Changes of nuclear structure induced by increasing temperatures

Despite the recent improvement in understanding the higher-order structure of chromatin fibers, the organization of interphase chromosomes in specific nuclear domains emerged only recently and it is still controversial. This study took advantage of an integrated approach using complementary techniques in order to investigate the structure and organization of chromatin in interphase nucleus. Native CHO-K1 cells were progressively heated from 310 K to 410 K and the effects of increasing temperatures on nuclear chromatin were analyzed in situ by means of cytometric and calorimetric techniques. Distribution and organization of chromatin domains were analyzed by Fluorescence microscopy, while the mean condensation of nuclear chromatin was measured by Differential scanning calorimetry. The results show as changes of nuclear structures (envelope and matrix, namely) affect significantly organization and condensation of in situ chromatin. Moreover when volume is modified by an external force (the temperature gradient in our case) we observe significant alterations of chromatin structure. These data are in accordance with the hypothesis of an inverse relationship between nuclear volume and chromatin condensation.     

Chromatin of Trypanosoma cruzi: in situ analysis revealed its unusual structure and nuclear organization

Chromatin of Trypanosoma cruzi is known to be organized in classical nucleosomal filaments, but surprisingly, these filaments do not fold in visible chromosomes and the nuclear envelope is preserved during cell division. Our hypothesis about the role of chromatin structure in regulating gene expression and, more generally, cell functioning, pressed us to verify if chromatin organization is modulated during the parasite life-cycle. To this end, we analyzed in situ the fine structural organization of T. cruzi chromatin by means of an integrated biophysical approach, using differential scanning calorimetry and fluorescence microscopy. We observed that logarithmic forms exhibit a less condensed chromatin with respect to the stationary ones. Thermal analysis revealed that parasite chromatin is organized in three main levels of condensation, barring from the polynucleosomal filament till to superstructured fibers. Besides, the fluorescence images of nuclei showed a characteristic chromatin distribution, with defined domains localized near to the nuclear envelope. While in stationary parasites, these regions are highly condensed, in logarithmic forms they unfold by extending themselves toward the center of nucleus. These observations suggest that, in comparison with higher eukaryotes, in T. cruzi the nuclear envelope plays an unusual and pivotal role in interphase and in mitosis.     

Chromatin Dynamics in Interphase Nuclei and Its Implications for Nuclear Structure

Translational dynamics of chromatin in interphase nuclei of living Swiss 3T3 and HeLa cells was studied using fluorescence microscopy and fluorescence recovery after photobleaching. Chromatin was fluorescently labeled using dihydroethidium, a membrane-permeant derivative of ethidium bromide. After labeling, a laser was used to bleach small (~0.4 μm radius) spots in the heterochromatin and euchromatin of cells of both types. These spots were observed to persist for >1 h, implying that interphase chromatin is immobile over distance scales 0.4 μm. Over very short times (<1 s), a partial fluorescence recovery within the spots was observed. This partial recovery is attributed to independent dye motion, based on comparison with results obtained using ethidium homodimer-1, which binds essentially irreversibly to nucleic acids. The immobility observed here is consistent with chromosome confinement to domains in interphase nuclei. This immobility may reflect motion-impeding steric interactions that arise in the highly concentrated nuclear milieu or outright attachment of the chromatin to underlying nuclear substructures, such as nucleoli, the nuclear lamina, or the nuclear matrix.     

Large-scale chromatin structural domains within mitotic and interphase chromosomes in vivo and in vitro

Higher-order chromatin structural domains approximately 130 nm in width are observed as prominent components of both Drosophila melanogaster and human mitotic chromosomes using buffer conditions which preserve chromosome morphology as determined by light microscopic comparison with chromosomes within living cells. Spatially discrete chromatin structural domains of similar size also exist as prominent components within interphase nuclei prepared under equivalent conditions. Examination of chromosomes during the anaphase-telophase transition suggests that chromosomes decondense largely through the progressive straightening or uncoiling of these large-scale chromatin domains. A quantitative analysis of the size distribution of these higher-order domains in telophase nuclei indicated a mean width of 126±36 nm. Three-dimensional views using stereopairs of chromosomes and interphase nuclei from 0.5 m thick sections suggest that these large-scale chromatin domains consist of 30 nm fibers packed by tight folding into larger, linear, fiber-like elements. Reduction in vitro of either polyamine or divalent cation concentrations within two different buffer systems results in a loss of these large-scale domains, with no higher-order chromatin organization evident above the 20–30 nm fiber. Under these conditions the DNA distribution within mitotic chromosomes and interphase nuclei appears significantly diffuse relative to the appearance by light microscopy within living cells, or, by electron microscopy, within cells fixed directly without permeabilization in buffer. These results suggest that these large-scale chromatin structural domains are fundamental elements of chromosome architecture in vivo.     

Location of nuclear antigen(s) recognized by DSB389 MAb, a monoclonal antibody against desmin, observed by confocal laser scanning fluorescence microscopy

The location of antigen(s) of DSB389 MAb, a monoclonal antibody which was raised against an intermediate filament protein, desmin, was investigated in HeLa S3 cells by indirect immuno-electron microscopy and by confocal laser scanning fluorescence microscopy. At interphase, the antigen(s) locates mainly in the intra-nuclear space adjacent to chromatin and sometimes near the nuclear periphery. At mitosis, they locate at the periphery of the chromosomes -first at each chromosome, then around the mass of chromosomes. The antigen(s) are thought to be a component of one type of nuclear matrix which is present outside both chromatin and chromosomes and which has some structural role(s) in organizing them in the nucleus or in the nuclear region.     

Super-resolution fluorescence microscopy as a tool to study the nanoscale organization of chromosomes

Chromatin organization spans a wide range of structural complexity. Substructures at the 10-200nm scale are poorly characterized, especially in living cells, due to the limitations of electron microscopy and standard optical microscopy. Recently developed super-resolution fluorescence microscopy methods represent an exciting opportunity to access those substructures, and recent progress with these techniques has yielded insights into chromatin organization at different condensation stages. Recent studies have focused on confronting the challenges that are specific to chromatin super-resolution imaging, such as the high packing density of mitotic chromosomes and difficulties in interpreting interphase chromatin images. Building on these first results and with ongoing rapid technical advances in super-resolution fluorescence imaging there is great potential to uncover new features with unprecedented detail.     

Common pathway of chromosome condensation in Mammalian cells

Chromatin folding in the interphase nucleus is not known. We compared the pattern of chromatin condensation in Indian muntjac, Chinese hamster ovary, murine pre B, and K562 human erythroleukemia cells during the cell cycle. Fluorescent microscopy showed that chromosome condensation follows a general pathway. Synchronized cells were reversibly permeabilized and used to isolate interphase chromatin structures. Based on their structures two major categories of intermediates were distinguished: (1) decondensed chromatin and (2) condensed chromosomal forms. (1) Chromatin forms were found between the G1 and mid-S phase involving veil-like, supercoiled, fibrous, ribboned structures; (2) condensing chromosomal forms appeared in the late-S, G2, and M phase, including strings, chromatin bodies, elongated pre-chromosomes, pre-condensed chromosomes, and metaphase chromosomes. Results demonstrate that interphase chromosomes are clustered in domains; condensing interphase chromosomes are linearly arranged. Our results raise questions related to telomer sequences and to the chemical nature of chromosome connectivity.     

SCFSlimb ubiquitin ligase suppresses condensin II–mediated nuclear reorganization by degrading Cap-H2

Condensin complexes play vital roles in chromosome condensation during mitosis and meiosis. Condensin II uniquely localizes to chromatin throughout the cell cycle and, in addition to its mitotic duties, modulates chromosome organization and gene expression during interphase. Mitotic condensin activity is regulated by phosphorylation, but mechanisms that regulate condensin II during interphase are unclear. Here, we report that condensin II is inactivated when its subunit Cap-H2 is targeted for degradation by the SCF^Slimb ubiquitin ligase complex and that disruption of this process dramatically changed interphase chromatin organization. Inhibition of SCF^Slimb function reorganized interphase chromosomes into dense, compact domains and disrupted homologue pairing in both cultured Drosophila cells and in vivo, but these effects were rescued by condensin II inactivation. Furthermore, Cap-H2 stabilization distorted nuclear envelopes and dispersed Cid/CENP-A on interphase chromosomes. Therefore, SCF^Slimb-mediated down-regulation of condensin II is required to maintain proper organization and morphology of the interphase nucleus.     

Interphase chromosomes undergo constrained diffusional motion in living cells

Background: Structural studies of fixed cells have revealed that interphase chromosomes are highly organized into specific arrangements in the nucleus, and have led to a picture of the nucleus as a static structure with immobile chromosomes held in fixed positions, an impression apparently confirmed by recent photobleaching studies. Functional studies of chromosome behavior, however, suggest that many essential processes, such as recombination, require interphase chromosomes to move around within the nucleus.Results: To reconcile these contradictory views, we exploited methods for tagging specific chromosome sites in living cells of Saccharomyces cerevisiae with green fluorescent protein and in Drosophila melanogaster with fluorescently labeled topoisomerase ll. Combining these techniques with submicrometer single-particle tracking, we directly measured the motion of interphase chromatin, at high resolution and in three dimensions. We found that chromatin does indeed undergo significant diffusive motion within the nucleus, but this motion is constrained such that a given chromatin segment is free to move within only a limited subregion of the nucleus. Chromatin diffusion was found to be insensitive to metabolic inhibitors, suggesting that it results from classical Brownian motion rather than from active motility. Nocodazole greatly reduced chromatin confinement, suggesting a role for the cytoskeleton in the maintenance of nuclear architecture.Conclusions: We conclude that chromatin is free to undergo substantial Brownian motion, but that a given chromatin segment is confined to a subregion of the nucleus. This constrained diffusion is consistent with a highly defined nuclear architecture, but also allows enough motion for processes requiring chromosome motility to take place. These results lead to a model for the regulation of chromosome interactions by nuclear architecture.     

Interphase chromosome structures of human cells

Major intermediates of chromosome condensation in erythroleukemia K562 cells are presented. Interphase chromatin structures became visible after reversal of permeabilization. Large-scale chromatin structures and the development of individual interphase chromosomes were observed by fluorescence microscopy. In the linear arrangement the following major intermediates of K562 chromatin condensation could be distinguished: (1) the most decondensed chromatin veil, (2) chromatin ribbon, (3) chromatin funnel, a new intermediate regarded as the earliest visible form of interphase chromosomes, (4) chromatin body, (5) 300 nm chromatin fiber, (6) u, v, or s forms of chromosomes, and (7) linear chromosomes. The observations made in nuclei of K562 cells conform to the model of helical coil chromosome condensation.     

Immunocytochemical localization of chromatin regions UV-microirradiated in S phase or anaphase. Evidence for a territorial organization of chromosomes during cell cycle of cultured Chinese hamster cells

Chinese hamster cells (M3-1 line) in S phase were laser-UV-microirradiated (lambda, 257 nm) at a small site of the nucleus. Cells were fixed either immediately thereafter or in subsequent stages of the cell cycle, including prophase and metaphase. The microirradiated chromatin was visualized by indirect immunofluorescence microscopy using antibodies specific for UV-irradiated DNA. During the whole post-incubation period (4-15 h) immunofluorescent labelling was restricted to a small part of the nucleus (means, 4.5% of the total nuclear area). In mitotic cells segments of a few chromosomes only were labelled. Following microirradiation of chromosome segments in anaphase, immunofluorescent labelling was observed over a small part of the resulting interphase nucleus. A territorial organization of interphase chromosomes, i.e. interphase chromosomes occupying distinct domains, has previously been demonstrated by our group for the nucleus of Chinese hamster cells in G1. Our present findings provide evidence that this organization pattern is maintained during the entire cell cycle.     

The positioning of rye homologous chromosomes added to wheat through the cell cycle in somatic cells untreated and treated with colchicine

The arrangement of chromosome pairs 5RL and 7R added to the wild type and the ph1b mutant line of hexaploid wheat are analyzed in 2N somatic root tip cells during the cell cycle relative to the arrangement that chromosomes 5RL show in 4N tapetal cells produced after colchicine treatment. Both homologous chromosome pairs are identified at interphase and mitosis by fluorescence in situ hybridization. In nuclei at interphase, chromosomes appear as discrete domains that show the Rabl orientation. Homologous chromosomes are predominantly non-associated and their positioning seems not to be influenced by the Ph1 gene that suppresses homoeologous meiotic pairing. This pattern of arrangement contrasts with the high level of somatic pairing that sister chromosomes show in the interphase that follows chromosome duplication induced by colchicine. Disruption of pairing observed in some 4N nuclei is produced at c-anaphase which suggests no topological redistribution of homologues during conformation of the new nucleus. Homologous chromosomes show no predominant arrangement in ellipsoidal metaphase plates, which contrasts with the preferential opposite location of homologues in human prometaphase rosettes. Differences between chromosomes in the variation of the length through the cell cycle and in the chromatin morphology when the Ph1 is absent suggest different patterns of chromatin condensation in both chromosomes.     

INDUCTION OF PROPHASE IN INTERPHASE NUCLEI BY FUSION WITH METAPHASE CELLS

Fusion of an interphase cell with a metaphase cell results in profound changes in the interphase chromatin that have been called "chromosome pulverization" or "premature chromosome condensation" In addition to the usual light microscopy, the nature of the changes has been investigated in the present study with electron microscopy and biochemical techniques Metaphase and interphase cells were mixed and fused at 37°C by means of ultraviolet-inactivated Sendai virus. After cell fusion, morphological changes in interphase nuclei occurred only in binucleate cells which contained one intact set of metaphase chromosomes Irrespective of the nuclear stage at the time of cell fusion, the morphologic changes that occurred 5–20 min later simulated very closely a sequence of events that characterizes the normal G₂-prophase transition. Radioautography revealed that, late in the process, substantial amounts of RNA and probably protein were transferred from the interphase nucleus into the cytoplasm of fused cells. Thus, the findings indicate the existence in metaphase cells of factor(s) which are capable of initiating biochemical and morphological events in interphase nuclei intrinsic to the normal mitotic process.