TY - JOUR
AU - Takeyasu, Kunio
AB - Abstract Genome function is closely linked to the higher-order chromatin structures. To reveal a structural basis for the interphase chromatin organization, the ‘on-substrate’ lysis procedure was applied to nuclei isolated from human HeLa cells, chicken erythrocyte cells and yeast Schizosaccharomyces pombe, which possessed different intrinsic properties of the genomes such as histone composition and inter-nucleosomal distance. The isolated nuclei on a coverslip were successively treated with a detergent and a high-salt solution to extract the nuclear membrane and the nucleoplasm, and therefore, atomic force microscopy (AFM) visualized the structural changes in response to the lysis procedure. After the nucleoplasm was extracted, AFM clarified that chromatin fibers, ∼40 nm in width, were partially released out of the nuclei and that the other chromatin still remaining in the nuclei was composed of granular structures with diameter of 80–100 nm. Thus, these results suggest that the ∼40 nm fiber would be a stable structural unit and fold the 80–100 nm granules into a one-step higher unit. A common mechanism could be implied regardless of the intrinsic properties of the eukaryotic genomes. chromatin fiber, nuclear isolation, on-substrate lysis, atomic force microscopy Introduction Eukaryotic genome is organized into the nucleus through several compaction steps. Core histones, H2A, H2B, H3 and H4, which are well conserved among eukaryotes, form a ‘beads-on-a-string’ structure of nucleosomes together with the genomic DNA. Linker histones, H1 and H5, are also found in eukaryotes and contribute to establish thicker 30–40 nm chromatin fibers [1–5], although they exhibit a large diversity in the amino acid sequences [6]. Interestingly, the linker histones do not always play a role in the genome packaging [7,8]. Nevertheless, the relationship between the genome structure and its function seems to be generally conserved among eukaryotes. In this study, we comparatively analyzed the chromatin folding with different intrinsic properties—genome size, histone composition and inter-nucleosomal distance. Namely, the nuclei isolated from human HeLa cells, chicken erythrocytes and yeast Schizosaccharomyces pombe were subjected to detailed structural analyses using a nano-scale imaging technique, atomic force microscopy (AFM). For this, we adopted the ‘on-substrate’ lysis procedure, which had been originally developed for subcellular fractionation of cultured cells [9]. This method successively removes the nuclear membrane and the nucleoplasm of the isolated nuclei, while the chromatin and nuclear scaffold remain on the substrate. After the lysis of the nuclei, the chromatin fibers were spread out of the nucleus, thus, allowing us to look at the chromatin structure at nano-scale. In the HeLa nucleus, 3.3 × 109 bp of genome DNA (haploid) forms nucleosomes in every ∼190 bp and also carries linker histones [10–12]. The chicken chromatin also contains both core and linker histones, forming a nucleosome in every ∼210 bp to fold the ∼1.1 × 109 bp genome [13–15]. On the contrary, S. pombe is a unicellular eukaryote with a smaller genome (1.2 × 107 bp), which is folded up via nucleosome structure (every 156 bp) [16], but without linker histone [17]. A direct comparison of the chromatin structures among various eukaryotes, which were prepared under the same condition, would significantly shed light on chromatin properties necessary for the folding. Materials and methods Nuclear isolation from HeLa cells HeLa S3 cells were cultured in the Dulbecco's modified Eagle medium supplemented with 10% foetal bovine serum in 5% CO2 at 37°C. The cultured cells were washed twice with phosphate-buffered saline (PBS). The cells were treated with buffer A [10 mM HEPES (pH 7.0), 100 mM NaCl, 3 mM MgCl2, 1 mM EGTA, 0.5% Triton X-100, 1 mM phenylmethylsulfonyl fluoride (PMSF)] at 4°C for 10 min. The nuclei were collected after a centrifugation (4000× g, 5 min) and resuspended in buffer A without Triton X-100. Nuclear isolation from chicken erythrocyte cells Chicken erythrocyte nuclei were isolated according to the procedure [18] with a slight modification. Chicken erythrocyte cells (Nippon Biotest Laboratory) were washed with buffer B [10 mM PIPES (pH 6.8), 100 mM NaCl, 340 mM sucrose, 1 mM EGTA, 1% β-mercaptoethanol, 1 mM PMSF). The nuclei were extracted by disrupting the cellular membrane by treating the cell suspension with buffer B containing 0.5% Nonidet P-40, and then collected after a centrifugation (8000× g, 5 min). The nuclei were stored in buffer B until used. Nuclear isolation from yeast cells S. pombe nuclei were isolated from the cells as described previously [19]. The wild-type 972 h- S. pombe haploid strain was used in this study. S. pombe cells were vegetatively grown in the YE medium at 30°C to a density of 1 × 107 cell ml−1. The cells were treated with 100 mM Tris–HCl (pH 8.0), 10 mM EDTA, 1% β-mercaptoethanol, 1 mM PMSF at 4°C for 30 min, and then spheroplasts were formed by an incubation with 0.2 mg ml−1 Zymolyase 100T (Seikagaku) in a spheroplast buffer [10 mM PIPES (pH 6.8), 0.1 mM CaCl2, 1.0 M sorbitol, 1 mM PMSF] for 60 min at 30°C. The spheroplasts were resuspended in a homogenization buffer [10 mM PIPES (pH 6.8), 15% Ficoll, 0.1 mM CaCl2, 1 mM PMSF] and then gently disrupted with a teflon pestle. The homogenate was loaded onto 10 mM PIPES (pH 6.8), 30% Ficoll, 0.1 mM CaCl2, 1 mM PMSF and then centrifuged at 26000 r.p.m. for 80 min at 4°C in a SW28 rotor. The precipitated crude nuclei was resuspended in the homogenization buffer and was purified by a centrifugation at 7000 r.p.m. for 30 min in 10 mM PIPES (pH 6.8), 0.1 mM CaCl2, 1 mM PMSF under a 15–60% percoll gradient, which removed the cell debris and the unlysed cells. AFM imaging The nuclei isolated from HeLa, chicken erythrocyte and yeast cells were resuspended in a nuclear buffer [10 mM PIPES (pH 6.8), 0.1 mM CaCl2, 1 mM PMSF]. Five microliters of the nuclear suspension was dropped onto a coverslip at 25°C for 10 min and subjected to a sequential treatment with 0.5% Triton X-100 and then 250 mM (NH4)2SO4 in the nuclear buffer. Then, the samples were fixed by 0.1% glutaraldehyde, rinsed with deionized water, and finally dried under nitrogen gas. AFM images were taken with an NVB100 under the control of the AC mode in air at 25°C (Olympus Optical). A silicon cantilever, OMCL-AC160TS-C2 (Olympus Optical), was routinely used for imaging. The typical resonance frequency of this cantilever was 300–400 kHz, and the normal scanning rate was 0.2–0.3 Hz. The images were captured in a 512 × 512 pixel format and analyzed by the software SPIP (Image Metrology A/S). The widths of the spread chromatin fibers in AFM images are affected by the size of the tips used. To avoid the broadening effect caused by the tip, the fiber widths were estimated at the half maximum height (FWHM: full width at half-maximum). This measurement is expected to subtract the broadening effect of the tip as described previously [20]. Indirect immunostaining for the isolated nuclei The immunostaining for the isolated nuclei was supposed to affect the surface structure. In this experiment, therefore, the immunostaining was employed for the same nucleus that had been used for AFM imaging. The nuclear lamina or the nuclear pore complex was stained to define the nuclear periphery of the isolated nuclei. After AFM imaging, the samples were treated with 1 mg ml−1 sodium borohydride at 25°C for 30 min, and then blocked with 1% BSA at 25°C for 30 min. The resultant samples were incubated with anti-lamin B1 (1:200 dilution, Zymed Laboratories) or anti-nuclear pore complex (1:200 dilution, Covance) for 2 h at 25°C. The specific binding of the primary antibody was detected by Cy3-conjugated anti-mouse IgG (1:2000 dilution, Sigma). For fluorescent microscopy, genomic DNA was counterstained by 0.2 µg ml−1 DAPI. Results FM and AFM analyses of HeLa nuclei The surface of the purified HeLa cell nucleus was relatively smooth before the detergent treatment, although many small projections and cavities were identified (Figs 1a and 1d). The isolated nuclei were successively subjected to the detergent treatment on the glass substrate to remove the nuclear membrane but not the nuclear lamina and the nuclear pore complex [9]. Fluorescence and atomic force microscopic observations revealed that the overall shape of the nucleus was not drastically changed after the detergent treatment (Figs 1b and 1e). After removal of the nucleoplasm by the high-salt treatment, another fibrous structure appeared (Fig. 2), although the overall staining of the nuclear lamina still remained (Figs 1c and 1f). A close examination with the statistical analysis of the nuclear structures after the high-salt treatment clarified that a granular structure with ∼80 nm width (Figs 2c and 2e), and fibers with different widths, ∼40 nm and ∼80 nm were frequently observed (Figs 2d and 2f). The thicker fibrous, but not granular, structure spread out of the nucleus, however, was not a single fiber but a bundle apparently formed by the assembly of several thin fibers (Fig. 2d), suggesting that the thinnest fiber of ∼40 nm width would be a structural unit of the HeLa chromatin. The granular structure was observed in the nucleus after the high-salt treatment, and no internal structure other than the granular structure could be recognized. These observations, therefore, suggest that the ∼40 nm fiber would be wound up to form a one-step higher unit, which was stabilized as ‘beads’ structure with a diameter of ∼80 nm in the nucleus, although it has not been clear as yet how the beads are built up from the ∼40 nm chromatin fiber. Fig. 1 View largeDownload slide Fluorescence and atomic force microscopic (AFM) analyses of the HeLa nucleus before and after the ‘on-substrate’ lysis. Fluorescent microscopy (FM) identified the localization of the nuclear lamina after isolation without detergent (a), after detergent treatment (b), and after high-salt treatment (c). These fluorescence micrographs were taken after the AFM observation and obtained from the same nuclei that were used for the AFM imaging. AFM visualized the isolated nucleus without detergent treatment (d), after the detergent treatment (e) and after the high-salt treatment (f). Scale bars indicate 2 µm. The isolated nucleus was ∼1200 nm in height (g), 900 nm after the detergent treatment (h), and 300 nm after the additional high-salt treatment (i). In these experiments, asynchronously cultured HeLa cells were used for FM and AFM analyses, and, therefore, the nuclear size varied depending upon the cell cycle (10-20 µm in diameter). Fig. 1 View largeDownload slide Fluorescence and atomic force microscopic (AFM) analyses of the HeLa nucleus before and after the ‘on-substrate’ lysis. Fluorescent microscopy (FM) identified the localization of the nuclear lamina after isolation without detergent (a), after detergent treatment (b), and after high-salt treatment (c). These fluorescence micrographs were taken after the AFM observation and obtained from the same nuclei that were used for the AFM imaging. AFM visualized the isolated nucleus without detergent treatment (d), after the detergent treatment (e) and after the high-salt treatment (f). Scale bars indicate 2 µm. The isolated nucleus was ∼1200 nm in height (g), 900 nm after the detergent treatment (h), and 300 nm after the additional high-salt treatment (i). In these experiments, asynchronously cultured HeLa cells were used for FM and AFM analyses, and, therefore, the nuclear size varied depending upon the cell cycle (10-20 µm in diameter). Fig. 2 View largeDownload slide Cross-section analyses of the HeLa nucleus and the fibers released out of the nucleus after the high-salt treatment. The high-salt treated HeLa nucleus (Fig. 1f; see also the inset figure) was rescanned for magnification and shown in different panels (a–d). A section profile obtained along X–Y line shows a typical granular structure in the nucleus (c), and the peak-to-peak distance between the granular structure was distributed from 60 to 120 nm with 85.5 ± 14.4 nm of the mean value (n = 61) (e). The thickness of the chromatin fibers released out of the nucleus varied possibly due to the assembly of thinner fibers (d). A section profile for the spread fibers was obtained along X–Y line (d), and a further statistical analysis indicated the thinnest fiber width was found to be ∼40 nm (44.3 ± 8.6 nm, n = 74) (f). Fig. 2 View largeDownload slide Cross-section analyses of the HeLa nucleus and the fibers released out of the nucleus after the high-salt treatment. The high-salt treated HeLa nucleus (Fig. 1f; see also the inset figure) was rescanned for magnification and shown in different panels (a–d). A section profile obtained along X–Y line shows a typical granular structure in the nucleus (c), and the peak-to-peak distance between the granular structure was distributed from 60 to 120 nm with 85.5 ± 14.4 nm of the mean value (n = 61) (e). The thickness of the chromatin fibers released out of the nucleus varied possibly due to the assembly of thinner fibers (d). A section profile for the spread fibers was obtained along X–Y line (d), and a further statistical analysis indicated the thinnest fiber width was found to be ∼40 nm (44.3 ± 8.6 nm, n = 74) (f). FM and AFM analyses of chicken erythrocyte nuclei Similar to the HeLa cell nucleus, the surface of the isolated chicken erythrocyte nucleus was smooth and no internal structure was observed without the detergent treatment (Figs 3a and 3d). After the detergent treatment, the nuclear height was greatly reduced from ∼500 nm to ∼100 nm (Figs 3g and 3h), although the overall shape of the nucleus and the immunostaining signal of nuclear lamina were preserved (Figs 3a and 3b). This clearly indicates that the nuclear membranes were extracted during this treatment. A successive treatment with high-salt revealed that many chromatin fibers also came out of the nucleus (Fig. 3f). Under this condition, the chicken erythrocyte nucleus kept the height constant (Figs 3h and 3i), although the nuclear proteins weakly associated with the chromatin and other inner nuclear architectures were expected to be removed by this treatment [9]. A statistical analyses of the chromatin revealed that the internal structure comprised ∼80 nm beads (89.8 ± 12.7 nm, n = 70; Figs 4c and 4e) and that ∼40 nm and ∼80 nm fibers were identified as major populations (Figs 4d and 4f). These results also suggest that, similar to the HeLa cell nucleus, the chicken erythrocyte nucleus has the ∼40 nm chromatin fiber and the 80 nm beads as stable structural units of its genome. Fig. 3 View largeDownload slide Fluorescence and AFM analyses of the chicken erythrocyte nucleus before and after the ‘on-substrate’ lysis. The nuclear lamina distribution was determined after isolation without detergent (a), after detergent (b) and after high-salt treatments (c). As is the case for Fig. 1, these fluorescence micrographs were taken after the AFM imaging and obtained from the same nuclei that were used for the AFM imaging. AFM visualized the isolated nuclei without detergent treatment (d), after the detergent treatment (e) and after the high-salt treatment (f). Scale bars indicate 1 µm. Section profiles along white lines in the panels d–f show that the nucleus was ∼400 nm in height (g), 100 nm after the detergent treatment (h) and 100 nm after the additional high-salt treatment (i). Fig. 3 View largeDownload slide Fluorescence and AFM analyses of the chicken erythrocyte nucleus before and after the ‘on-substrate’ lysis. The nuclear lamina distribution was determined after isolation without detergent (a), after detergent (b) and after high-salt treatments (c). As is the case for Fig. 1, these fluorescence micrographs were taken after the AFM imaging and obtained from the same nuclei that were used for the AFM imaging. AFM visualized the isolated nuclei without detergent treatment (d), after the detergent treatment (e) and after the high-salt treatment (f). Scale bars indicate 1 µm. Section profiles along white lines in the panels d–f show that the nucleus was ∼400 nm in height (g), 100 nm after the detergent treatment (h) and 100 nm after the additional high-salt treatment (i). Fig. 4 View largeDownload slide Cross-section analyses of the chicken erythrocyte nucleus and the chromatin fibers released out of the nucleus after the high-salt treatment. The high-salt treated chicken erythrocyte nucleus (a; see also Fig. 3f) was magnified (b–d). The AFM image on the erythrocyte nucleus after the detergent and high-salt treatments also revealed a typical granular structure of the nuclear chromatin (c). The high-magnification images were subjected to further quantitative analyses. A section profile was obtained along X–Y line in the panel, and the peak-to-peak distance between neighboring beads on the X–Y line was ∼80 nm (89.8 ± 12.7 nm, n = 70) (c and e). Chromatin fibers with various thicknesses were released (d). A section profile was obtained along X–Y line, and a histogram shows the widths distribution of the fibers (the average width: 46.4 ± 9.4 nm, n = 78) (f). Fig. 4 View largeDownload slide Cross-section analyses of the chicken erythrocyte nucleus and the chromatin fibers released out of the nucleus after the high-salt treatment. The high-salt treated chicken erythrocyte nucleus (a; see also Fig. 3f) was magnified (b–d). The AFM image on the erythrocyte nucleus after the detergent and high-salt treatments also revealed a typical granular structure of the nuclear chromatin (c). The high-magnification images were subjected to further quantitative analyses. A section profile was obtained along X–Y line in the panel, and the peak-to-peak distance between neighboring beads on the X–Y line was ∼80 nm (89.8 ± 12.7 nm, n = 70) (c and e). Chromatin fibers with various thicknesses were released (d). A section profile was obtained along X–Y line, and a histogram shows the widths distribution of the fibers (the average width: 46.4 ± 9.4 nm, n = 78) (f). FM and AFM analyses of yeast nuclei Since S. pombe lacks linker histone H1, the structural comparison of its genome and other eukaryotic genomes has great significance. In S. pombe, the staining pattern of DAPI and Mab414, which was a monoclonal antibody against the nuclear pore complex [21], showed that the ‘on-substrate’ procedure could be similarly applied to the yeast nuclei with 2–3 µm in size (Figs 5a, 5b, and 5c). AFM made it clear that, although the isolated yeast nucleus had a smooth nuclear envelope without the detergent treatment (Fig. 5d), the removal of the nuclear membranes by the detergent treatment greatly changed the surface morphology and reduced the nuclear height (Figs 5d, 5e, 5g, and 5h). The high-salt treatment also led the chromatin fiber spread (Fig. 5f), as observed in the nuclei isolated from HeLa cells (Fig. 2) and chicken erythrocytes (Fig. 4). The majority of the fiber widths was also ∼40 nm (Figs 6c and 6e), and the size of the beads inside the nucleus was ∼100 nm after this treatment (Figs 6b and 6d). Unlike the HeLa and chicken erythrocyte nuclei, the fiber spread in yeast required longer incubation under the same salt concentration, suggesting that the yeast chromosome is more tightly folded than the other two chromosomes. Fig. 5 View largeDownload slide Fluorescence and AFM analyses of the yeast nucleus before and after the ‘on-substrate’ lysis. The nuclear pore complex (NPC) distribution was determined after isolation without detergent (a), after detergent treatment (b) and after high-salt treatment (c). As is the case for Fig. 1, these fluorescence micrographs were taken after the AFM imaging and obtained from the same nuclei that were used for the AFM imaging. AFM visualized the isolated nuclei without detergent treatment (d), after the detergent treatment (e) and after the high-salt treatment (f). Scale bars indicate 1 µm. The nucleus was ∼400 nm in height (g) and gradually decreased in the process of the additional detergent (h; 100 nm) and the high-salt treatments (i; 50 nm). Fig. 5 View largeDownload slide Fluorescence and AFM analyses of the yeast nucleus before and after the ‘on-substrate’ lysis. The nuclear pore complex (NPC) distribution was determined after isolation without detergent (a), after detergent treatment (b) and after high-salt treatment (c). As is the case for Fig. 1, these fluorescence micrographs were taken after the AFM imaging and obtained from the same nuclei that were used for the AFM imaging. AFM visualized the isolated nuclei without detergent treatment (d), after the detergent treatment (e) and after the high-salt treatment (f). Scale bars indicate 1 µm. The nucleus was ∼400 nm in height (g) and gradually decreased in the process of the additional detergent (h; 100 nm) and the high-salt treatments (i; 50 nm). Fig. 6 View largeDownload slide Cross-section analyses of the yeast nucleus and the chromatin fibers released out of the nucleus after the high-salt treatment. Like HeLa and chicken nuclei, the yeast nucleus responded to the high-salt treatment; beads structures inside the nucleus became apparent and chromatin fibers were released. The high-salt treated nucleus (a; see also Fig. 5f) was magnified and shown (b and c). The high-magnification image was subjected to further quantitative analyses. A section profile along X–Y line (b) clarifies that a typical peak-to-peak distance between neighboring beads on the X–Y line was ∼100 nm (99.7 ± 17.2 nm, n = 57) (d). Chromatin fibers with various thicknesses were released (c). A section profile was obtained along X–Y line in the inset of the panel c. A histogram shows the widths distribution of the fibers (the average width: 45.2 ± 7.9 nm, n = 57) (e). Fig. 6 View largeDownload slide Cross-section analyses of the yeast nucleus and the chromatin fibers released out of the nucleus after the high-salt treatment. Like HeLa and chicken nuclei, the yeast nucleus responded to the high-salt treatment; beads structures inside the nucleus became apparent and chromatin fibers were released. The high-salt treated nucleus (a; see also Fig. 5f) was magnified and shown (b and c). The high-magnification image was subjected to further quantitative analyses. A section profile along X–Y line (b) clarifies that a typical peak-to-peak distance between neighboring beads on the X–Y line was ∼100 nm (99.7 ± 17.2 nm, n = 57) (d). Chromatin fibers with various thicknesses were released (c). A section profile was obtained along X–Y line in the inset of the panel c. A histogram shows the widths distribution of the fibers (the average width: 45.2 ± 7.9 nm, n = 57) (e). Discussion In a variety of microscopy works, specimen preparation procedures have been known to affect the cellular and molecular structures of biological materials. Any specimen preparation step, which includes a change in ionic compositions in the buffer solution, fixation and drying processes, has this inherent concern, when the real (physiological) structures in cells come to consideration. Among these factors, ionic strength significantly affects morphological changes in chromatin, because monovalent and divalent cations in solution act as general DNA counter-ions [22,23]. On the other hand, chromatin fibers are weakly associated with a variety of nuclear proteins in interphase nuclei. In spite of this dilemma, in order to evaluate widths of chromatin fibers, therefore, it is absolutely necessary to remove the nucleoplasm consisting of the nuclear proteins. In this study, the nuclei isolated from three eukaryotes were successively treated with the detergent and high-salt buffers, and then AFM visualized the spread chromatin fibers. The ‘on-substrate’ lysis procedure shows that chromatin fibers were spread after the nucleoplasm extraction (Figs 1, 3, and 5), suggesting that the nuclear proteins would be dissociated from the fibers by this treatment, although these spread fibers may not preserve ‘native’ chromatin fibers that exist in vivo. On the other hand, the fibers detected in this study are expected to reflect a ‘stable’ structural state governed by the physicochemical properties of chromatin fibers, because the chromatins from different species in different kingdoms have shown a very similar behavior [24–26, also this work]. Thus, a structural comparison of the released chromain fibers from different species with different intrinsic properties will provide a significant insight into the elucidation of the chromatin-folding mechanism, even after the high-salt treatment, as long as the spread chromatin fibers were prepared under the same conditions. Our comparative structural analyses on the nuclei from different eukaryotes identified a relatively ‘stable’ structural unit of the interphase chromosome. The high-salt treatment caused a release of 40 nm fibers from the nuclei of all eukaryotic cells tested (Table 1). Such 40 nm fibers were also identified in the partially relaxed plant chromosome and were looped out from the mitotic chromosome [27,28]. Therefore, the 40 nm fiber seems to be the thinnest chromatin structure relatively stable in the eukaryotic genome, although the intrinsic characteristics of chromatin, such as genome size, nucleosome spacing and histone composition vary among these eukaryotic species. It is of interest that Saccharomyces cerevisiae, in which a nucleosome-repeat length (165 bp) is shorter than those of higher eukaryotes (∼200 bp), forms a similar 30–40 nm chromatin fiber [29]. It was recently demonstrated that such 30–40 nm chromatin fibers can be reconstituted in vitro from super-coiled DNA, core histones and histone H1 [5]. Table 1 Summary of chromatin structure after the high-salt treatment   Beads sizea  Fiber widthsb  HeLa cell  85.5 ± 14.4 nm (n = 61)  44.3 ± 8.6 nm (n = 74)  Chicken erythrocyte  89.8 ± 12.7 nm (n = 70)  46.4 ± 9.4 nm (n = 78)  Yeast  99.7 ± 17.2 nm (n = 57)  45.2 ± 7.9 nm (n = 57)    Beads sizea  Fiber widthsb  HeLa cell  85.5 ± 14.4 nm (n = 61)  44.3 ± 8.6 nm (n = 74)  Chicken erythrocyte  89.8 ± 12.7 nm (n = 70)  46.4 ± 9.4 nm (n = 78)  Yeast  99.7 ± 17.2 nm (n = 57)  45.2 ± 7.9 nm (n = 57)  aPeak-to-peak distances between the neighboring beads inside nuclei. bFull width at half-maximum (FWHM) of the thinner fibers spread out of nuclei. View Large After the high-salt treatment, the 80–100 nm bead structures were also observed on the surface of HeLa, chicken and yeast nuclei (Figs 2c, 4c, 6b, and Table 1). Similar 80 nm beads structure had been reported in the previous studies using HeLa cells grown on the cover glass [9]. The beads with 80–100 nm width were also identified on the surface of the human mitotic chromosome under various conditions [30–32]. No structural population other than the 40 nm fiber and the 80 nm beads was clearly found, suggesting that the 40 nm fiber would be folded directly up to the 80–100 nm beads. A 30–100 kb chromosomal loop found between the nuclear scaffold attachments is important for the higher order organization of chromatin [33–37]. The yeast chromatin is more strongly and frequently associated with the matrix, and, thus, the loop size in yeast seems to be much smaller than that in mammalian chromatin [38]. An interesting fact is that, when a piece of S. pombe DNA was introduced into mouse cells by protoplast fusion, it associated with the mouse nucleoskeleton at intervals shorter than the host DNA and formed more condensed and narrower chromatin region in the host chromosomes [39]. The frequent and tight attachment to the matrix in yeast may provide an implication for the mechanism of differential responses of nuclei to the high-salt treatment; the yeast nuclei required approximately 30 min—three times longer than the others—to release the 40 nm chromatin fibers. Conclusion remarks We examined the chromatin and nuclear architectures of human HeLa cells, chicken erythrocyte cells and fission yeast cells to identify fundamental steps of the interphase genome folding. For this, the ‘on-substrate’ lysis procedure combined with AFM and FM was applied to the isolated nuclei, which exhibited the structural changes in response to the lysis procedure. In addition, a comparison of the fluorescent and atomic force microscopic images revealed the detailed structures of the nuclear periphery and thus could illuminate an apparent difference between the spread and the folded interphase chromatin. 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TI - Comparative structural biology of the genome: nano-scale imaging of single nucleus from different kingdoms reveals the common physicochemical property of chromatin with a 40 nm structural unit
JF - Journal of Electron Microscopy
DO - 10.1093/jmicro/dfi076
DA - 2006-01-01
UR - https://www.deepdyve.com/lp/oxford-university-press/comparative-structural-biology-of-the-genome-nano-scale-imaging-of-B4tn0qb0f5
SP - 31
EP - 40
VL - 55
IS - 1
DP - DeepDyve
ER -