Imaging Replicative Domains in Ultrastructurally Preserved Chromatin by Electron Tomography.

Principles of DNA folding in the cell nucleus and its dynamic transformations that occur during the fulfillment of basic genetic functions (transcription, replication, segregation, etc.) remain poorly understood, partially due to the lack of experimental approaches to high-resolution visualization of specific chromatin loci in structurally preserved nuclei. Here we present a protocol for the visualization of replicative domains in monolayer cell culture in situ, by combining EdU labeling of newly synthesized DNA with subsequent label detection with Ag-amplification of Nanogold particles and ChromEM staining of chromatin. This protocol allows for the high-contrast, high-efficiency pre-embedding labeling, compatible with traditional glutaraldehyde fixation that provides the best structural preservation of chromatin for room-temperature sample processing. Another advantage of pre-embedding labeling is the possibility to pre-select cells of interest for sectioning. This is especially important for the analysis of heterogeneous cell populations, as well as compatibility with electron tomography approaches to high-resolution 3D analysis of chromatin organization at sites of replication, and the analysis of post-replicative chromatin rearrangement and sister chromatid segregation in the interphase.

Lamin A as a Determinant of Mechanical Properties of the Cell Nucleus in Health and Disease.

One of the main factors associated with worse prognosis in oncology is metastasis, which is based on the ability of tumor cells to migrate from the primary source and to form secondary tumors. The search for new strategies to control migration of metastatic cells is one of the urgent issues in biomedicine. One of the strategies to stop spread of cancer cells could be regulation of the nuclear elasticity. Nucleus, as the biggest and stiffest cellular compartment, determines mechanical properties of the cell as a whole, and, hence, could prevent cell migration through the three-dimensional extracellular matrix. Nuclear rigidity is maintained by the nuclear lamina, two-dimensional network of intermediate filaments in the inner nuclear membrane (INM). Here we present the most significant factors defining nucleus rigidity, discuss the role of nuclear envelope composition in the cell migration, as well consider possible approaches to control lamina composition in order to change plasticity of the cell nucleus and ability of the tumor cells to metastasize.

Osteoclasts Differentiation from Murine RAW 264.7 Cells Stimulated by RANKL: Timing and Behavior.

The development of multi-nucleated cells is critical for osteoclasts (OCs) maturation and function. Our objective was to extend knowledge on osteoclastogenesis, focusing on pre-OC fusion timing and behavior. RAW 264.7 cells, which is a murine monocyte-macrophage cell line, provide a valuable and widely used tool for in vitro studies on osteoclastogenesis mechanisms. Cells were treated with the receptor activator of nuclear factor κ-B ligand (RANKL) for 1-4 days and effects on cell morphology, cytoskeletal organization, protein distribution, and OC-specific gene expression examined by TEM, immunofluorescence, and qPCR. Multinucleated cells began to appear at two days of Receptor Activator of Nuclear factor κ-B Ligand (RANKL) stimulation, increasing in number and size in the following days, associated with morphological and cytoskeletal organization changes. Interesting cellular extensions were observed in three days within cells labeled with wheat germ agglutinin (WGA)-Fluorescein isothiocyanate (FITC). The membrane, cytoplasmic, or nuclear distribution of RANK, TRAF6, p-p38, pERK1/2, and NFATc1, respectively, was related to OCs maturation timing. The gene expression for transcription factors regulating osteoclastogenesis (NFATc1, c-fos, RelA, MITF), molecules involved in RANKL-signaling transduction (TRAF6), cytoskeleton regulation (RhoA), fusion (DC-STAMP), migration (MMP9), and OC-specific enzymes (TRAP, CtsK), showed different trends related to OC differentiation timing. Our findings provide an integrated view on the morphological and molecular changes occurring during RANKL stimulation of RAW 264.7 cells, which are important to better understand the OCs' maturation processes.

Fiber-Like Organization as a Basic Principle for Euchromatin Higher-Order Structure.

A detailed understanding of the principles of the structural organization of genetic material is of great importance for elucidating the mechanisms of differential regulation of genes in development. Modern ideas about the spatial organization of the genome are based on a microscopic analysis of chromatin structure and molecular data on DNA-DNA contact analysis using Chromatin conformation capture (3C) technology, ranging from the "polymer melt" model to a hierarchical folding concept. Heterogeneity of chromatin structure depending on its functional state and cell cycle progression brings another layer of complexity to the interpretation of structural data and requires selective labeling of various transcriptional states under nondestructive conditions. Here, we use a modified approach for replication timing-based metabolic labeling of transcriptionally active chromatin for ultrastructural analysis. The method allows pre-embedding labeling of optimally structurally preserved chromatin, thus making it compatible with various 3D-TEM techniques including electron tomography. By using variable pulse duration, we demonstrate that euchromatic genomic regions adopt a fiber-like higher-order structure of about 200 nm in diameter (chromonema), thus providing support for a hierarchical folding model of chromatin organization as well as the idea of transcription and replication occurring on a highly structured chromatin template.

Long-term live cells observation of internalized fluorescent Fe@C nanoparticles in constant magnetic field.

BACKGROUND: Theranostics application of superparamagnetic nanoparticles based on magnetite and maghemite is impeded by their toxicity. The use of additional protective shells significantly reduced the magnetic properties of the nanoparticles. Therefore, iron carbides and pure iron nanoparticles coated with multiple layers of onion-like carbon sheath seem to be optimal for biomedicine. Fluorescent markers associated with magnetic nanoparticles provide reliable means for their multimodal visualization. Here, biocompatibility of iron nanoparticles coated with graphite-like shell and labeled with Alexa 647 fluorescent marker has been investigated. METHODS: Iron core nanoparticles with intact carbon shells were purified by magnetoseparation after hydrochloric acid treatment. The structure of the NPs (nanoparticles) was examined with a high resolution electron microscopy. The surface of the NPs was alkylcarboxylated and further aminated for covalent linking with Alexa Fluor 647 fluorochrome to produce modified fluorescent magnetic nanoparticles (MFMNPs). Live fluorescent imaging and correlative light-electron microscopy were used to study the NPs intracellular distribution and the effects of constant magnetic field on internalized NPs in the cell culture were analyzed. Cell viability was assayed by measuring a proliferative pool with Click-IT labeling. RESULTS: The microstructure and magnetic properties of superparamagnetic Fe@C core-shell NPs as well as their endocytosis by living tumor cells, and behavior inside the cells in constant magnetic field (150 mT) were studied. Correlative light-electron microscopy demonstrated that NPs retained their microstructure after internalization by the living cells. Application of constant magnetic field caused orientation of internalized NPs along power lines thus demonstrating their magnetocontrollability. Carbon onion-like shells make these NPs biocompatible and enable long-term observation with confocal microscope. It was found that iron core of NPs shows no toxic effect on the cell physiology, does not inhibit the cell proliferation and also does not induce apoptosis. CONCLUSIONS: Non-toxic, biologically compatible superparamagnetic fluorescent MFMNPs can be further used for biological application such as delivery of biologically active compounds both inside the cell and inside the whole organism, magnetic separation, and magnetic resonance imaging (MRI) diagnostics.

Heterochromatin restricts the mobility of nuclear bodies.

Nuclear bodies are relatively immobile organelles. Here, we investigated the mechanisms underlying their movement using experimentally induced interphase prenucleolar bodies (iPNBs). Most iPNBs demonstrated constrained diffusion, exhibiting infrequent fusions with other iPNBs and nucleoli. Fusion events were actin-independent and appeared to be the consequence of stochastic collisions between iPNBs. Most iPNBs were surrounded by condensed chromatin, while fusing iPNBs were usually found in a single heterochromatin-delimited compartment ("cage"). The experimentally induced over-condensation of chromatin significantly decreased the frequency of iPNB fusion. Thus, the data obtained indicate that the mobility of nuclear bodies is restricted by heterochromatin.

Interstitial telomeric repeats-associated DNA breaks.

During a cell's lifespan, DNA break formation is a common event, associated with many processes, from replication to apoptosis. Most of DNA breaks are readily repaired, but some are meant to persist in time, such as the chromosome ends, protected by telomeres. Besides them, eukaryotic genomes comprise shorter stretches of interstitial telomeric repeats. We assumed that the latter may also be associated with the formation of DNA breaks meant to persist in time. In zebrafish and mouse embryos, cells containing numerous breakage foci were identified. These breaks were not associated with apoptosis or replication, nor did they seem to activate DNA damage response machinery. Unlike short-living, accidental sparse breaks, the ones we found seem to be closely associated, forming discrete break foci. A PCR-based method was developed, allowing specific amplification of DNA regions located between inverted telomeric repeats associated with breaks. The cloning and sequencing of such DNA fragments were found to denote some specificity in their distribution for different tissue types and development stages.

Plant-specific 4/1 polypeptide interacts with an endoplasmic reticulum protein related to human BAP31.

MAIN CONCLUSION: The plant-specific 4/1 protein interacts, both in yeast two-hybrid system and in vitro, and co-localizes in plant cells with plant BAP-like protein, the orthologue of human protein BAP31. In yeast two-hybrid system, we identified a number of Nicotiana benthamiana protein interactors of Nt-4/1, the protein known to affect systemic transport of potato spindle tuber viroid. For one of these interactors, an orthologue of human B-cell receptor-associated protein 31 (BAP31) termed plant BAP-like protein (PBL), the ability to interact with Nt-4/1 was studied in greater detail. Analyses of purified proteins expressed in bacterial cells carried out in vitro with the surface plasmon resonance (SPR) spectroscopy revealed that the N. tabacum PBL (NtPBL) was able to interact with Nt-4/1 with high-affinity, and that their complex can form at physiologically relevant concentrations of both proteins. Subcellular localization studies of 4/1-GFP and NtPBL-mRFP transiently co-expressed in plant cells revealed the co-localization of the two fusion proteins in endoplasmic reticulum-associated bodies, suggesting their interaction in vivo. The N-terminal region of the Nt-4/1 protein was found to be required for the specific subcellular targeting of the protein, presumably due to a predicted amphipathic helix mediating association of the Nt-4/1 protein with cell membranes. Additionally, this region was found to contain a trans-activator domain responsible for the Nt-4/1 ability to activate transcription of a reporter gene in yeast.

Magnetocontrollability of Fe7C3@C superparamagnetic nanoparticles in living cells.

BACKGROUND: A new type of superparamagnetic nanoparticles with chemical formula Fe7C3@C (MNPs) showed higher value of magnetization compared to traditionally used iron oxide-based nanoparticles as was shown in our previous studies. The in vitro biocompatibility tests demonstrated that the MNPs display high efficiency of cellular uptake and do not affect cyto-physiological parameters of cultured cells. These MNPs display effective magnetocontrollability in homogeneous liquids but their behavior in cytoplasm of living cells under the effect of magnetic field was not carefully analyzed yet. RESULTS: In this work we investigated the magnetocontrollability of MNPs interacting with living cells in permanent magnetic field. It has been shown that cells were capable of capturing MNPs by upper part of the cell membrane, and from the surface of the cultivation substrate during motion process. Immunofluorescence studies using intracellular endosomal membrane marker showed that MNP agglomerates can be either located in endosomes or lying free in the cytoplasm. When attached cells were exposed to a magnetic field up to 0.15 T, the MNPs acquired magnetic moment and the displacement of incorporated MNP agglomerates in the direction of the magnet was observed. Weakly attached or non-attached cells, such as cells in mitosis or after cytoskeleton damaging treatments moved towards the magnet. During long time cultivation of cells with MNPs in a magnetic field gradual clearing of cells from MNPs was observed. It was the result of removing MNPs from the surface of the cell agglomerates discarded in the process of exocytosis. CONCLUSIONS: Our data allow us to conclude for the first time that the magnetic properties of the MNPs are sufficient for successful manipulation with MNP agglomerates both at the intracellular level, and within the whole cell. The structure of the outer shells of the MNPs allows firmly associate different types of biological molecules with them. This creates prospects for the use of such complexes for targeted delivery and selective removal of selected biological molecules from living cells.

Cytology of DNA Replication Reveals Dynamic Plasticity of Large-Scale Chromatin Fibers.

In higher eukaryotic interphase nuclei, the 100- to >1,000-fold linear compaction of chromatin is difficult to reconcile with its function as a template for transcription, replication, and repair. It is challenging to imagine how DNA and RNA polymerases with their associated molecular machinery would move along the DNA template without transient decondensation of observed large-scale chromatin "chromonema" fibers [1]. Transcription or "replication factory" models [2], in which polymerases remain fixed while DNA is reeled through, are similarly difficult to conceptualize without transient decondensation of these chromonema fibers. Here, we show how a dynamic plasticity of chromatin folding within large-scale chromatin fibers allows DNA replication to take place without significant changes in the global large-scale chromatin compaction or shape of these large-scale chromatin fibers. Time-lapse imaging of lac-operator-tagged chromosome regions shows no major change in the overall compaction of these chromosome regions during their DNA replication. Improved pulse-chase labeling of endogenous interphase chromosomes yields a model in which the global compaction and shape of large-Mbp chromatin domains remains largely invariant during DNA replication, with DNA within these domains undergoing significant movements and redistribution as they move into and then out of adjacent replication foci. In contrast to hierarchical folding models, this dynamic plasticity of large-scale chromatin organization explains how localized changes in DNA topology allow DNA replication to take place without an accompanying global unfolding of large-scale chromatin fibers while suggesting a possible mechanism for maintaining epigenetic programming of large-scale chromatin domains throughout DNA replication.

Mechanisms of nuclear lamina growth in interphase.

The nuclear lamina represents a multifunctional platform involved in such diverse yet interconnected processes as spatial organization of the genome, maintenance of mechanical stability of the nucleus, regulation of transcription and replication. Most of lamina activities are exerted through tethering of lamina-associated chromatin domains (LADs) to the nuclear periphery. Yet, the lamina is a dynamic structure demonstrating considerable expansion during the cell cycle to accommodate increased number of LADs formed during DNA replication. We analyzed dynamics of nuclear growth during interphase and changes in lamina structure as a function of cell cycle progression. The nuclear lamina demonstrates steady growth from G1 till G2, while quantitative analysis of lamina meshwork by super-resolution microscopy revealed that microdomain organization of the lamina is maintained, with lamin A and lamin B microdomain periodicity and interdomain gap sizes unchanged. FRAP analysis, in contrast, demonstrated differences in lamin A and B1 exchange rates; the latter showing higher recovery rate in S-phase cells. In order to further analyze the mechanism of lamina growth in interphase, we generated a lamina-free nuclear envelope in living interphase cells by reversible hypotonic shock. The nuclear envelope in nuclear buds formed after such a treatment initially lacked lamins, and analysis of lamina formation revealed striking difference in lamin A and B1 assembly: lamin A reassembled within 30 min post-treatment, whereas lamin B1 did not incorporate into the newly formed lamina at all. We suggest that in somatic cells lamin B1 meshwork growth is coordinated with replication of LADs, and lamin A meshwork assembly seems to be chromatin-independent process.

Interleukin-1 beta is an irradiation-induced stromal growth factor.

Gamma irradiation of tissues and organs leads to many pathological consequences due to the formation of reactive oxygen species, DNA damage and the subsequent massive death of cells. The therapeutic use of gamma irradiation in the treatment of cancer is based on its penetrating power and damaging effects on tumor cells. Other effects from the irradiation are unnoticeable in comparison. Moreover, the long-term consequences of gamma irradiation are still poorly understood. When a donor bone marrow plug is implanted under the renal capsule of a syngeneic animal, а hematopoietic ectopic focus is formed. The size of the focus is increased in mice that received irradiation compared to non-irradiated ones, regardless of the amount of time between irradiation and bone marrow plug implantation. Long-term repetitive injections of blood serum from irradiated mice given to syngeneic non-irradiated recipients of bone marrow plugs also lead to the formation of enlarged foci. Hence, the blood of irradiated animals must contain an activity that induces the growth of a hematopoietic microenvironment. It was previously shown that the bones of irradiated animals secrete a growth factor required to create stromal microenvironments. The identity of this factor has, until now, been difficult to obtain. We demonstrated that interleukin 1 beta (IL-1) stimulates the growth of murine bone marrow stromal cells in vitro and in vivo. It was shown that the expression of the Il1b gene and the secretion of its product, IL-1, were activated in bone cells long after total body gamma irradiation. Hence, IL-1, or proteins regulated by this cytokine, appears to be the same stromal growth factor previously observed in the serum of irradiated animals. Our data demonstrate several non-canonical functions of IL-1. In addition, the presence of up-regulated levels of IL-1 long after irradiation points to an unknown mechanism governing its gene expression.

Clonal composition of human multipotent mesenchymal stromal cells.

Multipotent mesenchymal stromal cells (MMSCs) are a heterogeneous population consisting of cells with a distinct proliferative potential. The aim of this study was to define clonal composition in MMSCs and trace the dynamics of individual clones in MMSC subpopulations with different proliferative potentials during the process of cultivation. The investigation was performed at single-cell level using genetically marked cells. Specifically, human bone marrow MMSCs were infected with a lentiviral vector-bearing marker gene. Integration site analysis was performed for clones at each passage by ligation-mediated polymerase chain reaction and Southern blot hybridization. Sibling connections between clones and clonal composition of MMSC culture at each passage were revealed. The MMSC population contained multiple, different, mainly small, clones. It was found that large long-living clones with a high, but limited proliferative potential could be detected rarely in MMSCs population. These data suggest that the human MMSC population does not fit the "stem cell" criteria, however, MMSCs may contain a subpopulation of large clones with a high proliferative potential.

Multipotent Mesenchymal Stromal Cells for the Prophylaxis of Acute Graft-versus-Host Disease-A Phase II Study.

The efficacy and the safety of the administration of multipotent mesenchymal stromal cells (MMSCs) for acute graft-versus-host disease (aGVHD) prophylaxis following allogeneic hematopoietic cell transplantation (HSCT) were studied. This prospective clinical trial was based on the random patient allocation to the following two groups receiving (1) standard GVHD prophylaxis and (2) standard GVHD prophylaxis combined with MMSCs infusion. Bone marrow MMSCs from hematopoietic stem cell donors were cultured and administered to the recipients at doses of 0.9-1.3 × 10(6)/kg when the blood counts indicated recovery. aGVHD of stage II-IV developed in 38.9% and 5.3% of patients in group 1 and group 2, respectively, (P = 0.002). There were no differences in the graft rejection rates, chronic GVHD development, or infectious complications. Overall mortality was 16.7% for patients in group 1 and 5.3% for patients in group 2. The efficacy and the safety of MMSC administration for aGVHD prophylaxis were demonstrated in this study.