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        Reversible Changes of Chromosome Structure upon Different Concentrations of Divalent Cations
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        Reversible Changes of Chromosome Structure upon Different Concentrations of Divalent Cations
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        Reversible Changes of Chromosome Structure upon Different Concentrations of Divalent Cations
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Abstract

The structural details of chromosomes have been of interest to researchers for many years, but how the metaphase chromosome is constructed remains unsolved. Divalent cations have been suggested to be required for the organization of chromosomes. However, detailed information about the role of these cations in chromosome organization is still limited. In the current study, we investigated the effects of Ca2+ and Mg2+ depletion and the reversibility upon re-addition of one of the two ions. Human chromosomes were treated with different concentrations of Ca2+and Mg2+. Depletion of Ca2+ and both Ca2+ and Mg2+ were carried out using 1, 2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid and ethylenediaminetetraacetic acid (EDTA), respectively. Chromosome structure was examined by fluorescence microscopy and scanning electron microscopy. The results indicated that chromosome structures after treatment with a buffer without Mg2+, after Ca2+ depletion, as well as after depletion of both Mg2+, and Ca2+, yielded fewer compact structures with fibrous chromatin than those without cation depletion. Interestingly, the chromatin of EDTA-treated chromosomes reversed to their original granular diameters after re-addition of either Mg2+ or Ca2+ only. These findings signify the importance of divalent cations on the chromosome structure and suggest the interchangeable role of Ca2+ and Mg2+.

Introduction

Understanding chromosome structure is the basis for determining the accurate segregation during cell division. Until now, how the metaphase chromosome is constructed remains unsolved. There is long-standing evidence that the integrity of condensed chromatin in the nucleus, as well as in metaphase chromosomes, depends on the binding of cations (Engelhardt, 2004). At high ionic concentrations, chromatin becomes granulated and the chromosomes become condensed. The most frequently observed granules have a diameter of about 35 nm (Caravaca et al., 2005), although the existence of 30 nm chromatin in native chromosomes is still controversial (Eltsov et al., 2008; Grigoryev & Woodcock, 2012; Nishino et al., 2012).

The ionic environment has frequently been considered to affect chromosome structure. Divalent cations, especially Ca2+ and Mg2+, have long been studied for their responsibility in chromosome organization. However, detailed information about their role in chromosome organization has not been confirmed. In this study, the effects of Ca2+ and Mg2+ concentrations on chromosome structure were investigated by using fluorescence microscopy and scanning electron microscopy (SEM). This study was particularly focused on the reversibility of the globular and fibrous chromatin structure. Furthermore, the reversibility from the Ca2+- and Mg2+-depleted chromosomes to the original structure upon the re-addition of each of the two cations was also assessed in detail, with information on the roles of divalent cations.

Materials and methods

Sample preparation

In this study, chromosomes from the HeLa S3 cell line established from human cervical adenocarcinoma were investigated. Chromosomes were isolated and suspended by the polyamine method (PA isolated chromosomes, Uchiyama et al., 2004, 2005; Hayashihara et al., 2008). The HeLa S3 cells were synchronized and treated with 75 mM KCl as the hypotonic treatment. The cell membranes were then lysed. The polyamine buffer was used for the chromosome isolation. Samples were then subjected to the multi-step centrifugation to obtain the isolated PA chromosomes (Uchiyama et al., 2004, 2005; Hayashihara et al., 2008).

To investigate the effect of Ca2+ concentration changes on chromosome structure, 20 μM 1, 2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) (Dojindo) was applied to chelate Ca2+ directly from isolated chromosomes. After Ca2+ depletion, some samples were stained with 4′,6-diamidino-2-phenylindole (DAPI) and observed with a fluorescence microscope (FM, Axioplan 2, Zeiss). The rest of the samples were subjected to preparation for SEM samples.

In addition to Ca2+ depletion, some chromosomes were treated with buffers containing 0 or 5 mM of Mg2+. Chromosomes were placed on coverslips, incubated on ice for 10 min, and then subjected to each of the two different buffers, XBE (10 mM HEPES, pH 7.7, 100 mM KCl, and 5 mM EGTA) and XBE5 (10 mM HEPES, pH 7.7, 5 mM MgCl2, 100 mM KCl, and 5 mM EGTA), for 30 min. Samples were then either stained with DAPI for fluorescence microscopic observation or subjected to preparation for SEM samples. A similar preparation was also performed in order to evaluate the effect of both Ca2+ and Mg2+ using 1 mM ethylenediaminetetraacetic acid (EDTA) (Dojindo).

SEM sample preparation and observation

Chromosomes on the coverslips were fixed with 2.5% glutaraldehyde diluted in XBE5 or XBE buffer and 0.2% tannic acid/XBE5 or XBE, postfixed with 2% OsO4/Milli-Q, and washed with Milli-Q water three times for 5 min each. They were subsequently dehydrated with 70% ethanol and twice with 100% ethanol, dried with a critical point dryer (JCPD-5, JEOL, Tokyo, Japan), coated with an OsO4 coater (HPC-1S; Vacuum Device Inc., Japan). The chromosomes were then observed in an SEM S-5200 (Hitachi, Tokyo, Japan) with an accelerating voltage of 10 kV in the secondary electron mode.

Results

The effects of Ca2+ and Mg2+ on chromosome structures observed by the fluorescence microscope

For evaluating the effect of Ca2+ and Mg2+, chromosomes were subjected to different Ca2+ and Mg2+ concentrations. Figure 1 shows the different chromosome structures under different Ca2+ and Mg2+ treatments. As a control, isolated HeLa S3 chromosomes were prepared using XBE5 buffer, which contains 5 mM Mg2+ without the addition of ion chelators (Fig. 1a). The 5 mM Mg2+ was set following the measurements of peak intensity levels during mitosis observed for nuclei and chromatin by secondary ion mass spectrometry reported by Strick et al. (2001). It is shown that the chromosome is highly condensed and compact. In contrast, when Ca2+ was depleted from the control chromosomes by using BAPTA, the chromosome structure changed; it became less condensed with a larger chromosome area, and some fibers dispersed out of the chromosome as shown in Figure 1b. Chromosomes were expanded more after Ca2+ depletion than they were with the control.

Fig. 1. Fluorescence micrographs showing the effects of Ca2+ and Mg2+ on chromosome structures. Chromosomes prepared with the buffer containing 5 mM Mg2+ without Ca2+-chelating agent as the control (a). Depletion of Ca2+ with 10 μM BAPTA (b), chromosome in the buffer without Mg2+ (c), and chromosome in the buffer with 1 mM EDTA (d) caused greater expansion of the chromosome. Bars: 1 µm.

Similar results were also obtained from the chromosomes treated with buffer without Mg2+ (Fig. 1c). The dispersed structure of the chromosomes treated with this buffer indicated that Mg2+ has an important role on the structural maintenance of the chromosomes.

We further assessed the importance of both Ca2+ and Mg2+ by treating the samples using EDTA, which has the ability to chelate both ions. The result is depicted in Figure 1d showing a highly altered chromosome structure. Even though the “X-shaped” structure could still be observed, it is clear that deformation of the chromosome has occurred. The chromosome area expanded, and the chromatin radiated out of the chromosomes.

The effects of Mg2+ and Ca2+ on chromosome and chromatin ultrastructure observed by SEM

The detailed ultrastructure of the chromosomes or chromatin structure treated with different Ca2+ and Mg2+ concentrations was gained by SEM, which provides an advantage of higher magnification and resolution. Consistent with FM results, SEM results also showed that the chromosome structure and the diameter of the chromatin granules/fibers varied with the different treatments. According to the data (Fig. 2), only the control with Ca2+ and Mg2+ achieved a condensed chromosome and globular chromatin surface structure (Figs. 2a, 2a’). Ca2+ depletion with Mg2+ (Figs. 2b, 2b’), treatment with the buffer without Mg2+ (but with Ca2+) (Figs. 2c, 2c’), and depletion of both Ca2+ and Mg2+ (Figs. 2d, 2d’) resulted in more fibrous chromatin structures. The diameters of chromatin granules in the control (Figs. 2a, 2a’) were larger than the diameter of chromatin fibers after BAPTA treatment (Figs. 2b, 2b’) illustrating the importance of Ca2+ in maintaining the compact chromosome structure.

Fig. 2. SEM micrographs depicting the effects of Ca2+ and Mg2+ on chromosome ultrastructure. Chromosomes prepared with the buffer containing 5 mM Mg2+ and Ca2+ as a control (a, a’) are compared with those treated with 10 µM BAPTA (b, b’), the buffer without Mg2+ (c, c’), and 1 mM EDTA (d, d’). a’–d’: Show the magnified images of the box in (a–d), respectively. Bars: 500 nm (ad) and 100 nm (a’–d’).

The chromosome structure also varied depending on Mg2+ concentration. Chromosomes maintained their structure when treated with a buffer containing 5 mM Mg2+ and dispersed when treated with a buffer without Mg2+. Chromosomes treated with a buffer containing 5 mM Mg2+ showed a more compact structure (Figs. 2a, 2a’), and the structure became more distorted when the chromosomes were treated with a buffer without Mg2+ (Figs. 2c, 2c’). Finally, both Ca2+ and Mg2+ were depleted by using EDTA, which has the affinity to chelate both ions. Based on the data, EDTA-treated chromosomes showed a similar less condensed and more fibrous chromatin structure than those treated with BAPTA (Ca2+ chelator) as well as chromosomes treated with the buffer without Mg2+.

The reversibility of chromosome structure upon Mg2+ and Ca2+ treatment

The effects of Ca2+ and Mg2+ depletion depicted above suggest that it is reasonable to estimate that both Ca2+ and Mg2+ played major roles in maintaining the structure of the chromosomes. Each ion depletion caused the structural deformation as shown by the fluorescence microscope and SEM. The highest expansion of the chromosome area was obtained from the chromosome treated with EDTA (Fig. 1d). To further assess the role of each ion, the re-addition of each ion to the EDTA-treated samples was carried out. Fluorescence microscopic observation clearly revealed that the severely altered chromosome structure after EDTA treatment (Fig. 3b) recovered its authentic form such as that observed in the control condition (Fig. 3a) after re-addition of either Ca2+ (Fig. 3c) or Mg2+ (Fig. 3d).

Fig. 3. Fluorescence micrographs indicating the reversibility of chromosome structure upon Ca2+ or Mg2+ re-addition. Chromosomes treated with EDTA showed the altered structure (b) compared with the control (a); however, the expanded structure could be recovered to its authentic structure after the re-addition of either Mg2+ (c) or Ca2+ (d). Bars: 1 µm.

Lastly, to further prove the reversibility of the EDTA-treated chromosome structure upon re-addition of only one cation, chromosomes were treated with different cation combinations. Figures 4a and 4a’ display the chromosome treated with a buffer with 5 mM Mg2+ as the control. Figures 4b and 4b’ display the chromosome treated with EDTA (Ca2+ and Mg2+ chelator). Figures 4c and 4c’ were the EDTA-treated chromosome followed with re-addition of 5 mM Mg2+ examined under SEM. It is clear that after re-addition of Mg2+, the less condensed EDTA-treated chromosome returned to its original condensed structure as shown by the control.

Fig. 4. SEM micrographs revealing the reversibility of chromosome ultrastructure upon 5 mM Mg2+ re-addition. (a and a’): Show the control. Chromosomes treated with EDTA showed the altered chromatin structure compared with the control (b, b’). However, the chromosome and chromatin structures could be recovered to their authentic structures by the re-addition of Mg2+ only (c, c’). a’–c’: Showed the magnified images of the box in (ac), respectively. Bars: 1 µm (ac) and 100 nm (a’–c’).

Not only did the appearance of the whole chromosome structure change, but also the EDTA-treated chromosomes showed changes within their chromatin structure, from more globular structures to more fibrous ones. Some fibers radiated from the chromosomes, and the diameter of the chromatin fibers also decreased. In contrast, after Mg2+ re-addition, the radiated fibers were not observed, and the chromosomes returned to their authentic compact structure. The reversibility of chromosome structure upon the depletion and re-addition of divalent cations observed by SEM showed the similar phenomenon with those observed by FM. It is thus further confirmed that the compaction of chromosome underwent a reversible manner under different ion concentrations.

Discussion

In this study, the effects of Ca2+ and Mg2+ on the chromosome structure was examined. The results demonstrated that the drastic changes of chromosome structures are caused by varying the Ca2+ and/or Mg2+ concentration, suggesting the importance of these divalent cations for the structural maintenance of the chromosome. The reversibility of chromosome structure and chromatin diameter under different Ca2+ concentrations has been described by Phengchat et al. (2016). After Ca2+ depletion by using BAPTA, chromatin granules diameter was decreased. Interestingly, after re-addition of Ca2+, the chromosome structure returned to its original form.

Similar to the effect of Ca2+, the changes of chromosome structure upon Mg2+ concentration were also reversible (Dwiranti et al., 2014). The chromosomes were shown as a compact structure with granular chromatin structure when treated with a buffer containing 5 mM Mg2+. However, when the buffer containing 0 mM Mg2+ was used, the chromosomes showed a more decondensed structure with a smaller diameter of chromatin fibers. Reversibility of these changes was shown after the re-addition of 5 mM Mg2+. The dispersed structure of chromatin fiber when it was treated with the buffer without Mg2+ was also shown by STEM tomography as previously reported (Dwiranti et al., 2016). Previous studies showed that high Mg2+ concentration (≥2 mM) preserved the heterochromatin in a condensed state (Adolph et al., 1986; Caravaca et al., 2005).

Previous work has demonstrated that chromatin architecture physically responds to environmental conditions, with condensation occurring in response to hyperosmotic conditions (Albiez et al., 2006). Another report indicated that in the presence of multivalent cations, high molecular weight DNA undergoes dramatic condensation to a compact, usually highly ordered toroidal structure, with experimental evidence showing that DNA condensation occurs when about 90% of its charge is neutralized by counterions (Bloomfield, 1997). Furthermore, Visvanathan et al. (2013) showed that divalent cations, but not monovalent cations, provoke chromatin compaction as evaluated by either confocal microscopy or FLIM-FRET. Another tool has also been used to reveal the role of divalent cations, a high-resolution scanning ion microprobe (UC-SIM) revealed a similar conclusion that Ca2+ and Mg2+, in particular, are essential for the condensation and the structural integrity of chromosomes (Levi-Setti et al., 2008).

In the current study, we further explore the role of these two ions by chelating them with EDTA. It is interesting that even though the structure of the chromosome is highly distorted after both Ca2+ and Mg2+ depletion using EDTA, its structure is reversible by the addition of only one of these two divalent cations. This result implies that both Ca2+ and Mg2+ have similar functions in maintaining the chromosome structure. The transition between globular and fibrous structures of the chromatin and the compact/expanded chromosome structures upon the presence or absence of the Ca2+ and Mg2+ strongly suggest that chromatin has a large amount of conformational freedom, allowing dynamic unfolding and refolding and that the charge interactions play an essential role in maintaining the chromosomes as described by Poirier et al. (2002). As mentioned above, the most probable mechanism is by neutralizing the negative charge of the DNA and thus chromatin fiber.

The issue of neutralization, by cations, of the excess negative charge of DNA, contributed by the backbone phosphate groups to allow chromatin compaction, has been reviewed and discussed in detail by Strick et al. (2001) on mammalian chromosomes. Interestingly, in dinoflagellate chromosomes, where histones and therefore nucleosomes are absent, it has been shown that the cations may interact directly with DNA (Herzog & Soyer, 1983; Levvi-Setti et al., 2008). These findings further support that positive divalent cations (Ca2+ and Mg2+) play major roles in the neutralization of negatively charged DNA in chromosomes of both primitive and advanced eukaryotes. Such neutralization is needed to prevent repulsive Coulomb forces between DNA.

Conclusion

In conclusion, chromosome structure was affected by divalent cations (Ca2+ and Mg2+). An adequate concentration of divalent cations is required for the proper regulation of chromosome organization and maintenance of its higher-order structure. Both Ca2+ and Mg2+ are important for chromosome organization. In addition, changes of chromosome structure upon different concentrations of divalent cations are reversible.

Author ORCIDs

Astari Dwiranti, 0000-0002-0105-2748; Hideaki Takata, 0000-0001-9436-1517; Kiichi Fukui, 0000-0002-9156-819X.

Acknowledgments

We acknowledge Prof. Susumu Uchiyama for his support, guidance, and suggestions for this work. Parts of the present experiment were carried out at the Research Center for Ultra-High Voltage Electron Microscopy, Osaka University. We are grateful to Dr. Tomoki Nishida and Mr. Toshiaki Hasegawa for their technical support. This study was financially supported by a grant from the Japanese Ministry of Education, Culture, Sports, Science, and Technology (No. 212 480 40 and No. 252 520 64) to K. F.

Author Contributions

K.F. conceived and designed the research, A.D. conducted experiments under the guidance of H.T. and K.F., A.D. analyzed the data under the guidance of H.T. and K.F., and A.D. wrote the paper.

References

Adolph, KW, Kreisman, LR & Kuehn, RL (1986). Assembly of chromatin fibers into metaphase chromosomes analyzed by transmission electron microscopy and scanning electron microscopy. Biophys J 49, 221231.
Albiez, H, Cremer, M, Tiberi, C, Vecchio, L, Schermelleh, L, Dittrich, S, Küpper, K, Joffe, B, Thormeyer, T, von Hase, J, Yang, S, Rohr, K, Leonhardt, H, Solovei, I, Cremer, C, Fakan, S & Cremer, T (2006). Chromatin domains and the interchromatin compartment form structurally defined and functionally interacting nuclear networks. Chrom Res 14, 707733.
Bloomfield, VA (1997). DNA condensation by multivalent cations. Biopolymers 44, 269282.
Caravaca, JM, Caño, S, Gállego, I & Daban, J (2005). Structural elements of bulk chromatin within metaphase chromosomes. Chrom Res 13, 725743.
Dwiranti, A, Hamano, T, Takata, H, Nagano, S, Guo, H, Onishi, K, Wako, T, Uchiyama, S & Fukui, K (2014). The effect of magnesium ions on chromosome structure as observed by helium ion. Microscopy Microsc 20, 184188.
Dwiranti, A, Takata, H, Uchiyama, S & Fukui, K (2016). The effect of magnesium ions on chromosome structure as observed by scanning electron microscopy (SEM) and scanning electron microscope (STEM) tomography. Chrom Sci 19, 1923.
Eltsov, M, Maclellan, KM, Maeshima, K, Frangakis, AS & Dubochet, J (2008) Analysis of cryo-electron microscopy images does not support the existence of 30-nm chromatin fibers in mitotic chromosomes in situ. Proc Natl Acad Sci 105, 1973219737.
Engelhardt, M (2004). Condensation of chromatin in situ by cation-dependent charge shielding and aggregation. Biochem Biophys Res Com 324, 12101214.
Grigoryev, SA & Woodcock, CL (2012). Chromatin organization—the 30 nm fiber. Exp Cell Res 318, 14481455.
Hayashihara, K, Uchiyama, S, Kobayashi, S, Yanagisawa, M, Matsunaga, S & Fukui, K (2008). Isolation method for human metaphase chromosomes. Protoc Exchange doi:10.1038/nprot.2008.166.
Herzog, M & Soyer, M (1983). The native structure of dinoflagellate chromosomes and their stabilization by Ca2+ and Mg2+ cations. Eur J Cell Biol 30, 3341.
Levi-Setti, R, Gavrilova, KL & Rizzo, PJ (2008). Divalent cation distribution in dinoflagellate chromosomes imaged by high-resolution ion probe mass spectrometry. Eur J Cell Biol 87, 963976.
Nishino, Y, Eltsov, M, Joti, Y, Ito, K, Takata, H, Takahashi, Y, Hihara, S, Frangakis, AS, Imamoto, N, Ishikawa, T & Maeshima, K (2012). Human mitotic chromosomes consist predominantly of irregularly folded nucleosome fibres without a 30-nm chromatin structure. EMBO J 31, 16441653.
Phengchat, R, Takata, H, Morii, K, Inada, N, Murakoshi, H, Uchiyama, S & Fukui, K (2016). Calcium ions function as a booster of chromosome condensation. Sci Rep 6, 38281. DOI: 10.1038/srep38281.
Poirier, MG, Monhait, T & Marko, JF (2002). Reversible hypercondensation and decondensation of mitotic chromosomes studied using combined chemical-micromechanical techniques. J Cell Biochem 85, 422434.
Strick, R, Strissel, PL, Gavrilov, K & Levi-Setti, R (2001). Cation-chromatin binding as shown by ion microscopy is essential for the structural integrity of chromosomes. J Cell Biol 155, 899910.
Uchiyama, S, Kobayashi, S, Takata, H, Ishihara, T, Sone, T, Matsunaga, S & Fukui, K (2004). Protein composition of human metaphase chromosomes analyzed by two-dimensional electrophoreses. Cytogenet Genome Res 107, 4954.
Uchiyama, S, Kobayashi, S, Takata, H, Ishihara, T, Hori, N, Higashi, T, Hayashihara, K, Sone, T, Higo, D, Nirasawa, T, Takao, T, Matsunaga, S & Fukui, K (2005). Proteome analysis of human metaphase chromosomes. J Biol Chem 280, 1699417004.
Visvanathan, A, Ahmed, K, Even-Faitelson, L, Lleres, D, Bazett-Jones, DP & Lamond, A (2013). Modulation of higher order chromatin conformation in mammalian cell nuclei can be mediated by polyamines and divalent cations. PLoS ONE 8, e67689.