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Although cancer is an age-related disease, how the processes of aging contribute to cancer progression is not well understood. In this study, we uncovered how mouse B cell lymphoma develops as a consequence of a naturally aged system. We show here that this malignancy is associated with an age-associated clonal B cell (ACBC) population that likely originates from age-associated B cells. Driven by c-Myc activation, promoter hypermethylation and somatic mutations, IgM+ ACBCs clonally expand independently of germinal centers and show increased biological age. ACBCs become self-sufficient and support malignancy when transferred into young recipients. Inhibition of mTOR or c-Myc in old mice attenuates pre-malignant changes in B cells during aging. Although the etiology of mouse and human B cell lymphomas is considered distinct, epigenetic changes in transformed mouse B cells are enriched for changes observed in human B cell lymphomas. Together, our findings characterize the spontaneous progression of cancer during aging through both cell-intrinsic and microenvironmental changes and suggest interventions for its prevention.
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Sensory hair cells of the cochlea are essential for hearing, relying on the mechanosensitive stereocilia bundle at their apical pole for their function. Polycystic Kidney and Hepatic Disease 1-Like 1 (PKHD1L1) is a stereocilia protein required for normal hearing in mice, and for the formation of the transient stereocilia surface coat, expressed during early postnatal development. While the function of the stereocilia coat remains unclear, growing evidence supports PKHD1L1 as a human deafness gene. In this study we carry out in depth characterization of PKHD1L1 expression in mice during development and adulthood, analyze hair-cell bundle morphology and hearing function in aging PKHD1L1-defficient mouse lines, and assess their susceptibility to noise damage. Our findings reveal that PKHD1L1-deficient mice display no disruption to bundle cohesion or tectorial membrane attachment-crown formation during development. However, starting from 6 weeks of age, PKHD1L1-defficient mice display missing stereocilia and disruptions to bundle coherence. Both conditional and constitutive PKHD1L1 knock-out mice develop high-frequency hearing loss progressing to lower frequencies with age. Furthermore, PKHD1L1-deficient mice are susceptible to permanent hearing loss following moderate acoustic overexposure, which induces only temporary hearing threshold shifts in wild-type mice. These results suggest a role for PKHD1L1 in establishing robust sensory hair bundles during development, necessary for maintaining bundle cohesion and function in response to acoustic trauma and aging.
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Mammalian sperm show an unusual and heavily compacted genomic packaging state. In addition to its role in organizing the compact and hydrodynamic sperm head, it has been proposed that sperm chromatin architecture helps to program gene expression in the early embryo. Scores of genome-wide surveys in sperm have reported patterns of chromatin accessibility, nucleosome localization, histone modification, and chromosome folding. Here, we revisit these studies in light of recent reports that sperm obtained from the mouse epididymis are contaminated with low levels of cell-free chromatin. In the absence of proper sperm lysis, we readily recapitulate multiple prominent genome-wide surveys of sperm chromatin, suggesting that these profiles primarily reflect contaminating cell-free chromatin. Removal of cell-free DNA, and appropriate lysis conditions, are together required to reveal a sperm chromatin state distinct from most previous reports. Using ATAC-seq to explore relatively accessible genomic loci, we identify a landscape of open loci associated with early development and transcriptional control. Histone modification and chromosome folding profiles also strongly support the hypothesis that prior studies suffer from contamination, but technical challenges associated with reliably preserving the architecture of the compacted sperm head prevent us from confidently assaying true localization patterns for these epigenetic marks. Together, our studies show that our knowledge of chromosome packaging in mammalian sperm remains largely incomplete, and motivate future efforts to more accurately characterize genome organization in mature sperm.
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Cromatina , Espermatozoides , Masculino , Animales , Ratones , Espermatozoides/metabolismo , Cromatina/metabolismo , Cromatina/genética , Ensamble y Desensamble de Cromatina , Código de Histonas , Histonas/metabolismoRESUMEN
Usher syndrome type 1 F (USH1F), caused by mutations in the protocadherin-15 gene (PCDH15), is characterized by congenital deafness, lack of balance, and progressive blindness. In hair cells, the receptor cells of the inner ear, PCDH15 is a component of tip links, fine filaments which pull open mechanosensory transduction channels. A simple gene addition therapy for USH1F is challenging because the PCDH15 coding sequence is too large for adeno-associated virus (AAV) vectors. We use rational, structure-based design to engineer mini-PCDH15s in which 3-5 of the 11 extracellular cadherin repeats are deleted, but which still bind a partner protein. Some mini-PCDH15s can fit in an AAV. An AAV encoding one of these, injected into the inner ears of mouse models of USH1F, produces a mini-PCDH15 which properly forms tip links, prevents the degeneration of hair cell bundles, and rescues hearing. Mini-PCDH15s may be a useful therapy for the deafness of USH1F.
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Oído Interno , Síndromes de Usher , Animales , Ratones , Cadherinas/metabolismo , Oído Interno/metabolismo , Células Ciliadas Auditivas/metabolismo , Audición/genética , Síndromes de Usher/genética , Síndromes de Usher/terapia , Proteínas Relacionadas con las Cadherinas/metabolismoRESUMEN
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.
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Cromatina , Tomografía con Microscopio Electrónico , Núcleo Celular/genética , Cromatina/genética , Cromosomas , ADN/química , InterfaseRESUMEN
Excess noise damages sensory hair cells, resulting in loss of synaptic connections with auditory nerves and, in some cases, hair-cell death. The cellular mechanisms underlying mechanically induced hair-cell damage and subsequent repair are not completely understood. Hair cells in neuromasts of larval zebrafish are structurally and functionally comparable to mammalian hair cells but undergo robust regeneration following ototoxic damage. We therefore developed a model for mechanically induced hair-cell damage in this highly tractable system. Free swimming larvae exposed to strong water wave stimulus for 2 hr displayed mechanical injury to neuromasts, including afferent neurite retraction, damaged hair bundles, and reduced mechanotransduction. Synapse loss was observed in apparently intact exposed neuromasts, and this loss was exacerbated by inhibiting glutamate uptake. Mechanical damage also elicited an inflammatory response and macrophage recruitment. Remarkably, neuromast hair-cell morphology and mechanotransduction recovered within hours following exposure, suggesting severely damaged neuromasts undergo repair. Our results indicate functional changes and synapse loss in mechanically damaged lateral-line neuromasts that share key features of damage observed in noise-exposed mammalian ear. Yet, unlike the mammalian ear, mechanical damage to neuromasts is rapidly reversible.
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Sistema de la Línea Lateral/lesiones , Mecanorreceptores/fisiología , Mecanotransducción Celular , Sinapsis/fisiología , Pez Cebra/lesiones , Animales , Fenómenos Biomecánicos , Células Ciliadas Auditivas/fisiología , Sistema de la Línea Lateral/fisiología , Pez Cebra/fisiologíaRESUMEN
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.
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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.
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Plaquetas/metabolismo , Púrpura Trombocitopénica Idiopática/sangre , Púrpura Trombocitopénica Idiopática/tratamiento farmacológico , Receptores Fc/administración & dosificación , Proteínas Recombinantes de Fusión/administración & dosificación , Trombopoyetina/administración & dosificación , Adulto , Plaquetas/patología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Púrpura Trombocitopénica Idiopática/patologíaRESUMEN
The contribution of nucleoli to the cellular stress response has been discussed for over a decade. Stress-induced inhibition of RNA polymerase I-dependent transcription is hypothesized as a possible effector program in such a response. In this study, we report a new mechanism by which ribosomal DNA transcription can be inhibited in response to cellular stress. Specifically, we demonstrate that mild hypoosmotic stress induces stabilization of R loops in ribosomal genes and thus provokes the nucleoli-specific DNA damage response, which is governed by the ATM- and Rad3-related (ATR) kinase. Activation of ATR in nucleoli strongly depends on Treacle, which is needed for efficient recruitment/retention of TopBP1 in nucleoli. Subsequent ATR-mediated activation of ATM results in repression of nucleolar transcription.
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Proteínas de la Ataxia Telangiectasia Mutada/fisiología , Proteínas Portadoras/genética , Nucléolo Celular/metabolismo , ADN Ribosómico/genética , Proteínas de Unión al ADN/genética , Silenciador del Gen , Proteínas Nucleares/genética , Presión Osmótica , Estructuras R-Loop , Transcripción Genética/fisiología , Animales , Línea Celular , Nucléolo Celular/efectos de los fármacos , Supervivencia Celular , Roturas del ADN de Doble Cadena , Daño del ADN , Replicación del ADN , Dactinomicina/farmacología , Activación Enzimática/efectos de los fármacos , Técnicas de Inactivación de Genes , Histonas/metabolismo , Humanos , Soluciones Hipotónicas/farmacología , Ratones , Proteínas Nucleares/fisiología , Fosfoproteínas/fisiología , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Transcripción Genética/efectos de los fármacosRESUMEN
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.
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Rastreo Celular/métodos , Colorantes Fluorescentes/química , Nanopartículas de Magnetita/química , Apoptosis , Línea Celular Tumoral , Supervivencia Celular , Endocitosis , Óxido Ferrosoférrico/química , Grafito/química , Humanos , Luz , Campos Magnéticos , Nanopartículas de Magnetita/toxicidad , Imagen Óptica/métodos , Tamaño de la Partícula , Propiedades de SuperficieRESUMEN
Mitochondria are important regulators of tumour growth and progression due to their specific role in cancer metabolism and modulation of apoptotic pathways. In this paper we describe that mitochondria-targeted antioxidant SkQ1 designed as a conjugate of decyl-triphenylphosphonium cation (TPP+) with plastoquinone, suppressed the growth of fibrosarcoma HT1080 and rhabdomyosarcoma RD tumour cells in culture and tumour growth of RD in xenograft nude mouse model. Under the same conditions, no detrimental effect of SkQ1 on cell growth of primary human subcutaneous fibroblasts was observed. The tumour growth suppression was shown to be a result of the antioxidant action of low nanomolar concentrations of SkQ1. We have revealed significant prolongation of mitosis induced by SkQ1 in both tumour cell cultures. Prolonged mitosis and apoptosis could be responsible for growth suppression after SkQ1 treatment in RD cells. Growth suppression in HT1080 cells was accompanied by the delay of telophase and cytokinesis, followed by multinuclear cells formation. The effects of SkQ1 on the cell cycle were proved to be at least partially mediated by inactivation of Aurora family kinases. ABBREVIATIONS: TPP+: Triphenylphosphonium cation; ROS: Reactive oxygen species; mtROS: Mitochondrial reactive oxygen species; NAC: N-acetyl-L-cysteine; DCFH-DA: Dichlorodihydrofluorescein diacetate; APC: Anaphase promoting complex; ABPs: Actin-binding proteins; DMEM: Dulbecco's modified Eagle media; SDS: sodium dodecyl sulfate; HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid.
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Antioxidantes/farmacología , Fibrosarcoma/patología , Mitocondrias/metabolismo , Plastoquinona/análogos & derivados , Rabdomiosarcoma/patología , Citoesqueleto de Actina/efectos de los fármacos , Citoesqueleto de Actina/metabolismo , Animales , Aurora Quinasa B/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Forma de la Célula/efectos de los fármacos , Modelos Animales de Enfermedad , Ratones Desnudos , Mitocondrias/efectos de los fármacos , Mitosis/efectos de los fármacos , Plastoquinona/farmacología , Proteína de Retinoblastoma/metabolismoRESUMEN
The Nicotiana tabacum 4/1 protein (Nt-4/1) of unknown function expressed in plant vasculature has been shown to localize to cytoplasmic bodies associated with endoplasmic reticulum. Here, we analyzed molecular interactions of an Nt-4/1 mutant with a deletion of 90 N-terminal amino acid residues (Nt-4/1d90) having a diffuse GFP-like localization. Upon transient co-expression with VAP27, a membrane protein known to localize to the ER, ER-plasma membrane contact sites and plasmodesmata, Nt-4/1d90 was concentrated around the cortical ER tubules, forming a network matching the shape of the cortical ER. Additionally, in response to mechanical stress, Nt-4/1d90 was re-localized to small spherical bodies, whereas the subcellular localization of VAP27 remained essentially unaffected. The Nt-4/1d90-containing bodies associated with microtubules, which underwent noticeable bundling under the conditions of mechanical stress. The Nt-4/1d90 re-localization to spherical bodies could also be induced by incubation at an elevated temperature, although under heat shock conditions the re-localization was less efficient and incomplete. An Nt-4/1d90 mutant, which had phosphorylation-mimicking mutations in a predicted cluster of four potentially phosphorylated residues, was found to both inefficiently re-localize to spherical bodies and tend to revert back to the initial diffuse localization. The presented data show that Nt-4/1 has a potential for response to stresses that is manifested by its deletion mutant Nt-4/1d90, and this response can be mediated by protein dephosphorylation.
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Espacio Intracelular/metabolismo , Nicotiana , Proteínas de Plantas/metabolismo , Eliminación de Secuencia , Estrés Mecánico , Secuencia de Aminoácidos , Microtúbulos/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/genética , Transporte de ProteínasRESUMEN
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.
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Roturas del ADN de Doble Cadena , Secuencias Repetitivas de Ácidos Nucleicos/genética , Telómero/genética , Animales , Apoptosis , Cromatina/metabolismo , Fragmentación del ADN , Reparación del ADN , Replicación del ADN , Embrión de Mamíferos/metabolismo , Embrión no Mamífero/metabolismo , Histonas/metabolismo , Ratones , Especificidad de Órganos , Pez Cebra/embriologíaRESUMEN
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.
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Técnicas Citológicas/métodos , Nanopartículas de Magnetita/química , Microscopía Fluorescente/métodos , Línea Celular Tumoral , Citoesqueleto/química , Citoesqueleto/metabolismo , Endocitosis/fisiología , Humanos , Espacio Intracelular/química , Magnetismo , Microscopía Electrónica de TransmisiónRESUMEN
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.
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Cromatina/metabolismo , Replicación del ADN , Animales , Células CHO , Línea Celular , Núcleo Celular , Cricetulus , Interfase , Imagen de Lapso de TiempoRESUMEN
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.
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Interfase , Lámina Nuclear/metabolismo , Animales , Células Cultivadas , Cricetulus , Humanos , Ratones , Lámina Nuclear/química , PorcinosRESUMEN
Superparamagnetic carbon-encapsulated iron carbide nanoparticles (NPs), Fe7C3@C, with unique properties, were produced from pure ferrocene by high pressure-high temperature synthesis. These NPs combine the merits of nanodiamonds and SPIONs but lack their shortcomings which limit their use for biomedical applications. Investigation of these NPs by X-ray diffraction, electron microscopy techniques, X-ray spectroscopic and magnetic measurement methods has demonstrated that this method of synthesis yields NPs with perfectly controllable physical properties. Using magnetic and subsequent fractional separation of magnetic NPs from residual carbon, the aqueous suspensions of Fe7C3@C NPs with an average particle size of â¼25 nm were prepared. The suspensions were used for in vitro studies of the interaction of Fe7C3@C NPs with cultured mammalian cells. The dynamics of interaction of the living cells with Fe7C3@C was studied by optical microscopy using time-lapse video recording and also by transmission electron microscopy. Using novel highly sensitive cytotoxicity tests based on the cell proliferation assay and long-term live cell observations it was shown that the internalization of Fe7C3@C NPs has no cytotoxic effect on cultured cells and does not interfere with the process of their mitotic division, a fundamental property that ensures the existence of living organisms. The influence of NPs on the proliferative activity of cultured cells was not detected as well. These results indicate that the carbon capsules of Fe7C3@C NPs are air-tight which could offer great opportunities for future use of these superparamagnetic NPs in biology and medicine.
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The current progress in the study of the spatial organization of interphase chromosomes became possible owing to the development of the chromosome conformation capture (3C) protocol. The crucial step of this protocol is the proximity ligation-preferential ligation of DNA fragments assumed to be joined within nuclei by protein bridges and solubilized as a common complex after formaldehyde cross-linking and DNA cleavage. Here, we show that a substantial, and in some cases the major, part of DNA is not solubilized from cross-linked nuclei treated with restriction endonuclease(s) and sodium dodecyl sulphate and that this treatment neither causes lysis of the nucleus nor drastically affects its internal organization. Analysis of the ligation frequencies of the mouse ß-globin gene domain DNA fragments demonstrated that the previously reported 3C signals were generated predominantly, if not exclusively, in the insoluble portion of the 3C material. The proximity ligation thus occurs within the cross-linked chromatin cage in non-lysed nuclei. The finding does not compromise the 3C protocol but allows the consideration of an active chromatin hub as a folded chromatin domain or a nuclear compartment rather than a rigid complex of regulatory elements.