate effectively. Although no DNA remained, as judged by the negative DAPI staining, the structure of these chromosomes was similar to undigested ones. The chromosomal material was homogeneous.


Fig. 7

Ultrastructural organization of irradiated metaphase cell treated before extraction with DNase I. Bars=1μm.


4 Discussion

Native chromatin is highly sensitive to the different agents used during isolation of interphase nuclei and mitotic chromosomes. The application of chromatin stabilization methods would allow chromosomes to be manipulated in vitro without loss of their structure or biochemical composition (Burakov and Chentsov, 2002). Here we describe the method of mitotic chromosome photostabilization: after irradiation, the chromosomes acquire resistance to deproteinization (2M NaCl) and decondensation (5mM TEA–HCl solution).

Enzyme treatments elucidated the role of different chromatin constituents in the induction of chromosome resistance. RNase and DNase treatments had no effect on the resistance of photostabilized chromosomes to decondensation, but proteinase treatment affected the morphology dramatically under decondensing conditions. These results suggest that chromosome proteins are mainly responsible for irradiation-induced chromatin resistance. This corroborates our earlier observations on chromatin in isolated interphase nuclei (Sheval et al., 2002; Prusov et al., 2003).

As shown by electron microscopy, irradiation cannot completely preserve chromosome morphology: after salt extraction the chromosomes swell and take on the appearance of a dense meshwork. To reveal the loci of highest photostabilized chromatin concentration, we analyzed color-coded images. This approach revealed thick fibers of photostabilized material inside the chromosome arms. The distance between their axial regions was 60–120nm, providing a rough estimate of the thickness of the photostabilized fibrillar structures. The main structural complexes in permeabilized chromosomes are represented by 40–60nm fibers (chromonemata) and it is tempting to assume that these fibers are selectively stabilized by irradiation.

Numerous authors have described chromonemal complexes in mitotic chromosomes in situ (Sparvoli et al., 1965; Chentsov et al., 1984; Zatsepina et al., 1983; Belmont et al., 1989; Hao et al., 1990, 1994; Iwano et al., 1997) and in permeabilized cells in vitro (Marsden and Laemmli, 1979; Laughlin et al., 1982; Belmont et al., 1989; Burakov and Chentsov, 2002). The structural organization of chromonema is unknown and at present there are two main models. (1) In partially decondensed in vitro chromosomes, each chromonema consists of a thread of globules – elementary chromomeres, closely juxtaposed (Zatsepina et al., 1983). Decondensed in vitro chromomeres resemble rosettes, consisting of an electron dense core and DNA loops radiating from it (Zatsepina et al., 1983; Tikhonenko et al., 1984). Rosette-like complexes resembling decondensed chromomeres were obtained after partial deproteinization of condensed chromatin from interphase nuclei (Sonnenbichler, 1969; Comings and Okada, 1976; Prusov et al., 1983; Ascoli et al., 1988) and mitotic chromosomes (Okada and Comings, 1979). In this model, the loop domains are considered to result from chromomere unfolding induced by extensive deproteinization (Zatsepina et al., 1983). Indeed, chromonemata and chromomeres are not resistant to high salt extraction, so some of the extracted non-histone proteins could be involved in the maintenance of chromonema integrity (Sheval et al., 2002). (2) Another set of data suggests that chromonemal complexes are formed as a result of irregular folding, twisting and aggregation of the 30-nm fibers at physiological ionic strength (Marsden and Laemmli, 1979; Belmont and Bruce, 1994). Interestingly, circular or linearized plasmids introduced into solution of cationic silanes become condensed in vitro and form rosettes, which closely resemble the rosette-like structures formed by native chromatin (Fang and Hoh, 1999). In this connection, recent data showing that bivalent cations are essential for the structural integrity of chromosomes are of great importance (Strick et al., 2001). These cations might promote the folding of loop domains into regular chromomeres. This reconciles the two models in their structural aspects, but the biochemical nature of the compacting agents is different – structural proteins and cations.

The discrete high-density zones in the axial regions of photostabilized chromonemata may correspond to the cores of elementary chromomeres. Since proteins play the major role in induced chromatin resistance, it can be assumed that the stabilized chromomere cores contain structural proteins that maintain the integrity of these complexes. These proteins are not scaffold proteins because the complexes are not seen in extracted non-irradiated cells (i.e. the putative chromomere-organizing proteins are extracted by 2M NaCl). Moreover, extracted cells contain no complexes resembling chromosome scaffolds. Antibody staining allows chromosome scaffolds to be visualized in similar preparations from HeLa, L929 and XL2 cells (manuscript in preparation); it appears that scaffolds are not revealed in our photographs because of the low sensitivity of the visualization approaches used.

The photostabilization of different chromosome domains has some peculiarities. The material of the chromosome arms seems to be a relatively homogeneous meshwork, but the centromeric regions in both the initial and color-coded images contain noticeably higher densities of photostabilized material. Increased intercalation of ethidium bromide into centromeric heterochromatin could account for this phenomenon. However, in permeabilized cells and cytogenetic preparations, chromosomes are stained homogeneously by ethidium bromide. This suggests that the centromeric regions (heterochromatic C-bands, according to cytogenetic terminology) contain a higher concentration of structural proteins than the chromosome arms (euchromatic G- and R-bands). This agrees with data showing that the centromeric chromatin of mouse chromosomes contains more non-histone proteins (Matsukuma and Utakoji, 1977). According to published data, the loop domain in centromeric heterochromatin is very small (Strissel et al., 1996), and therefore scaffold components might play the major role in centromeric chromatin organization. But these centromeric blocks are seen only after irradiation; on serial sections of non-irradiated extracted cells there are no aggregates that can be identified as centromeric chromatin remnants. Hence, the stabilized proteins are not scaffolding proteins. On the other hand, the centromeres have a fundamentally different organization from the chromosome arm material: they do not contain the main higher-level chromatin structures, chromonemata. However, threads of dense material are observed inside photostabilized centromeres after high salt extraction. This suggests that the centromeric DNA is packed into higher-order complexes; it is impossible to exclude the possibility that these complexes are identical to chromonemata. In any case, it is tempting to assume that the increased protein concentration is involved in the generation of C-banding.

In summary, the approach used here (chromatin stabilization by light irradiation in the presence of ethidium bromide) allows macromolecular chromatin complexes (chromonema and centromeric heterochromatin) in mitotic chromosomes to be stabilized. In their stabilized form, these complexes resist the actions of strong deproteinization and decondensation. This makes it possible to isolate chromatin complexes from mitotic chromosomes in nearly native form.

Acknowledgements

We thank V.V. Krugljakov for technical assistance and Dr. N.E. Yelina for reading this manuscript and helpful discussion. The work was supported by the Russian Foundation for Basic Research (grants 04-04-49359, 04-04-48541 and 03-04-48955).

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Received 10 June 2004; accepted 30 July 2004

doi:10.1016/j.cellbi.2004.07.013


ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
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