Experimental Visualization of Chromoneme as a Higher Level of Chromatin Compactization in the Mitotic Chromosome

We succeeded to visualize the chromoneme or a filamentous chromatin structure, with the mean thickness 0.1–0.2 μm, as a higher level of chromatin compactization in animal and plant cells at different stages of chromosome condensation at mitotic prophase and during chromatid decondensation at telophase. Under the natural conditions, chromoneme elements are not detected in the most condensed chromatin of metaphase chromosomes on ultrathin sections. We studied the ultrastructure and behavior of the chromatin of mitotic chromosomes in situ in cultured mouse L-197 cells under the conditions selectively demonstrating the chromoneme structure of the mitotic chromosomes in the presence of Ca²⁺. Loosely packaged dense chromatin bands, ca. 100 nm in diameter, chromonemes, were detected in chromosome arms in a solution containing 3 mM CaCl₂. When transferred in a hypotonic solution containing 10 mM tris-HCl, these chromosomes swelled, lost the chromoneme level of structure, and rapidly transformed in loose aggregates of elementary DNP fibrils, 30 nm in diameter. After this decondensation in the low ionic strength solution, the chromoneme structure of mitotic chromosomes was restored when they were transferred in a Ca₂₊ containing solution. The morphological characteristics of the chromoneme and pattern of its packaging in the chromosome were preserved. However, when the mitotic cells with chromosomes, in which the chromoneme structure was visualized with the help of 3 mM CaCl₂, were treated with a photosensitizer, ethidium bromide, and illuminate with a light with the wavelength 460 nm, chromatic decondensation under the hypotonic solution was not observed. The chromoneme elements in a stabilized chromatin of the mitotic chromosome preserved specific interconnection and the general pattern of their packaging in the chromatid was also preserved. The chromoneme elements in the chromosomes stabilized by light preserved their density and diameter even in a 0.6 M NaCl solution, which normally leads to chromoneme destruction. An even more rigid treatment of the stabilized chromosomes with a 2 M NaCl solution, which normally fully decondenses the chromosomes, made it possible to detect a 3D reticular skeleton devoid of any axial structures. __________ Translated from Ontogenez, Vol. 36, No. 5, 2005, pp. 323–332. Original Russian Text Copyright ? 2005 by Burakov, Tvorogova, Chentsov.     

Stabilization of chromonema--one of the highest compactization levels--by visible light in the presence of ethidium bromide

This paper deals with the ultrastructure and behavior of interphase chromatin and metaphase chromosomes of L-197 culture cells under experimental conditions, which help to reveal the chromonemal level of chromosomal structure after the treatment of living cells with 0.1% Triton X-100 and 3 mM CaCl2. In these conditions, the chromonemata can be seen as dense chromatin fibers with thickness about 100 nm. Such chromosomes, whose chromonemal substructure after the treatment with hypotonic solution (10 mM Tris-HCl), look like loose chromosomal bodies composed of elementary 30 nm DNP fibrils. On the other hand, if chromosomes, in which chromonemal levels were revealed by 3 mM CaCl2, were treated with etidium bromide and then illuminated by light with length wave about 460 nm, no chromosomal decondensation in hypotonic conditions is observed. Chromonemata in chromosomes stabilized by light retain their density and dimensions. It is very important that chromonemata in stabilizated chromatin of metaphase chromosome keep specific connections between themselves and also general trend in their composition inside the chromosome. Thus, we have found conditions for observation of chromonemal elements in metaphase chromosome, providing the possibility for future three-dimensional investigation of chromonema packing in mitotic chromosomes.     

小鼠生精细胞染色质与染色体构筑的扫描电镜研究（简报）

It remains unclear about the intermediate construction of chromosome due to its highly compact nature and the limitation in methods. The present study was designed to investigate the construction of chromatin and mitotic chromosome in situ with scanning electron microscopy. Mouse testes were selected as the material, because of in which the spermatogenic cells divide actively and successively to form the sperm. Such a feature would be able to study the structure of mammalian chromatin and chromosomes along with the change of nuclear cycle. The animal were perfused with 200 ml of 0.075 mol/L KCl hypotonic solution to remove blood and placed for 15-20 min on ice followed by 0.5% glutaraldehyde and 0.5% formaldehyde for fixing. Through treated by the routine process of fractured and freeze dried with t-butyl alcohol, the specimens were then coated with a 3 nm thick platinum and observed with Hitachi S-430 scanning electron microscopy. It was found that the hypotonic treatment with 0.075 mol/L KCl solution was suit for demonstrating the nuclear structure, when the organelles were well preserved. The chromatin fibers of 10-30 nm and 80-125 nm in diameter could be recognized in the interphase nuclei, which were arranged losely at the region of euchromatin, and folded with each other into chromatin masses at the region of heterochromatin, while the chromatin fibers with the diameter of 80-125 nm often could be viewed on the mitotic chromosomes. Since its presence in interphase nuclei and mitotic chromosomes, it was considered that the chromatin fibers with 80-125 nm in diameter might play a role in the condensation of chromosome, serve as a type of the intermediate structure.     

小鼠生精细胞染色质与染色体构筑的扫描电镜研究(简报)

正常情况下,染色质和染色体在细胞内呈高度致密状态,在光镜和透射电镜下常呈浓染的斑块状。由于方法学上的困难,至今对染色质乃至染色体的微细结构,仍缺乏清楚的了解。特别是关于染色质如何凝缩形成染色体方面,现仍存在有争论。扫描电镜的冷冻割断技术,曾被用于对游离细胞间期核染色质的观察,并取得了较好的     

Chromonema and chromomere

A study of ultrathin sections of normal Chinese hamster cells and cells treated with decreasing concentrations of bivalent cations (Ca²⁺ and Mg²⁺) in situ revealed several discrete levels of compaction of DNA-nucleoprotein (DNP) fibrils in mitotic chromosomes and the chromatin of interphase nuclei. At concentrations ranging from 3 mM CaCl₂ and 1 mM MgCl₂ to ten times less, the chromosomes are found to contain fibrous elements (chromonemata) about 100 nm in diameter. As Ca²⁺ concentration is gradually decreased to 0.2–0.1 mM, the chromosomes decondense into a number of discrete chromatin structures, the chromomeres. As decondensation proceeds, these chromomeres acquire a rosettelike structure with DNP fibrils radiating from an electron-dense core. Upon complete decondensation of chromosomes, individual chromomeres persist only in the centromeric regions. The following levels of DNP compaction in mitotic chromosomes are suggested: a 10-nm nucleosomal fibril, a 25-nm nucleomeric fibril, and the chromonema, a fibrous structure, about 100 nm in diameter, composed of chromomeres. Interphase nuclei also contain structures which are morphologically similar to the chromomeres of mitotic chromosomes.     

Ultrastructure of the mitotic chromosomes in pig embryonic kidney cells during their reversible artificial decondensation in vivo

Using methods of in vivo observation and ultrathin sectioning, it is shown that chromosomes of metaphase PE cells, previously treated with diluted Henk's solutions (70, 30 and 15%), undergo some structural transitions resulting in the formation of micronuclei. At the early stages of hypotonic treatment chromosomes are seen considerably swollen and losing the higher levels of organization, including the chromonema and chromomeres. The chromosomal bodies are formed by DNP fibers 10-25 nm in diameter making loops radiating from the central part of the chromatids. Chromosomes are capable of recondensing from this state by consecutive reconstitution of G-bands, chromomeres and the chromonema. The subsequent secondary decondensation of chromosomes is analogous to telophase decondensation at the normal mitosis, but it results in the formation of a great number of small nuclei (micronuclei). The chromatin structure in micronuclei as well as their ability to synthesize RNA and to replicate DNA show these effects to be reversible. It has been suggested that the loop organization of DNP may be essential for sustaining the structural integrity of the mitotic chromosome.     

Chromosome organization revealed upon the decondensation of telophase chromosomes inAllium

Chromosomes of root tip cells ofAllium cepa andAllium sativum were studied in early, middle and late telophase to examine the organization of mitotic chromosomes, taking advantage of the naturally occurring chromosome dispersion during the process of decondensation in telophase. Longitudinal and transverse sections of telophase chromosomes viewed under the transmission electron microscope showed that mitotic chromosomes inAllium were composed of helically coiled 400–550 nm chromatin fibres. In some regions of the longitudinal sections, these chromatin fibres were seen to be orientated parallel to one another but formed roughly a right angle to the long axis of the chromosome. In transverse sections, the telophase chromosome appeared to have a hollow centre encircled by the 400–550 nm chromatin fibre which in turn was a hollow tube structure formed by the coiling of a thinner fibre of 170–200 nm. In addition, cross views of chromatin fibres of 170–200 nm and 50–70 nm were also identified in telophase chromosome preparations. These two organizational levels of chromatin fibres also showed a hollow centre. The process of decondensation of telophase chromosomes is described, and some morphological characteristics associated with the activities of chromosome decondensation are analysed. Based on the observations made onAllium chromosomes in this study, various models of chromosome organization are discussed.     

Stabilization of non-histone proteins by copper ions does not alter chromatin properties of polythene chromosomes of <Emphasis Type="Italic">Chironomus plumosus</Emphasis>

Our previous study showed that the body of polythene chromosomes can be identified even after removal of all histones and DNA in the presence of 2 mM CuCl₂; this suggested that copper ions stabilized the bonds between non-histone proteins. In this study we tried to find out if copper ions bind with non-histone proteins reversibly or irreversibly. It is shown that the bodies of normal chromosomes and chromosomes stabilized by 2 mM CuCl₂ swell with partial disappearance of the banding pattern in a hypotonic solution (0.055 M NaCl) without copper ions. The selective removal of bivalent cations by 10 mM EDTA solution resulted in decondensation of normal polythene and stabilized chromosomes. The treatment of nuclear protein matrix of polythene chromosomes preparations with 10 mM EDTA resulted in the swelling of polythene chromosome body and disappearance of the banding pattern but their morphological organization maintained.     

Ultrastructural composition of nonhistone chromosomal skeleton at different stages of mitosis in situ

In interphase cells of the SPEV culture treated with Triton X-100, 2 M NaCl, and DNAse, in the presence of 2 mM CuCl₂, we clearly revealed a stabilized nuclear protein material (NPM) composed of a peripheral lamina, residual nucleolus, and internal fibrillar network. This network is formed by thin fibrils 10–20 nm in diameter, which are also revealed in the nonhistone matrix of mitotic chromosomes at all stages of mitosis. In mitotic chromosomes, NPM is represented as a network of the 10–20-nm-thick fibrils without any features of the central-axial structures. Beginning from the middle prophase, it is possible to see approached sister chromatids in contact with each other in certain sites, similar to centromeres. At these sites, the thickness of fibrils increases up to 40–50 nm, whereas the fibrils themselves are disposed more tightly; this structure can be seen in the chromosome until telophase. At the end of telophase, the decondensation of chromosomes and formation of two new nuclei whose NPM is analogous to NPM of usual interphase nucleus are observed. Thus, the NPM elements can perform the role of a skeleton in both the interphase nucleus and mitotic chromosomes.     

A differential staining technique for simultaneous visualization of mitotic spindle and chromosomes in mammalian cells

A new technique has been devised for staining the mitotic spindle in mammalian cells while preserving spindle structure and chromosome number. The cells are trypsinized and fixed with a 3:1 methanol:acetic acid solution containing 4 mM MgCl2 and 1.5 mM CaCl2 at room temperature. The cells are then placed on slides and treated with 5% perchloric acid before staining with a 10% acetic acid solution containing safranin O and brilliant blue R. The preserved spindles appear dark blue against a light cytoplasmic background with chromosomes stained bright red. Individual chromosomes and chromatids are clearly visible. Positioning of the chromosomes relative to the spindle apparatus is readily ascertained allowing easy study of mitotic spindle and chromosome behavior.     

The dynamics of the structural reconstruction of mitotic chromosomes after their artificial decondensation in vitro

The treatment of isolated metaphase chromosomes with 5 mM Tris buffer caused their decondensation into DNP fibers 10 nm in diameter. The following increase in CaCl2 concentration induced the transition of nucleosomic DNP fibers into DNP fibers 20 nM and 40-50 nM in diameter, and the recovery of the whole chromosomes. However, in the similar conditions, the typical chromosomes (threads about 100 nm thick), chromomeres and G-bands were not reconstructed. According to these data, we assume that DNP threads 40-50 nm in diameter may be artificial (i.e. "pseudochromonemes"). The treatment of isolated chromosomes with 0.35 and 0.6 M NaCl prevents from formation of nucleomeric and pseudochromomeric fibers, although bodies of chromosomes can be recovered after the removal of HMG and H1 proteins. These observations point to a high stability of chromosomal fasteners providing the structural integrity of mitotic chromosomes.     

Differential decondensation of mitotic chromosomes in SPEV tissue cultured cells induced by repeated hypotonic shock

A new method of differential decondensation of mitotic chromosomes has been proposed by means of repeated treatment of live cells with 15% Hanks' balanced salt solution. The procedure of cell treatment includes three stages: the first hypotonic shock, cultivation in isotonic medium, and the second hypotonic shock. As a result, after a standard methanol-acetic acid fixation and Giemsa staining some discrete Giemsa-positive globules are revealed in mitotic chromosomes. Such globules are symmetrically arranged in axial regions of sister chromatids. The comparative analysis of marker chromosomes has revealed a topological conformity of these globules to G-bands of chromosomes. It has been shown that it is the first hypotonic shock that triggers induction of structural modification of chromatin in interphase nuclei and in mitotic chromosomes. Of interest is the fact that the effect of the first shock is prolonged in time and is realized during at least one cell cycle, with the normal structure of mitotic chromosomes being restored after S-phase of the successive cell cycle.     

Reversible hypercondensation and decondensation of mitotic chromosomes studied using combined chemical-micromechanical techniques

We show that the chromatin in mitotic chromosomes can be drastically overcompacted or unfolded by temporary shifts in ion concentrations. By locally 'microspraying' reactants from micron-size pipettes, while simultaneously monitoring the size of and tension in single chromosomes, we are able to quantitatively study the dynamics of these reactions. The tension in a chromosome is monitored through observation and calibration of bending of the glass pipettes used to manipulate the chromosomes. For concentrations > 500 mM of NaCl and > 200 mM of MgCl2, we find that the initially applied tensions of approximately 500 pN relax to zero and that mitotic chromatin temporarily disperses in agreement with previous work (Maniotis et al. [1997] J. Cell. Biochem. 65:114-130). This unfolding occurs in about 1 s, and is reversible once the charge density is returned to physiological levels, if the exposure is not longer than approximately 1 min. Low concentrations of NaCl (< 30 mM) also induces a decrease in tension and increase in size. We observe this swelling to be isotropic in experiments on chromosomes under zero tension, a behavior inconsistent with the existence of a well-defined central chromosome 'scaffold'. By contrast 10 mM of divalent cations (MgCl2 and CaCl2) induces an extremely rapid and reversible increase in tension and a reduction in the size of mitotic chromosomes. Hexaminecobalt trichloride (trivalent cation) has the same effect as MgCl2 and CaCl2, except the magnitude of force increase and size change are much larger. Hexaminecobalt trichloride reduces mitotic chromosomes to 65% of their original volume, indicating that at least 1/3 of their apparent volume is aqueous solution. These results indicate that chromatin inside mitotic chromatids has a large amount of conformational freedom allowing dynamic unfolding and refolding and that charge interactions play a central role in maintaining mitotic chromosome structure.     

Proteolysis of mitotic chromosomes induces gradual and anisotropic decondensation correlated with a reduction of elastic modulus and structural sensitivity to rarely cutting restriction enzymes

The effect of nonspecific proteolysis on the structure of single isolated mitotic newt chromosomes was studied using chromosome elastic response as an assay. Exposure to either trypsin or proteinase K gradually decondensed and softened chromosomes but without entirely eliminating their elastic response. Analysis of chromosome morphology revealed anisotropic decondensation upon digestion, with length increasing more than width. Prolonged protease treatment resulted only in further swelling of the chromosome without complete dissolution. Mild trypsinization induced sensitivity of chromosome elasticity to five- and six-base-specific restriction enzymes. These results, combined with previous studies of effects of nucleases on mitotic chromosome structure, indicate that mild proteolysis gradually reduces the density of chromatin-constraining elements in the mitotic chromosome, providing evidence consistent with an anisotropically folded "chromatin network" model of mitotic chromosome architecture.     

A Differential Staining Technique for Simultaneous Visualization of Mitotic Spindle and Chromosomes in Mammalian Cells

A new technique has been devised for staining the mitotic spindle in mammalian cells while preserving spindle structure and chromosome number. The cells are trypsinized and fixed with a 3:1 methanobacetic acid solution containing 4 mM MgCl₂ and 1.5 mM CaCl₂ at room temperature. The cells are then placed on slides and treated with 5% perchloric acid before staining with a 10% acetic acid solution containing safranin O and brilliant blue R. The preserved spindles appear dark blue against a light cytoplasmic background with chromosomes stained bright red. Individual chromosomes and chromatids are clearly visible. Positioning of the chromosomes relative to the spindle apparatus is readily ascertained allowing easy study of mitotic spindle and chromosome behavior.     

Association of BAF53 with mitotic chromosomes

The conversion of mitotic chromosome into interphase chromatin consists of at least two separate processes, the decondensation of the mitotic chromosome and the formation of the higher-order structure of interphase chromatin. Previously, we showed that depletion of BAF53 led to the expansion of chromosome territories and decompaction of the chromatin, suggesting that BAF53 plays an essential role in the formation of higher-order chromatin structure. We report here that BAF53 is associated with mitotic chromosomes during mitosis. Immunostaining with two different anti-BAF53 antibodies gave strong signals around the DNA of mitotic preparations of NIH3T3 cells and mouse embryo fibroblasts (MEFs). The immunofluorescent signals were located on the surface of mitotic chromosomes prepared by metaphase spread. BAF53 was also found in the mitotic chromosome fraction of sucrose gradients. Association of BAF53 with mitotic chromosomes would allow its rapid activation on the chromatin upon exit from mitosis.     

Non-histone skeleton of mitotic chromosome in situ

Studying giant nuclei of Chironomus plumosus in situ (Makarov, Chentsov, 2010), we concluded that polythene chromosome structure appears after 2 M NaCl and DNase treatment in presence of 2 mM CuCl2. Cu2+ -ions may stabilize bonds between specific non-histone components, arranged into non-histone matrix of polythene chromosome. Here, we investigated the non-histone matrix of pig embryo mitotic chromosomes in situ, using 2 mM CuCl2-stabilization method. In 2 mM CuCl2-stabilized cells the residual chromosome body (non-histone matrix) could be visualized in every stage of mitosis. Mitotic chromosome non-histone matrix had the same reaction on preliminary hypotonic treatment as normal chromosome: different decondensation of non-histone material was observed. Topoisomerase IIalpha and SMC 1 had uniform localization inside chromosomal body and did not form any axial structures.     

Differences in the structural organization of the G- and R-bands detectable during the differential decondensation of chromosomes

The ultrastructure of G- and R-bands in differentially decondensed chromosomes of Chinese hamster was studied with a gradual decrease in CaCl2 concentration in the medium. The gradual reduction of CaCl2 concentration leads to the decondensation of compact G-bands into chromonemes, chromomeres and further into DNP-fibrils. In the complete local decondensation zones (R-bands), the DNP-fibril orientation is parallel to the chromosome longitudinal axis. These zones have no lateral loops or chromomeres. Thus, different chromosome regions corresponding to G- and R-bands possess different sensibility to the decondensing action. Following the complete decondensation in the calcium-free medium chromosomes can be "reconstructed" by adding Ca2+. The data obtained permit to suggest a "fastener" model of the mitotic chromosome organization in which the chromosome represents an hierarchy of discrete structures--G-bands, chromomeres, nucleomeres (superbeads) and nucleosomes. The structural integrity of these levels is supported by specific protein "fasteners".     

Mechanical continuity and reversible chromosome disassembly within intact genomes removed from living cells

Chromatin is thought to be structurally discontinuous because it is packaged into morphologically distinct chromosomes that appear physically isolated from one another in metaphase preparations used for cytogenetic studies. However, analysis of chromosome positioning and movement suggest that different chromosomes often behave as if they were physically connected in interphase as well as mitosis. To address this paradox directly, we used a microsurgical technique to physically remove nucleoplasm or chromosomes from living cells under isotonic conditions. Using this approach, we found that pulling a single nucleolus or chromosome out from interphase or mitotic cells resulted in sequential removal of the remaining nucleoli and chromosomes, interconnected by a continuous elastic thread. Enzymatic treatments of interphase nucleoplasm and chromosome chains held under tension revealed that mechanical continuity within the chromatin was mediated by elements sensitive to DNase or micrococcal nuclease, but not RNases, formamide at high temperature, or proteases. In contrast, mechanical coupling between mitotic chromosomes and the surrounding cytoplasm appeared to be mediated by gelsolin-sensitive microfilaments. Furthermore, when ion concentations were raised and lowered, both the chromosomes and the interconnecting strands underwent multiple rounds of decondensation and recondensation. As a result of these dynamic structural alterations, the mitotic chains also became sensitive to disruption by restriction enzymes. Ion-induced chromosome decondensation could be blocked by treatment with DNA binding dyes, agents that reduce protein disulfide linkages within nuclear matrix, or an antibody directed against histones. Fully decondensed chromatin strands also could be induced to recondense into chromosomes with pre-existing size, shape, number, and position by adding anti-histone antibodies. Conversely, removal of histones by proteolysis or heparin treatment produced chromosome decondensation which could be reversed by addition of histone H1, but not histones H2b or H3. These data suggest that DNA, its associated protein scaffolds, and surrounding cytoskeletal networks function as a structurally-unified system. Mechanical coupling within the nucleoplasm may coordinate dynamic alterations in chromatin structure, guide chromosome movement, and ensure fidelity of mitosis. J. Cell. Biochem. 65:114–130. © 1997 Wiley-Liss, Inc.