O. A. Zhironkina

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

AbstractMain conclusionThe 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.

BackgroundA 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.ResultsIn 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.ConclusionsOur 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.

Magnet-induced behavior of Iron Carbide (Fe7C3@C) Nanoparticles in the Cytoplasm of Living Cells

I. Alieva, I. Kireev, A. Rakhmanina, A. Garanina, O. Strelkova, O. Zhironkina, V. Cherepaninets, V. Davydov, V. Khabashesku, V. Agafonov, R. Uzbekov Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, Russia L. F. Vereshchagin Institute for High Pressure Physics of the RAS, Troitsk, Russia Center for Technology Innovation, Baker Hughes Inc., Houston, TX, 77040, USA GREMAN, UMR CNRS 7347, Universite Francois Rabelais, 37200 Tours, France Faculte de Medecine, Universite Francois Rabelais, Tours, France Faculty of Bioengineering and Bioinformatics, Moscow State University, Moscow, Russia rustem.uzbekov@univ-tours.fr, viatcheslav.agafonov@univ-tours.fr

Cohesion peculiarities in eu- and heterochromatin in human cells

Sister chromatids are considered to be held together from just after replication until the beginning of compaction in prophase via a specific complex—cohesin consisting of Smc1-Smc3 dimer and two additional subunits Scc1 and Scc3, the process being referred to as “cohesion.” We have characterized peculiarities of binding of the cohesin complex with early and late replicating chromatin at various stages of the cell cycle in human cell lines HeLa and HT1080 using structured illumination microscopy and immune electron microscopy. It has been shown that cohesion in heterochromatic domains is evidently provided without the participation of cohesins, while the majority of cohesins bound to chromatin provides cohesion and subdomain organization in euchromatin.

Overcoming steric hindrances during replication of peripheral heterochromatin

Due to a tight attachment of peripheral heterochromatin to the nuclear lamina its replication is connected with inevitable topological hindrances. Additional hindrances are caused by high stability of the lamina that complicates the access of replication factors to DNA duplication sites under conditions of highly condensed matrix with limited mobility. The work focuses on detailed study of structural organization and dynamics of the lamina in respect to replication of peripheral heterochromatin that is attached to it. The study of mobile properties of lamins at various stages of the S-phase using live cell imaging and super-resolution microscopy showed the absence of the dependence of lamins’ mobility on replicative status of attached heterochromatin. These data confirm the hypothesis on regulation of linkage between chromatin and lamina at the level of molecular intermediates. It has been shown at the ultrastructural level that possible temporary disruption in molecular bonds between the lamina and peripheral chromatin during replication does not cause movement of replicated domains from the nuclear periphery.

Interstitial telomeric repeats-associated DNA breaks

ABSTRACT During a cells 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.

High expression levels and nuclear localization of novel Danio rerio ncRNA transcribed from a genomic region containing repetitive elements

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.