Dna organization in histone-free nuclei at various stages of rat spermatogenesis

Electron microscopy analysis of DNA organization in histone-depleted nuclei of rat spermatogonial cells was performed. It was shown that the rosette-like structures are one of the forms of the loop organization of the meiotic cell nuclear DNA. Ions of bivalent metal play an important role in stabilizing the rosette-like structures of meiotic cells. A scheme for the restructuring of the mitotic organization of nuclear DNA into the meiotic one is suggested.     

Structural-functional organization of DNA in the interphase nucleus. Structural aspects

The role of residual nuclear structures (structures persisting upon the treatment of nuclei with a non-ionic detergent, nucleases and 2 M NaCl) in the spatial organization of DNA in the interphase nucleus has been considered. Experimental works that have engendered the concept of loop level of DNA organization in the nucleus are discussed. A comparison is made of the loop-domain and rosette-like patterns of DNA organization in the interphase nucleus.     

Isolation of rosette-like structures from partially deproteinized chromatin in rat hepatocytes

The structure of partial deproteinized rat hepatocyte chromatin has been studied. Depending on the magnesium concentration the chromatin of isolated nuclei is present in the two conditions: diffuse (at 0-1.5 mM MgCl2) and condensed (at 2-5 mM MgCl2). The main components of nuclei with condensed chromatin are chromomers--globular structures about 100 nm in diameter. By treating such nuclei with heparin and dextransulfate one can observe a rosette-like structure with lateral loops having the following parameters: the length of the loops, 15-20 micron; the number of loops, 15-30. The rosette-like structures are sensitive to endogenous nuclease and DNase 1, but not to RNase. Pronase or higher concentration of polyanions give rise to unfolding of the rosette-like structures. The rosette structures cannot be isolated from the nuclei with diffuse chromatin. On the basis of these observations a hypothesis of chromatin structural organization in the interphase nucleus is proposed, and the connection of the rosette-like structures with some structural levels of chromatin organization is discussed.     

Comparative analysis of nucleotide sequences of chromosomal DNA isolated from nuclear envelopes and cores of rosette-like structures of murine interphase chromosomes]

Six DNA fragments of interphase chromosomes isolated from nuclear envelopes of murine hepatocytes were cloned and sequenced. Analysis of their structural-functional organization suggests that these fragments are highly specified protein-nonencoding fractions of a eukaryotic genome. In the evolutionary process, they appear already in archaebacteria and may be "ancestral" for DNA sequences involved in structuring chromosomal domains (rosette-like structures) of tissue-specific genes. In their composition, these fragments have nucleotide sequences homologous to the repeats of the SINE and LINE families and to the satellite DNA of murine centromeres.     

Interactions between the Su(Hw) and Mod(mdg4) proteins required for gypsy insulator function

The gypsy insulator is thought to play a role in nuclear organization and the establishment of higher order chromatin domains by bringing together several individual insulator sites to form rosette-like structures in the interphase nucleus. The Su(Hw) and Mod(mdg4) proteins are components of the gypsy insulator required for its effect on enhancer-promoter interactions. Using the yeast two-hybrid system, we show that the Mod(mdg4) protein can form homodimers, which can then interact with Su(Hw). The BTB domain of Mod(mdg4) is involved in homodimerization, whereas the C-terminal region of the protein is involved in interactions with the leucine zipper and adjacent regions of the Su(Hw) protein. Analyses using immunolocalization on polytene chromosomes confirm the involvement of these domains in mediating the interactions between these proteins. Studies using diploid interphase cells further suggest the contribution of these domains to the formation of rosette-like structures in the nucleus. The results provide a biochemical basis for the aggregation of multiple insulator sites and support the role of the gypsy insulator in nuclear organization.     

DNA-containing polygonal structures detected in somatic nuclei of Bursaria ovata during preparation to cryptobiosis

Supramolecular chromatin organization of the somatic nucleus (macronucleus--Ma) was studied in a free-living unicellular eukaryotic organism, the ciliate Bursaria ovata Beers 1952, at two late successive stages of its encystment (in the state of preparation to cryptobiosis). A modified Miller's method (Sergejeva et al., 1987) and the same technique in combination with high resolution DNA autoradiography were used. In chromatin spread preparations of Ma, not labeled by 3H-thymidine, numerous electron dense structures (rounded, stick-like and polygonal) were revealed, along with rarely occurring typical supramolecular chromatin structures, such as nucleosomic and not nucleosomic threads, nucleomeres, chromomeres, rosette-like looping chromatin, and electron dense chromonemes (Fig. 1). For DNA visualizing in the revealed polygonal structures, the vegetative cells (trophonts) of B. ovata were inoculated into the culture medium, containing 3H-thymidine and food (ciliates Paramecium caudatum). Here, the ciliates passed through 3-4 successive cell division cycles, thus progressively accumulating the radioactive DNA precursor in Ma. After washing the ciliates in 3H-thymidine-free culture medium, the process of their encystment was induced, and Ma were isolated by hand from the ciliates being at two late successive stages of encystment. Isolated Ma were dispersed in the low ionic solutions, as described elsewhere (Sergejeva et al., 1987). The carbon shadowed electron grids, that contained spread Ma preparations, were individually coated with photographic emulsion, according to the loop interference method (Angelier et al., 1976a; Bouteille, 1976). After a 6 month exposure at 4 degrees C, thymidine incorporation was revealed in fibril crowds, rosette-like structures (Fig. 2), and crystal-like plates of different size and morphology (Fig. 3). In all our experiments, non-specific localization of radioactive DNA precursor was not observed. The above data confirm undoubtedly our earlier assumption (Sergejeva et al., 1987; Sergejeva, Bobyleva, 1988) that the Ma chromatin of Bursaria may undergo crystallization during encystment, i.e. in the state of preparation to cryptobiosis. The present data enable us first to suggest that the looping rosette-like chromatin may be transformed into crystal-like structures ("exotic liquid crystal structures") by means of a peculiar loop packing within the limits of an individual resette (Fig. 2-4), these structural transformations taking place without any visible loop destruction. In this paper, we first describe new morphological types of polygonal plates, differing from those earlier reported elsewhere for the Ma of Bursaria (Sergejeva, Bobylova, 1988), and also from the plates earlier described in studies on liquid crystals both in vivo and in vitro (see: Gianonni et al., 1969; Lerman, 1974; Livolant, 1991: Leforestier et al., 1993, 1997, 1999). The technical approaches used in the present work enabled us to obtain, for the first time, a direct evidence of the presence of DNA in the crystallized structures of somatic nuclei of ciliates during their preparation to cryptobiosis, the DNA-containing polygonal structures being highly morphologically diverse. Further studies into the reasons of this diversity are needed.     

Postnatal development of the rabbit pineal gland. A light- and electron-microscopic study.

The development of the rabbit pineal gland has been studied by light and electron microscopy from the 1st to the 120th postnatal day. After 24 h of postnatal life, the pineal parenchyma is highly cellular, showing two identifiable cell types: pinealocytes I and II. Immature type II pinealocytes arrange either in cellular cords or clusters or forme rosette-like structures. At the 5th postnatal day, corticomedullar differentiation is established. Rosette-like structures and cellular cords are absent from the cortex. Along the postnatal period, nuclei of pinealocytes are set apart due to cytoplasmic widening and development of cell processes. These structures pervade the cellular cords and rosette-like structures formed by immature type II pinealocytes. Rosette-like structures are no longer seen beyond the 30th postnatal day, and cords of type II pinealocytes from the 90th postnatal day on. At this time, the rabbit pineal gland is considered to be histologically mature.     

An improved protocol that induces human embryonic stem cells to differentiate into neural cells in vitro

Human embryonic stem (ES) cells have the capacity for self-renewal and are able to differentiate into any cell type. However, obtaining high-efficient neural differentiation from human ES cells remains a challenge. This study describes an improved 4-stage protocol to induce a human ES cell line derived from a Chinese population to differentiate into neural cells. At the first stage, embryonic bodies (EBs) were formed in a chemically-defined neural inducing medium rather than in traditional serum or serum-replacement medium. At the second stage, rosette-like structures were formed. At the third stage, the rosette-like structures were manually selected rather than enzymatically digested to form floating neurospheres. At the fourth stage, the neurospheres were further differentiated into neurons. The results show that, at the second stage, the rate of the formation of rosette-like structures from EBs induced by noggin was 88+/-6.32%, higher than that of retinoic acid 55+/-5.27%. Immunocytochemistry staining was used to confirm the neural identity of the cells. These results show a major improvement in obtaining efficient neural differentiation of human ES cells.     

Dystroglycan is required for proper retinal layering

Dystroglycan (DG) is a transmembrane receptor linking the extracellular matrix to the internal cytoskeleton. Its structural function has been mainly characterized in muscle fibers, but DG plays signaling and developmental roles also in different tissues and cell types. We have investigated the effects of dystroglycan depletion during eye development of Xenopus laevis. We have injected a specific morpholino (Mo) antisense oligonucleotide in the animal pole of one dorsal blastomere of embryos at four cells stage. Mo-mediated loss of DG function caused disruption of the basal lamina layers, increased apoptosis and reduction of the expression domains of specific retinal markers, at early stages. Later in development, morphants displayed unilateral ocular malformations, such as microphtalmia and retinal delayering with photoreceptors and ganglion cells scattered throughout the retina or aggregated in rosette-like structures. These results recall the phenotypes observed in specific human diseases and suggest that DG presence is crucial at early stages for the organization of retinal architecture.     

Double-stranded DNA organization in bacteriophage heads: an alternative toroid-based model.

Studies of the organization of double-stranded DNA within bacteriophage heads during the past four decades have produced a wealth of data. However, despite the presentation of numerous models, the true organization of DNA within phage heads remains unresolved. The observations of toroidal DNA structures in electron micrographs of phage lysates have long been cited as support for the organization of DNA in a spool-like fashion. This particular model, like all other models, has not been found to be consistent will all available data. Recently we proposed that DNA within toroidal condensates produced in vitro is organized in a manner significantly different from that suggested by the spool model. This new toroid model has allowed the development of an alternative model for DNA organization within bacteriophage heads that is consistent with a wide range of biophysical data. Here we propose that bacteriophage DNA is packaged in a toroid that is folded into a highly compact structure.     

ARF6 Promotes the Formation of Rac1 and WAVE-Dependent Ventral F-Actin Rosettes in Breast Cancer Cells in Response to Epidermal Growth Factor

Coordination between actin cytoskeleton assembly and localized polarization of intracellular trafficking routes is crucial for cancer cell migration. ARF6 has been implicated in the endocytic recycling of surface receptors and membrane components and in actin cytoskeleton remodeling. Here we show that overexpression of an ARF6 fast-cycling mutant in MDA-MB-231 breast cancer-derived cells to mimick ARF6 hyperactivation observed in invasive breast tumors induced a striking rearrangement of the actin cytoskeleton at the ventral cell surface. This phenotype consisted in the formation of dynamic actin-based podosome rosette-like structures expanding outward as wave positive for F-actin and actin cytoskeleton regulatory components including cortactin, Arp2/3 and SCAR/WAVE complexes and upstream Rac1 regulator. Ventral rosette-like structures were similarly induced in MDA-MB-231 cells in response to epidermal growth factor (EGF) stimulation and to Rac1 hyperactivation. In addition, interference with ARF6 expression attenuated activation and plasma membrane targeting of Rac1 in response to EGF treatment. Our data suggest a role for ARF6 in linking EGF-receptor signaling to Rac1 recruitment and activation at the plasma membrane to promote breast cancer cell directed migration.     

High content,size and distribution of single-stranded DNA in the mitochondria of Chenopodium album (L.)

Mitochondrial (mt) DNA of higher plants is unique in its large size and complexity. We report here a hitherto unknownfeature, the presence of large quantities of single-stranded (ss) DNA. About 2.0-8.5% of the chromosomal mtDNA from a suspension culture (depending on the growth stage) and 6.5% of the chromosomal mtDNA from whole plants of Chenopodium album were found to be in ss form by dot-blot hybridization after neutral transfer. Similar amounts of ss mtDNA were observed by binding of the single-strand binding (SSB) protein of Escherichia coli under the electron microscope. Significantly less ssDNA was found in plastids of C. album and in E. coli cells. We observed ss regions between 100 and 22 800 bases distributed in the mt genome spaced from 0.5-100 kb apart. After pulsed-field gel electrophoresis (PFGE), the well-bound fraction of mtDNA (found to consist of circular, sigma-shaped and rosette-like molecules), contained the major part of ssDNA as opposed to the migrating linear molecules. Digestion of mtDNA by ss-specific nucleases followed by PFGE mobilized all well-bound DNA and correspondingly increased the quantity of migrating linear DNA molecules. The implications of ssDNA for the structural organization on plant mt genomes are discussed.     

Nuclear matrix involvement in sperm head structural organization

Summary— In the sperm nuclei the DNA is packaged into a highly condensed form and is not organized into nucleosome and solenoid but is bound and stabilized mainly by the protamines that arrange the DNA in an almost crystalline state. As demonstrated for somatic cells, the sperm DNA has been reported to be organized in loop domains attached to the nuclear matrix structures. However, the possible role of the sperm head matrix in maintaining the loop organization in absence of a typical nucleosomal structures has not been fully elucidated. By using in situ nick translation at confocal and electron microscope level, we analyzed the organization of the DNAprotamine complex and its association with the sperm nuclear matrix. The data obtained indicate that the chromatin organization in sperm nuclei is maintained during the sperm condensation by means of interactions with the nuclear matrix at fixed sites. The fine stucture of sperm nucleus and of sperm nuclear matrix, investigated on sections and replicas of freeze-fractured specimens, suggests that the lamellar array, observed by freeze-fracturing in the sperm nuclei, could depend on the inner matrix which presents a regular organization of globular structures possibly involved in the maintenance of chromatin domains in highly condensed sperm nuclei also.     

Identification of a rosette-like agent as Sphaerothecum destruens, a multi-host fish pathogen

A recent threat to European fish diversity was attributed to an infectious pathogen, a rosette-like intracellular parasite carried by invasive cyprinids. Here we show that the emerging rosette-like agent is Sphaerothecum destruens, originally found to be responsible for disease outbreaks in salmon in the United States. Sequencing of the ribosomal internal transcribed spacer (ITS) DNA highlights some level of geographical isolation. Unlike the situation in the United States, its occurrence in invasive fishes presents a risk of spread from wild invasive populations to sympatric populations of susceptible native fish and as such represents a risk for fisheries, as movement of fish for stocking purposes is common practice.     

Organization of higher-level chromatin structures (chromomere,chromonema and chromatin block) examined using visible light-induced chromatin photo-stabilization

The method of chromatin photo-stabilization by the action of visible light in the presence of ethidium bromide was used for investigation of higher-level chromatin structures in isolated nuclei. As a model we used rat hepatocyte nuclei isolated in buffers which stabilized or destabilized nuclear matrix. Several higher-level chromatin structures were visualized: 100nm globules-chromomeres, chains of chromomeres-chromonemata, aggregates of chromomeres-blocks of condensed chromatin. All these structures were completely destroyed by 2M NaCl extraction independent of the matrix state, and DNA was extruded from the residual nuclei (nuclear matrices) into a halo. These results show that nuclear matrix proteins do not play the main role in the maintenance of higher-level chromatin structures. Preliminary irradiation led to the reduction of the halo width in the dose-dependent manner. In regions of condensed chromatin of irradiated nucleoids there were discrete complexes consisting of DNA fibers radiating from an electron-dense core and resembling the decondensed chromomeres or the rosette-like structures. As shown by the analysis of proteins bound to irradiated nuclei upon high-salt extraction, irradiation presumably stabilized the non-histone proteins. These results suggest that in interphase nuclei loop domains are folded into discrete higher-level chromatin complexes (chromomeres). These complexes are possibly maintained by putative non-histone proteins, which are extracted with high-salt buffers from non-irradiated nuclei.     

Nuclear organization in crucifer genomes: nucleolus-associated telomere clustering is not a universal interphase configuration in Brassicaceae

Arabidopsis thaliana has become a major plant research model, where interphase nuclear organization exhibits unique features, including nucleolus-associated telomere clustering. The chromocenter (CC)-loop model, or rosette-like configuration, describes intranuclear chromatin organization in Arabidopsis as megabase-long loops anchored in, and emanating from, peripherally positioned CCs, with those containing telomeres associating with the nucleolus. To investigate whether the CC-loop organization is universal across the mustard family (crucifers), the nuclear distributions of centromeres, telomeres and nucleoli were analyzed by fluorescence in situ hybridization in seven diploid species (2n = 10–16) representing major crucifer clades with an up to 26-fold variation in genome size (160–4260 Mb). Nucleolus-associated telomere clustering was confirmed in Arabidopsis (157 Mb) and was newly identified as the major nuclear phenotype in other species with a small genome (215–381 Mb). In large-genome species (2611–4264 Mb), centromeres and telomeres adopted a Rabl-like configuration or dispersed distribution in the nuclear interior; telomeres only rarely associated with the nucleolus. In Arabis cypria (381 Mb) and Bunias orientalis (2611 Mb), tissue-specific patterns deviating from the major nuclear phenotypes were observed in anther and stem tissues, respectively. The rosette-like configuration, including nucleolus-associated telomere clustering in small-genome species from different infrafamiliar clades, suggests that genomic properties rather than phylogenetic position determine the interphase nuclear organization. Our data suggest that nuclear genome size, average chromosome size and degree of longitudinal chromosome compartmentalization affect interphase chromosome organization in crucifer genomes.     

The podosomes of Rous sarcoma virus transformed chondrocytes show a peculiar ultrastructural organization

The ultrastructure of F-actin-containing punctate adhesion structures (podosomes) and of their rosette-like clusters has been studied by transmission electron microscopy in Rous sarcoma virus transformed chick embryo chondrocytes. Peculiar "glove finger" invaginations were found to take origin from the ventral membrane at sites of close contact; they were directed toward the center of the cell perpendicularly from the substratum. These new structures may be the sites where the local release of proteases takes place at the side of cell-to-substratum adhesion in podosome-bearing cells. The cytoplasmic face of glove finger invaginations and of the plasma membrane at cell-to-cell contact is lined by thick accumulations of microfilamentous material.     

ACTH-induced internalization of plasma membrane in xanthophores of the goldfish, Carassius auratus L.

The ACTH-induced pigment dispersion in primary cultures and in suspensions of xanthophores of the goldfish, $\text{Carassius}\text{auratus}$ L., has been shown to include the formation of rosette-like membranous structures and plasma membrane invaginations (pits). The rosettes and pits are probably similar structures sectioned at different angles. Horseradish peroxidase studies demonstrate that these structures are eventually converted into small spherical vesicles and long smooth elements, similar in appearance to spherical and “tubular” endoplasmic reticulum, respectively. In addition, these studies show that once vesicles are formed they are no longer continuous to the outside of the cell.     

Preexisting nuclear architecture defines the intranuclear location of herpesvirus DNA replication structures.

Herpes simplex virus DNA replication proteins localize in characteristic patterns corresponding to viral DNA replication structures in the infected cell nucleus. The intranuclear spatial organization of the HSV DNA replication structures and the factors regulating their nuclear location remain to be defined. We have used the HSV ICP8 DNA-binding protein and bromodeoxyuridine labeling as markers for sites of herpesviral DNA synthesis to examine the spatial organization of these structures within the cell nucleus. Confocal microscopy and three-dimensional computer graphics reconstruction of optical series through infected cells indicated that viral DNA replication structures extend through the interior of the cell nucleus and appear to be spatially separate from the nuclear lamina. Examination of viral DNA replication structures in infected, binucleate cells showed similar or virtually identical patterns of DNA replication structures oriented along a twofold axis of symmetry between many of the sister nuclei. These results demonstrate that HSV DNA replication structures are organized in the interior of the nucleus and that their location is defined by preexisting host cell nuclear architecture, probably the internal nuclear matrix.