Mitochondrial Transplantation Therapy Ameliorates Muscular Dystrophy in mdx Mouse Model.

Duchenne muscular dystrophy is caused by loss of the dystrophin protein. This pathology is accompanied by mitochondrial dysfunction contributing to muscle fiber instability. It is known that mitochondria-targeted in vivo therapy mitigates pathology and improves the quality of life of model animals. In the present work, we applied mitochondrial transplantation therapy (MTT) to correct the pathology in dystrophin-deficient mdx mice. Intramuscular injections of allogeneic mitochondria obtained from healthy animals into the hind limbs of mdx mice alleviated skeletal muscle injury, reduced calcium deposits in muscles and serum creatine kinase levels, and improved the grip strength of the hind limbs and motor activity of recipient mdx mice. We noted normalization of the mitochondrial ultrastructure and sarcoplasmic reticulum/mitochondria interactions in mdx muscles. At the same time, we revealed a decrease in the efficiency of oxidative phosphorylation in the skeletal muscle mitochondria of recipient mdx mice accompanied by a reduction in lipid peroxidation products (MDA products) and reduced calcium overloading. We found no effect of MTT on the expression of mitochondrial signature genes (Drp1, Mfn2, Ppargc1a, Pink1, Parkin) and on the level of mtDNA. Our results show that systemic MTT mitigates the development of destructive processes in the quadriceps muscle of mdx mice.

Plasma Membrane Blebbing Is Controlled by Subcellular Distribution of Vimentin Intermediate Filaments.

The formation of specific cellular protrusions, plasma membrane blebs, underlies the amoeboid mode of cell motility, which is characteristic for free-living amoebae and leukocytes, and can also be adopted by stem and tumor cells to bypass unfavorable migration conditions and thus facilitate their long-distance migration. Not all cells are equally prone to bleb formation. We have previously shown that membrane blebbing can be experimentally induced in a subset of HT1080 fibrosarcoma cells, whereas other cells in the same culture under the same conditions retain non-blebbing mesenchymal morphology. Here we show that this heterogeneity is associated with the distribution of vimentin intermediate filaments (VIFs). Using different approaches to alter the VIF organization, we show that blebbing activity is biased toward cell edges lacking abundant VIFs, whereas the VIF-rich regions of the cell periphery exhibit low blebbing activity. This pattern is observed both in interphase fibroblasts, with and without experimentally induced blebbing, and during mitosis-associated blebbing. Moreover, the downregulation of vimentin expression or displacement of VIFs away from the cell periphery promotes blebbing even in cells resistant to bleb-inducing treatments. Thus, we reveal a new important function of VIFs in cell physiology that involves the regulation of non-apoptotic blebbing essential for amoeboid cell migration and mitosis.

Expression, Intracellular Localization, and Maturation of Cysteine Cathepsins in Renal Embryonic and Cancer Cell Lines.

Cysteine cathepsins play an important role in tumor development and metastasis. The expression of these enzymes is often increased in many types of tumor cells. Cysteine cathepsins contribute to carcinogenesis through a number of mechanisms, including proteolysis of extracellular matrix and signaling molecules on the cell surface, as well as degradation of transcription factors and disruption of signaling cascades in the cell nucleus. Distinct oncogenic functions have been reported for several members of the cysteine cathepsin family in various types of cancer, but a comparative study of all eleven cysteine cathepsins in one experimental model is still missing. In this work, we assessed and compared the expression, localization, and maturation of all eleven cysteine cathepsins in embryonic kidney cells HEK293 and kidney cancer cell lines 769-P and A-498. We found that the expression of cathepsins V, B, Z, L, and S was 3- to 9-fold higher in kidney tumor cells than in embryonic cells. We also showed that all cysteine cathepsins were present in varying amounts in the nucleus of both embryonic and tumor cells. Notably, more than half of the cathepsin Z or K and over 88% of cathepsin F were localized in tumor cell nuclei. Moreover, mature forms of cysteine cathepsins were more prevalent in tumor cells than in embryonic cells. These results can be further used to develop novel diagnostic tools and may assist in the investigation of cysteine cathepsins as potential therapeutic targets.

Effect of Large-Conductance Calcium-Dependent K+ Channel Activator NS1619 on Function of Mitochondria in the Heart of Dystrophin-Deficient Mice.

Dystrophin-deficient muscular dystrophy (Duchenne dystrophy) is characterized by impaired ion homeostasis, in which mitochondria play an important role. In the present work, using a model of dystrophin-deficient mdx mice, we revealed decrease in the efficiency of potassium ion transport and total content of this ion in the heart mitochondria. We evaluated the effect of chronic administration of the benzimidazole derivative NS1619, which is an activator of the large-conductance Ca2+-dependent K+ channel (mitoBKCa), on the structure and function of organelles and the state of the heart muscle. It was shown that NS1619 improves K+ transport and increases content of the ion in the heart mitochondria of mdx mice, but this is not associated with the changes in the level of mitoBKCa protein and expression of the gene encoding this protein. The effect of NS1619 was accompanied by the decrease in the intensity of oxidative stress, assessed by the level of lipid peroxidation products (MDA products), and normalization of the mitochondrial ultrastructure in the heart of mdx mice. In addition, we found positive changes in the tissue manifested by the decrease in the level of fibrosis in the heart of dystrophin-deficient animals treated with NS1619. It was noted that NS1619 had no significant effect on the structure and function of heart mitochondria in the wild-type animals. The paper discusses mechanisms of influence of NS1619 on the function of mouse heart mitochondria in Duchenne muscular dystrophy and prospects for applying this approach to correct pathology.

Colocalization Analysis of Cytoplasmic Actin Isoforms Distribution in Endothelial Cells.

Actin cytoskeleton is an essential component of living cells and plays a decisive role in many cellular processes. In mammals, ß- and γ-actin are cytoplasmic actin isoforms in non-muscle cells. Despite minor differences in the amino acid sequence, ß- and γ-actin localize in different cell structures and perform different functions. While cytoplasmic ß-actin is involved in many intracellular processes including cell contraction, γ-actin is responsible for cell mobility and promotes tumor transformation. Numerous studies demonstrate that ß- and γ-actin are spatially separated in the cytoplasm of fibroblasts and epithelial cells; this separation is functionally determined. The spatial location of ß/γ-actin in endothelial cells is still a subject for discussion. Using super-resolution microscopy, we investigated the ß/γ-actin colocalization in endotheliocytes and showed that the ß/γ-actin colocalization degree varies widely between different parts of the marginal regions and near the cell nucleus. In the basal cytoplasm, ß-actin predominates, while the ratio of isoforms evens out as it moves to the apical cytoplasm. Thus, our colocalization analysis suggests that ß- and γ-actin are segregated in the endotheliocyte cytoplasm. The segregation is greatly enhanced during cell lamella activation in the nocodazole-induced endothelial barrier dysfunction, reflecting a different functional role of cytoplasmic actin isoforms in endothelial cells.

A mechanism of self-lipid endocytosis mediated by the receptor Mincle.

Cellular lipid uptake (through endocytosis) is a basic physiological process. Dysregulation of this process underlies the pathogenesis of diseases such as atherosclerosis, obesity, diabetes, and cancer. However, to date, only some mechanisms of lipid endocytosis have been discovered. Here, we show a previously unknown mechanism of lipid cargo uptake into cells mediated by the receptor Mincle. We found that the receptor Mincle, previously shown to be a pattern recognition receptor of the innate immune system, tightly binds a range of self-lipids. Moreover, we revealed the minimal molecular motif in lipids that is sufficient for Mincle recognition. Superresolution microscopy showed that Mincle forms vesicles in cytoplasm and colocalizes with added fluorescent lipids in endothelial cells but does not colocalize with either clathrin or caveolin-1, and the added lipids were predominantly incorporated in vesicles that expressed Mincle. Using a model of ganglioside GM3 uptake in brain vessel endothelial cells, we show that the knockout of Mincle led to a dramatic decrease in lipid endocytosis. Taken together, our results have revealed a fundamental lipid endocytosis pathway, which we call Mincle-mediated endocytosis (MiME), and indicate a prospective target for the treatment of disorders of lipid metabolism, which are rapidly increasing in prevalence.

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.

RNA Aptamers for Theranostics of Glioblastoma of Human Brain.

Conventional approaches for studying and molecular typing of tumors include PCR, blotting, omics, immunocytochemistry, and immunohistochemistry. The last two methods are the most used, as they enable detecting both tumor protein markers and their localizations within the cells. In this study, we have investigated a possibility of using RNA aptamers, in particular, 2'-F-pyrimidyl-RNA aptamer ME07 (48 nucleotides long), specific to the receptor of epidermal growth factor (EGFR, ErbB1, Her1), as an alternative to monoclonal antibodies for aptacytochemistry and aptahistochemistry for human glioblastoma multiforme (GBM). A specificity of binding of FAM-ME07 to the receptor on the tumor cells has been demonstrated by flow cytometry; an apparent dissociation constant for the complex of aptamer - EGFR on the cell has been determined; a number of EGFR molecules has been semi-quantitatively estimated for the tumor cell lines having different amount of EGFR: A431 (106 copies per cell), U87 (104 copies per cell), MCF7 (103 copies per cell), and ROZH, primary GBM cell culture derived from patient (104 copies per cell). According to fluorescence microscopy, FAM-ME07 interacts directly with the receptors on A431 cells, followed by its internalization into the cytoplasm and translocation to the nucleolus; this finding opens a possibility of ME07 application as an escort aptamer for a delivery of therapeutic agents into tumor cells. FAM-ME07 efficiently stains sections of GBM clinical specimens, which enables an identification of EGFR-positive clones within a heterogeneous tumor; and providing a potential for further studying animal models of GBM.

Treacle and TOPBP1 control replication stress response in the nucleolus.

Replication stress is one of the main sources of genome instability. Although the replication stress response in eukaryotic cells has been extensively studied, almost nothing is known about the replication stress response in nucleoli. Here, we demonstrate that initial replication stress-response factors, such as RPA, TOPBP1, and ATR, are recruited inside the nucleolus in response to drug-induced replication stress. The role of TOPBP1 goes beyond the typical replication stress response; it interacts with the low-complexity nucleolar protein Treacle (also referred to as TCOF1) and forms large Treacle-TOPBP1 foci inside the nucleolus. In response to replication stress, Treacle and TOPBP1 facilitate ATR signaling at stalled replication forks, reinforce ATR-mediated checkpoint activation inside the nucleolus, and promote the recruitment of downstream replication stress response proteins inside the nucleolus without forming nucleolar caps. Characterization of the Treacle-TOPBP1 interaction mode leads us to propose that these factors can form a molecular platform for efficient stress response in the nucleolus.

Mapping mechanical properties of living cells at nanoscale using intrinsic nanopipette-sample force interactions.

Mechanical properties of living cells determined by cytoskeletal elements play a crucial role in a wide range of biological functions. However, low-stress mapping of mechanical properties with nanoscale resolution but with a minimal effect on the fragile structure of cells remains difficult. Scanning Ion-Conductance Microscopy (SICM) for quantitative nanomechanical mapping (QNM) is based on intrinsic force interactions between nanopipettes and samples and has been previously suggested as a promising alternative to conventional techniques. In this work, we have provided an alternative estimation of intrinsic force and stress and demonstrated the possibility to perform qualitative and quantitative analysis of cell nanomechanical properties of a variety of living cells. Force estimation on decane droplets with well-known elastic properties, similar to living cells, revealed that the forces applied using a nanopipette are much smaller than in the case using atomic force microscopy. We have shown that we can perform nanoscale topography and QNM using a scanning procedure with no detectable effect on live cells, allowing long-term QNM as well as detection of nanomechanical properties under drug-induced alterations of actin filaments and microtubulin.

Suppression of liquid-liquid phase separation by 1,6-hexanediol partially compromises the 3D genome organization in living cells.

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.

Propagation of Mitochondria-Derived Reactive Oxygen Species within the Dipodascus magnusii Cells.

Mitochondria are considered to be the main source of reactive oxygen species (ROS) in the cell. It was shown that in cardiac myocytes exposed to excessive oxidative stress, ROS-induced ROS release is triggered. However, cardiac myocytes have a network of densely packed organelles that do not move, which is not typical for the majority of eukaryotic cells. The purpose of this study was to trace the spatiotemporal development (propagation) of prooxidant-induced oxidative stress and its interplay with mitochondrial dynamics. We used Dipodascus magnusii yeast cells as a model, as they have advantages over other models, including a uniquely large size, mitochondria that are easy to visualize and freely moving, an ability to vigorously grow on well-defined low-cost substrates, and high responsibility. It was shown that prooxidant-induced oxidative stress was initiated in mitochondria, far preceding the appearance of generalized oxidative stress in the whole cell. For yeasts, these findings were obtained for the first time. Preincubation of yeast cells with SkQ1, a mitochondria-addressed antioxidant, substantially diminished production of mitochondrial ROS, while only slightly alleviating the generalized oxidative stress. This was expected, but had not yet been shown. Importantly, mitochondrial fragmentation was found to be primarily induced by mitochondrial ROS preceding the generalized oxidative stress development.

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.

Chromatin Trapping of Factors Involved in DNA Replication and Repair Underlies Heat-Induced Radio- and Chemosensitization.

Hyperthermia has been used as an adjuvant treatment for radio- and chemotherapy for decades. In addition to its effects on perfusion and oxygenation of cancer tissues, hyperthermia can enhance the efficacy of DNA-damaging treatments such as radiotherapy and chemotherapy. Although it is believed that the adjuvant effects are based on hyperthermia-induced dysfunction of DNA repair systems, the mechanisms of these dysfunctions remain elusive. Here, we propose that elevated temperatures can induce chromatin trapping (c-trapping) of essential factors, particularly those involved in DNA repair, and thus enhance the sensitization of cancer cells to DNA-damaging therapeutics. Using mass spectrometry-based proteomics, we identified proteins that could potentially undergo c-trapping in response to hyperthermia. Functional analyses of several identified factors involved in DNA repair demonstrated that c-trapping could indeed be a mechanism of hyperthermia-induced transient deficiency of DNA repair systems. Based on our proteomics data, we showed for the first time that hyperthermia could inhibit maturation of Okazaki fragments and activate a corresponding poly(ADP-ribose) polymerase-dependent DNA damage response. Together, our data suggest that chromatin trapping of factors involved in DNA repair and replication contributes to heat-induced radio- and chemosensitization.

Interaction of two strongly divergent archaellins stabilizes the structure of the Halorubrum archaellum.

Halophilic archaea from the genus Halorubrum possess two extraordinarily diverged archaellin genes, flaB1 and flaB2. To clarify roles for each archaellin, we compared two natural Halorubrum lacusprofundi strains: One of them contains both archaellin genes, and the other has the flaB2 gene only. Both strains synthesize functional archaella; however, the strain, where both archaellins are present, is more motile. In addition, we expressed these archaellins in a Haloferax volcanii strain from which the endogenous archaellin genes were deleted. Three Hfx. volcanii strains expressing Hrr. lacusprofundi archaellins produced functional filaments consisting of only one (FlaB1 or FlaB2) or both (FlaB1/FlaB2) archaellins. All three strains were motile, although there were profound differences in the efficiency of motility. Both native and recombinant FlaB1/FlaB2 filaments have greater thermal stability and resistance to low salinity stress than single-component filaments. Functional supercoiled Hrr. lacusprofundi archaella can be composed of either single archaellin: FlaB2 or FlaB1; however, the two divergent archaellin subunits provide additional stabilization to the archaellum structure and thus adaptation to a wider range of external conditions. Comparative genomic analysis suggests that the described combination of divergent archaellins is not restricted to Hrr. lacusprofundi, but is occurring also in organisms from other haloarchaeal genera.

Labyrinthula diatomea n. sp.-A Labyrinthulid Associated with Marine Diatoms.

Labyrinthulomycetes are mostly fungus-like heterotrophic protists that absorb nutrients in an osmotrophic or phagotrophic manner. Members of order Labyrinthulida produce unique membrane-bound ectoplasmic networks for movement and feeding. Among the various types of labyrinthulids' food substrates, diatoms play an important role due to their ubiquitous distribution and abundant biomass. We isolated and cultivated new diatom consuming Labyrinthulida strains from shallow coastal marine sediments. We described Labyrinthula diatomea n. sp. that differs from all known labyrinthulids in both molecular and morphological features. We provided strain delimitation within the genus Labyrinthula based on ITS sequences via haplotype network construction and compared it with previous phylogenetic surveys.

Mechanisms of increased mitochondria-dependent necrosis in Wiskott-Aldrich syndrome platelets.

Wiskott-Aldrich syndrome (WAS) is associated with thrombocytopenia of unclear origin. We investigated real-time cytosolic calcium dynamics, mitochondrial membrane potential and phoszphatidylserine (PS) exposure in single fibrinogen-bound platelets using confocal microscopy. The WAS platelets had higher resting calcium levels, more frequent spikes, and their mitochondria more frequently lost membrane potential followed by PS exposure (in 22.9% of platelets vs 3.9% in controls; P<0.001) after the collapse of the last mitochondria. This phenomenon was inhibited by the mitochondrial permeability transition pore inhibitor cyclosporine A, as well by xestospongin C and lack of extracellular calcium. Thapsigargin by itself caused accelerated cell death in the WAS platelets. The number of mitochondria was predictive of PS exposure: 33% of platelets from WAS patients with fewer than five mitochondria exposed PS, while only 12% did among those that had five or more mitochondria. Interestingly, healthy donor platelets with fewer mitochondria also more readily became procoagulant upon PAR1/PAR4 stimulation. Collapse of single mitochondria led to greater cytosolic calcium increase in WAS platelets if they had one to three mitochondria compared with platelets containing higher numbers. A computer systems biology model of platelet calcium homeostasis showed that smaller platelets with fewer mitochondria could have impaired calcium homeostasis because of higher surface-to-volume ratio and greater metabolic load, respectively. There was a correlation (C=0.81, P<0.02) between the mean platelet size and platelet count in the WAS patients. We conclude that WAS platelets readily expose PS via a mitochondria-dependent necrotic mechanism caused by their smaller size, which could contribute to the development of thrombocytopenia.

Analysis of novel hyperosmotic shock response suggests 'beads in liquid' cytosol 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 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.