Experimental visualization of chromoneme as one of the higher levels 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 microm, 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 chromoeneme 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 chromosome 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 photosensbilizer, 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 their general pattern of packaging in in the chromatic 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.     

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.     

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.     

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.     

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.     

Localization of anti-topoisomerase IIα antibodies under normal conditions and after artificial chromosome condensation and decondensation

By means of immunofluorescence method, localization of DNA-topoisomerase IIα (Topo IIα) in interphase nuclei and chromosomes at different stages of mitosis was studied in situ under normal conditions and after treatment with condensing and decondensing solutions. In non-isolated mitotic M-HeLa cell chromosomes, Topo IIα was uniformly distributed along chromatids after fixation and permeabilization in situ. After treatment of cells with decondensing solutions (10 mM Tris; 0.1 mM CaCl₂ in 10 mM Tris; 0.3 mM CaCl₂ in 10 mM Tris; 15% DMEM; 75 mM KCl), Topo IIα was evenly distributed along chromatids in prophase, prometaphase and metaphase; its concentration was the highest in the pericentromere region. After treatment of cells with condensing solutions containing 0.7 mM, 1 mM, 2 mM or 3 mM CaCl₂ in 10 mM Tris, Topo IIα was not detected in prophase, metaphase and anaphase. However, in late telophase anti-Topo IIα antibodies were found in reforming nuclei under identical conditions. After sequential treatment with condensing and decondensing solutions, the distribution patterns of Topo IIα in chromosomes were the same as after treatment with only decondensing solutions. In anaphase and telophase, Topo IIα was evenly distributed along chromatids, while in prophase, prometaphase and metaphase it was predominantly localized in the pericentromere region. After the treatment of cells with condensing solutions chromosome staining was not observed, apparently due to “masking” of binding sites for anti-Topo IIα antibodies. Homogenous distribution of Topo IIα along chromatids in non-isolated chromosomes was preserved after the treatment of cells with hypotonic solutions; however, under these conditions Topo IIα concentration was higher in centromeres.     

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     

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.     

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.     

红翅皱膝蝗减数分裂染色体的螺旋与轴结构

用低渗处理和苯酚品红染色,在经过卡诺液(甲醇3∶冰醋酸1)固定和未经固定的红翅皱膝蝗减数分裂染色体上都看到了螺旋结构。观察和测量结果表明,每条染色单体都是由430nm左右的染色线螺旋形成的。由染色线到染色体的压缩率为4∶1。低渗处理后固定的材料经过银染,则显示了染色体轴结构。同样,未经低渗处理直接固定的材料银染时也出现了轴结构。银染的轴结构位于每个染色单体的中央,并贯穿整个染色单体。在光镜下,这个轴并不是直径均一的棒状结构,而似乎是由许多大小相近的颗粒相连而成。本文对染色体结构的有关模型、骨架和轴结构的真实性以及轴和螺旋的关系等问题进行了讨论。     

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.     

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.     

Chromatin structure during the prereplicative phases in the life cycle of mammalian cells

The object of this study was to determine the kinetics of chromosome decondensation during the G₁ period of the HeLa cell cycle. HeLa cells synchronized in the G₁ period following the reversal of mitotic block were fused with Colcemid-arrested mitotic HeLa cells at 1.5, 3, 5, and 7 h after the reversal of N₂O block. The resulting prematurely condensed chromosomes (PCC) were classified into six categories depending on the degree of their condensation. The frequency of occurrence of each category was plotted as a function of time after mitosis. The results of this study indicate that the process of chromosome decondensation, initiated during the telophase of mitosis continues throughout the G₁ period without any interruption, thus the chromatin reaches an ultimate state of decondensation by the end of G₁ period, when DNA synthesis is initiated.     

Superstructures of wet inactive chromatin and the chromosome surface

Superpacking of chromatin and the surface features of metaphase chromosomes have been studied by SiO replication of wet, unstained, and unfixed specimens in an exceedingly thin (≤ 1 nm) aqueous layer, keeping them wet. Hydrophilic Formvar substrates allow controlled thinning of the aqueous layer covering the wet specimens. Whole mounts of chromatin and chromosomes were prepared by applying a microsurface spreading method to swollen nuclei and mitotic cells at metaphase. The highest level of nucleosome folding of the inactive chromatin in chicken erythrocytes and rat liver nuclei is basically a second-order superhelical organization (width 150–200 nm, pitch distance 50–150 nm) of the elementary nucleosome filament. In unfavorable environments (as determined by ionic agents, fixative, and dehydrating agents) this superstructure collapses into chains of superbeads and beads. Formalin (10%) apparently attacks at discrete sites of chromatin, which are then separated into superbeads. The latter consist of 4–6 nucleosomes and seemingly correspond to successive turns of an original solenoidal coil (width 30–35 nm), which forms the superhelical organization. When this organization is unfolded, eg, in 1–2 mM EDTA, DNAse-sensitive filaments (diameter 1.7 nm) are seen to be wrapped around the nucleosomes. The wet chromosomes in each metaphase spread are held to each other by smooth microtubular fibers, 20–30 nm in diameter. Before they enter into a chromsome, these fibers branch into 9–13 protofilaments, each 5 nm wide. The chromosome surface contains a dense distribution of subunits about 10–25 nm in diameter. This size distribution corresponds to that of nucleosomes and their superbeads. Distinct from this beaded chromosome surface are several smooth, 23–30-nm-diameter fibers, which are longitudinal at the centromere and seem to continue into the chromatid structure. The surface replicas of dried chromosomes do not show these features, which are revealed only in wet chromosomes.     

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.     

The integrity of the histone-DNA complex in chromatin fibres is not necessary for the maintenance of the shape of mitotic chromosomes

In this study we addressed the question of whether scaffold structures produced from purified mitotic chromosomes are an artefact of dehistonization, and whether the integrity of the chromatin fibres is necessary for the maintenance of the well-known shape of mitotic chromosomes. Purified mitotic chromosomes from Friend erythroleukemia cells were treated either with increasing NaCl concentrations up to 500 mM, or with 6 M urea in the presence or absence of 10 mM 2-mercaptoethanol. The main criterion for the intactness of the overall chromosome shape as seen by electron microscopy was the characteristic X-or U-like appearance with clearly discernable chromatid axes. Histone H1 is known to be essential for the integrity of chromatin fibres. Its removal in sucrose gradients containing 500 mM NaCl did not lead to loss of the overall chromosome shape. However, treatment of chromosomes in sucrose gradients containing 10 mM 2-mercaptoethanol and 6 M urea led to loss of the structure probably due to dissociation (or denaturation) of shape-determining (scaffolding) components. Under these conditions most of the histones remained bound to the chromosomes, and the fibres in this chromatin material, after removal of excess urea and 2-mercaptoethanol, still showed condensation of the nucleosome filaments into the characteristic fibre structures upon increasing ionic strength. Our observations are compatible with the model that specific non-histone components, independently of histone-DNA interactions, organize or stabilize the structure of metaphase chromosomes.     

Mitotic Chromosome Structure: Reproducibility of Folding and Symmetry between Sister Chromatids

Mitotic chromosome structure and pathways of mitotic condensation remain unknown. The limited amount of structural data on mitotic chromosome structure makes it impossible to distinguish between several mutually conflicting models. Here we used a Chinese hamster ovary cell line with three different lac operator-tagged vector insertions distributed over an ∼1 μm chromosome arm region to determine positioning reproducibility, long-range correlation in large-scale chromatin folding, and sister chromatid symmetry in minimally perturbed, metaphase chromosomes. The three-dimensional positions of these lac operator-tagged spots, stained with lac repressor, were measured in isolated metaphase chromosomes relative to the central chromatid axes labeled with antibodies to topoisomerase II. Longitudinal, but not axial, positioning of spots was reproducible but showed intrinsic variability, up to ∼300 nm, between sister chromatids. Spot positions on the same chromatid were uncorrelated, and no correlation or symmetry between the positions of corresponding spots on sister chromatids was detectable, showing the absence of highly ordered, long-range chromatin folding over tens of mega-basepairs. Our observations are in agreement with the absence of any regular, reproducible helical, last level of chromosome folding, but remain consistent with any hierarchical folding model in which irregularity in folding exists at one or multiple levels.     

Molecular mechanisms of the chromosome condensation and decondensation cycle in mammalian cells

The chromosomes undergo a condensation-decondensation cycle within the life cycle of mammalian cells. Chromosome condensation is a complex and critical event that is necessary for the equal distribution of genetic material between the two daughter cells. Although chromosome condensation-decondensation and segregation is mechanistically complex, it proceeds with high fidelity during the eukaryotic cell division cycle. Cell fusion studies have indicated the presence of chromosome condensation factors in mammalian cells during mitosis. If extracts from mitotic cells are injected into immature oocytes of Xenopus laevis, they induce meiotic maturation (i.e. germinal vesicle breakdown and chromosome condensation) within 2–3 hours. Recently, we showed that the maturation-promoting activity of the mitotic cell extracts is inactivated by certain protein factors present in cells during the G₁ period. The activity of the G₁ factors coincides with the process of chromosome decondensation that begins at telophase and continues throughout the G₁ period. These studies have revealed that the mitotic factors and the G₁ factors play a pivotal role in the regulation of condensation and decondensation of chromosomes. Furthermore, our studies strongly suggest that nonhistone protein phosphorylation and dephosphorylation may mediate chromosome condensation and decondensation, respectively.