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Liquid–liquid phase separation (LLPS) contributes to the spatial and functional segregation of molecular processes within the cell nucleus. However, the role played by LLPS in chromatin folding in living cells remains unclear. Here, using stochastic optical reconstruction microscopy (STORM) and Hi-C techniques, we studied the effects of 1,6-hexanediol (1,6-HD)-mediated LLPS disruption/modulation on higher-order chromatin organization in living cells. We found that 1,6-HD treatment caused the enlargement of nucleosome clutches and their more uniform distribution in the nuclear space. At a megabase-scale, chromatin underwent moderate but irreversible perturbations that resulted in the partial mixing of A and B compartments. The removal of 1,6-HD from the culture medium did not allow chromatin to acquire initial configurations, and resulted in more compact repressed chromatin than in untreated cells. 1,6-HD treatment also weakened enhancer-promoter interactions and TAD insulation but did not considerably affect CTCF-dependent loops. Our results suggest that 1,6-HD-sensitive LLPS plays a limited role in chromatin spatial organization by constraining its folding patterns and facilitating compartmentalization at different levels.

Hypothesis Magnetic liposomes are shown to release the entrapped dye once modulated by low frequency AC MF. The mechanism and effectiveness of MF application should depend on lipid composition, magnetic nanoparticles (MNPs) properties, temperature and field parameters. Experiments The study was performed using liposomes of various lipid composition and embedded hydrophobic MNPs. The liposomes structural changes were studied by the transmission electron microscopy (TEM) and attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy and the leakage was monitored by the fluorescent dye release. Findings Magnetic liposomes exposure to the AC MF resulted in the clustering of the MNPs in the membranes and disruption of the lipid packaging. Addition of cholesterol diminished the dye release from the saturated lipid-based liposomes. Replacement of the saturated lipid for unsaturated one also decreased the dye release. The dye release depended on the strength, but not the frequency of the field. Thus, the oscillating motion of MNPs in AC MF ruptures the gel phase membranes of saturated lipids. As the temperature increases the disruption also increases. In the liquid crystalline membranes formed by unsaturated lipids the deformations and defects created by mechanical motion of the MNPs are more likely to heal and results in decreased release.

Magnetic nanoparticles are widely used in various fields of biomedicine. They can combine both therapy and diagnostics modalities thus opening many opportunities for their application. In our work, we have evaluated their ability to work as contrast agents for magnetic resonance imagining and drug delivery systems for photosensitizers. We have shown that after formation of human serum albumin retains ability to bind photosensitizers and they do not lose their optical properties. To obtain stable water solution suitable for long term storage we have additionally modified human serum albumin with polyethylene glycol. It was shown that such modification allows to increase overall stability over time.

Liquid-liquid phase separation (LLPS) contributes to the spatial and functional segregation of molecular processes. However, the role played by LLPS in chromatin folding in living cells remains unclear. Here, using stochastic optical reconstruction microscopy (STORM) and Hi-C techniques, we studied the effects of 1,6-hexanediol (1,6-HD)-mediated LLPS modulation on higher-order chromatin organization in living cells. We found that 1,6-HD treatment caused the enlargement of nucleosome nanodomains and their more uniform distribution in the nuclear space. At a megabase-scale, chromatin underwent moderate but irreversible perturbations that resulted in the partial mixing of A and B compartments. The removal of 1,6-HD from the culture medium did not allow chromatin to acquire initial configurations, but increased further mixing of the chromatin compartments and resulted in more compact repressed chromatin than in untreated cells. 1,6-HD treatment also weakened enhancer-promoter interactions but did not considerably affect CTCF-dependent loops. Our results suggest that 1,6-HD-sensitive LLPS plays a limited role in chromatin spatial organization by constraining its folding patterns and facilitating compartmentalization at different levels.

The overwhelming majority of investigations on mitochondrial morphology were performed using S. cerevisiae. In this study we showed the benefits of applying new model organisms including petite-negative D . magnusii and Y . lipolytica yeasts for visualization of mitochondrial fragmentation. Normally giant D . magnusii cells and filament-like Y . lipolytica cells contain the highly structured mitochondrial reticulum. Oxidative stress mediated by tert -butyl hydroperoxide triggered mitochondrial fragmentation in yeasts. In D. magnusii mitochondrial fragmentation was also induced by impairing the oxidative phosphorylation system. Higher prooxidant concentrations caused cell death. Cationic lipophilic antioxidant SkQ1 acted downstream of the excessive ROS production and prevented partially or almost totally oxidative stress and related mitochondrial fragmentation and cell death. We believe that utility of D . magnusii and Y . lipolytica yeasts as a “living test tube” would be useful for providing new information concerning the interplay between mitochondrial dynamics and mitochondrial dysfunction, cell cycle, aging, mitophagy and cell death.

In this paper, we investigated the delivery efficiency of doxorubicin by magnetite nanoparticles with different shape to LNCaP and PC-3 prostate cancer cell lines. Cubic and spherical nanoparticles of magnetite were synthesized in organic medium and hydrophilized by non-ionic surfactant Pluronic F127—polyethylene-polypropylene oxide polymer. Doxorubicin was loaded into hydrophobic region of polymeric shell. We have observed that cytotoxicity and distribution of doxorubicin in cells changed significantly in case of drug loaded into nanoparticles in comparison with free doxorubicin. We have shown that this change is due to two main reasons: (1) slower internalization of nanoparticles by cells compared to free doxorubicin and (2) slow and incomplete release of doxorubicin from nanoparticle polymer shell. Interestingly, nanoparticle shape influenced cytotoxicity and the dynamics of drug accumulation inside cancer cells. We have found that doxorubicin-loaded cubic nanoparticles were more toxic for both cell lines compared to spherical ones. Moreover, doxorubicin from cubic nanoparticles accumulated in cells faster than the drug loaded in spherical nanoparticles. So, our work shows that for efficient drug delivery, not only size and coating should be taken into account but also the shape of initial core as it plays an important role in nanoparticle interaction with cells.

Proteins can aggregate in response to stresses, including hyperosmotic shock. Formation and disassembly of aggregates is a relatively slow process. We describe a novel instant response of the cell to hyperosmosis, during which chaperones and other proteins form numerous foci with properties uncharacteristic of classical aggregates. These foci appeared/disappeared seconds after shock onset/removal, in close correlation with cell volume changes. Genome-wide and targeted testing revealed chaperones, metabolic enzymes, P-body components and amyloidogenic proteins in the foci. Most of these proteins can form large assemblies and for some, the assembled state was pre-requisite for participation in foci. A genome-wide screen failed to identify genes whose absence prevented foci participation by Hsp70. Shapes of and interconnections between foci, revealed by super-resolution microscopy, indicated that the foci were compressed between other entities. Based on our findings, we suggest a new model of cytosol architecture as a collection of numerous gel-like regions suspended in a liquid network. This network is reduced in volume in response to hyperosmosis and forms small pockets between the gel-like regions., Summary: We describe a novel cellular response to hyperosmotic shock – rapid reversible formation of foci by various proteins. Data on foci behavior suggest a novel ‘beads in liquid’ cytosolic structure.

Proteins can aggregate in response to stresses, including hyperosmotic shock. Formation and disassembly of aggregates is a relatively slow process. We describe a novel instant response of the cell to hyperosmosis, during which chaperones and other proteins form numerous foci with properties uncharacteristic of classical aggregates. These foci appeared/disappeared seconds after shock onset/removal, in close correlation with cell volume changes. Genome-wide and targeted testing revealed chaperones, metabolic enzymes, P-body components and amyloidogenic proteins in the foci. Most of these proteins can form large assemblies and for some, the assembled state was pre-requisite for participation in foci. A genome-wide screen failed to identify genes whose absence prevented foci participation by Hsp70. Shapes of and interconnections between foci revealed by super-resolution microscopy indicated that the foci were compressed between other entities. Based on our findings, we propose a new model of the cytosol architecture as a collection of numerous of gel-like regions suspended in a liquid network. This network is reduced in volume in response to hyperosmosis and forms small pockets between the gel-like regions.

We show here that mitochondria-targeted antioxidant composed of plastoquinone conjugated through hydrocarbon linker with cationic rhodamine 19 (SkQR1) protected against nuclear DNA damage induced by gamma radiation in K562 erythroleukemia cells. We also demonstrate that SkQR1 prevented the early (1 h postirradiation) accumulation of phosphorylated histone H2AX (γ-H2AX) an indicator of DNA double-strand break formation, as well as the radiation-induced increase in chromosomal aberrations. These data suggested that nuclear DNA damage induced by gamma radiation may be mediated by mitochondrial reactive oxygen species (ROS) production. We show that SkQR1 suppressed delayed accumulation of ROS 32 h after irradiation probably by inhibiting mitochondrial ROS-induced ROS release mechanisms. This suggests that mitochondria-targeted antioxidants may protect cells from the late consequences of radiation exposure related to delayed oxidative stress. We have previously reported that SkQRl is the substrate of multidrug resistance pump P-glycoproten (Pgp 170) and selectively protects Pgp 170-negative cells against oxidative stress. In line with this finding, we demonstrate here that SkQR1 did not protect Pgp170-positive K562 subline against DNA damage induced by gamma radiation. The selective radioprotection of normal Pgp 170-negative cells by mitochondria-targeted antioxidants could be a promising strategy to increase the efficiency of radiotherapy for multidrug-resistant tumors.

Noncoding and repetitive sequences make up a large part of the genome of high eukaryotes, but the elucidation of their roles and mechanisms of action are poorly understood. In this work, we found that interstitial telomeric repeats in the genome of Danio rerio colocalize with repetitive elements, including hAT and EnSpm, which are widely represented in vertebrate genomes. The investigation of a genomic region containing two pairs of these repeats located in close proximity showed that the area is transcribed. RNA-dependent structures containing this sequence were identified in D. rerio fibroblast nuclei, which indicates the functional importance of genomic repetitive elements or their transcripts.

We have studied how various drugs increasing the rate of nicotinic acetylcholine receptors (nAChRs) lateral diffusion affect the depression of ACh-induced current in land snail Helix lucorum neurons responsible for defensive behavior. The acetylcholine (ACh) iontophoretic application protocol imitated the behavioral habituation protocol for the intact animal. We found that the drugs decreasing cholesterol level in cell membranes as methyl-β-cyclodextrin 1 mM and Ro 48-8071 2 µM, and polyclonal antibodies to actin-binding proteins as spectrin 5 µg/ml and merlin 2.5 µg/ml have changed the dynamic of ACh-current depression. The nAChRs lateral diffusion coefficient was obtained by fluorescence recovery after photobleaching. A curve fitting model specially created for analysis of short-term choline sensitivity depression in snail neurons helped us evaluate separately the contribution of nAChRs lateral diffusion, their endocytosis and exocytosis to observed effects during electrophysiological experiments. Taken together, we hypothesize that nAChRs lateral diffusion plays an important role in the cellular correlate of habituation in land snail Helix lucorum neurons.

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. Electronic supplementary material The online version of this article (doi:10.1186/s12951-016-0219-4) contains supplementary material, which is available to authorized users.

he electron microscopic study of the structure of the motility apparatus of the archaea Halobacterium salinarium 4W12 and Natronobacterium magadii confirmed our earlier observation that the motility apparatus of halobacteria contains an intracellular disk-shaped lamellar structure (DLS). Polar cap structures (PCSs) isolated from the halobacterium were preliminarily identified as the DLSs. The PCSs in complexes with flagella were also isolated from the haloalkaliphilic bacterium N. magadii. The specific structure of the archaeal motility apparatus is discussed.

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: 100 nm globules—chromomeres, chains of chromomeres—chromonemata, aggregates of chromomeres—blocks of condensed chromatin. All these structures were completely destroyed by 2 M 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.

The dynamics of chondriome changes in oogenesis of the sea urchinParacentrotus lividuswere studied by electron microscopy. An oocyte-enriched fraction obtained by gonad mechanical dissociation without protease treatment was used. The shape, size and arrangement of mitochondria (Mt) in cells were quantitatively analysed on the basis of data from reconstruction experiments, with serial sections performed using a specific computer program. At all stages of oogenesis, the chondriome was shown to consist of rod-shaped Mt of various lengths and also of small amounts of globular Mt about 0.3 μm in diameter. Chondriome transformation during oogenesis is shown to involve the following processes: (1) a 64-fold increase in number of Mt, with the ratio of cytoplasm to Mt volume quite constant in the course of oogenesis; (2) an increase in length of Mt to a maximum of 1.54 μm in medium oocytes and successive considerable mitochondrial division; (3) changes in Mt ultrastructure; and (4) a clustering of Mt. In a mature egg, the modal value of Mt length was reduced and, unlike the oocytes, was more homogeneous, and the Mt were completely clustered.

A method was developed for stabilizing mitotic chromosomes. Light irradiation of permeabilized cells in a low concentration of ethidium bromide made chromatin resistant to high salt concentrations and decondensing buffer. This resistance was abolished by proteinase treatment, but not by DNase or RNase treatment. In photostabilized and extracted chromosomes, chromatin appeared as thick fibers with discrete high electron density regions. These stabilized structures might correspond to the higher-level structures (chromonemata) observed in native chromatin. Moreover, the electron density was higher in the centromeric regions than the chromosome arm material. Thus, the method allows chromatin substructures (chromonemata and centromeric heterochromatin) to be stabilized inside mitotic chromosomes.

Fixation of DNA and proteins in the isolated rat hepatocyte nuclei stained with ethidium bromide and irradiated with visible light was analyzed in this study. It was shown that irradiation results in the following modifications of higherlevel nucleoprotein complexes of interphase chromatin: (1) the complexes acquire resistance to decondensing treatments, which may be indicative of the formation of links between proteins or proteins and DNA in the chromatin; (2) the linking rate for both DNA and proteins is dose dependent; (3) the irradiation induces intermolecular link formation between DNA molecules, which brings about an increase in the average molecular weight of DNA fragments; (4) some modifications (dimerization, etc.) of histones and nonhistone proteins occur; and (5) histone proteins are not effectively cross-linked to DNA. The structural stabilization of interphase chromatin is possibly mediated by free radical–based mechanisms, whereas disulfide bonds seem to play no significant role in the crosslinking.

On incubation of 7-day-old wheat seedlings in the presence of [3H]thymidine, the radioactivity incorporated into coleoptile DNA is found to be localized mainly (greater than 95%) in the fraction of heavy mitochondrial DNA (H-mt DNA; rho = 1.716 gm/cm3). Upon long (48-72 h) incubation of cut-off seedlings in water, the amount of this DNA shows a dramatic increase and corresponds to about 10% of the total coleoptile DNA. H-mtDNA is represented by open circular molecules with a contour length varying from 0.12 to 0.6 microns. The functional role of this DNA is still unknown.

Cell culture density is shown to alter the parameters characterizing phagocytic activity of cells in vitro. Phagocytosis index (PI, mean number of beads per cell in the bead-containing population) and phagocytosis percent (PP, percentage of bead-containing cells in cell population under study) for IC-21 macrophages incubated in the presence of non-opsonized 2-μm fluorescent latex beads were determined using fluorescent microscopy and ImageJ software specially adapted for the purpose. Under control conditions (DMEM without serum), increase in cell culture density was accompanied with a decrease of both parameters of the phagocytic activity. At a mean density of 4 cells/105 μm2 (9 cells per a viewfield) PI was 7.1 ± 0.2 beads/cell and at 20 cells/105 μm2 (40 cells per a viewfield) PI dropped to 4.6 ± 0.1 beads/cell. PP was less sensitive, varied in the range of 95–100% but also decreased as the cell density grew. At any density, PI was 1.5–2 times higher than the expected value (number of beads per µm2 × cell contour area); apparently this divergence can be accounted for by cell locomotion and capture of a larger number of beads than could drop onto a motionless cell with a constant contour area. Increase in cell density was also accompanied by a decrease of the cell contour area (Sc), which amounted to 750 ± 16 μm2 at a density of 4 cells/105 μm2 and 346 ± 4 μm2 at a density of 20 cells/105 μm2. As the bead concentration was the same in all experiments, density-dependent decrease in PI and PP may be related with the observed decrease in cell contour area. Yet, the bead number per cell area unit (PI/Sc) was bigger at higher density and PI/Sc was higher in cells with smaller Sc. Thus, individual (specific) activity of the cells did not lessen with an increase of the cell culture density in the range studied (4–20 cells/105 μm2). Reduction of the cell contour area may reflect alteration in cell adhesion to the substrate as well as competitive relations between adhesion and phagocytic processes. The data obtained imply that cell culture density has to be controlled as a factor notably altering the phagocytic activity parameters. The effects of serum, methyl-β-cyclodextrin, and carbenoxolon reported earlier [Golovkina et al. 2009. Biol membrany. 26 (5), 379–386] are re-evaluated and confirmed here.