Induction of DNA single- and double-strand breaks by excited intra- or extracellular green fluorescent protein.

Green fluorescent protein (GFP) has opened vast new avenues in studies of live cells and is generally perceived as a benign, nontoxic and harmless fluorescent tag. We demonstrat that excited GFP is capable of inducing substantial DNA damage in cells expressing fusion proteins. In the presence of GFP, even low doses of blue light (12 μJ) induce single strand breaks (SSBs).

The method utilized to purify the SARS-CoV-2 N protein can affect its molecular properties.

One of the main structural proteins of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the nucleocapsid protein (N). The basic function of this protein is to bind genomic RNA and to form a protective nucleocapsid in the mature virion. The intrinsic ability of the N protein to interact with nucleic acids makes its purification very challenging.

Translocation of chromatin proteins to nucleoli-The influence of protein dynamics on post-fixation localization.

It is expected that the subnuclear localization of a protein in a fixed cell, detected by microscopy, reflects its position in the living cell. We demonstrate, however, that some dynamic nuclear proteins can change their localization upon fixation by either crosslinking or non-crosslinking methods. We examined the subnuclear localization of the chromatin architectural protein HMGB1, linker histone H1, and core histone H2B in cells fixed by formaldehyde, glutaraldehyde, glyoxal, ethanol, or zinc salts.

STRIDE-a fluorescence method for direct, specific in situ detection of individual single- or double-strand DNA breaks in fixed cells.

We here describe a technique termed STRIDE (SensiTive Recognition of Individual DNA Ends), which enables highly sensitive, specific, direct in situ detection of single- or double-strand DNA breaks (sSTRIDE or dSTRIDE), in nuclei of single cells, using fluorescence microscopy. The sensitivity of STRIDE was tested using a specially developed CRISPR/Cas9 DNA damage induction system, capable of inducing small clusters or individual single- or double-strand breaks. STRIDE exhibits significantly higher sensitivity and specificity of detection of DNA breaks than the commonly used terminal deoxynucleotidyl transferase dUTP nick-end labeling assay or methods based on monitoring of recruitment of repair proteins or histone modifications at the damage site (e.

The subcellular localization of bHLH transcription factor TCF4 is mediated by multiple nuclear localization and nuclear export signals.

Transcription factor 4 (TCF4) is a class I basic helix-loop-helix (bHLH) transcription factor which regulates the neurogenesis and specialization of cells. TCF4 also plays an important role in the development and functioning of the immune system. Additionally, TCF4 regulates the development of Sertoli cells and pontine nucleus neurons, myogenesis, melanogenesis and epithelial-mesenchymal transition.

Lattice Shrinkage by Incorporation of Recombinant Starmaker-Like Protein within Bioinspired Calcium Carbonate Crystals.

The biological mediation of mineral formation (biomineralization) is realized through diverse organic macromolecules that guide this process in a spatial and temporal manner. Although the role of these molecules in biomineralization is being gradually revealed, the molecular basis of their regulatory function is still poorly understood. In this study, the incorporation and distribution of the model intrinsically disordered starmaker-like (Stm-l) protein, which is active in fish otoliths biomineralization, within calcium carbonate crystals, is revealed.

Multiple sequences orchestrate subcellular trafficking of neuronal PAS domain-containing protein 4 (NPAS4).

Neuronal Per-Arnt-Sim (PAS) domain-containing protein 4 (NPAS4) is a basic helix-loop-helix (bHLH)-PAS transcription factor first discovered in neurons in the neuronal layer of the mammalian hippocampus and later discovered in pancreatic β-cells. NPAS4 has been proposed as a therapeutic target not only for depression and neurodegenerative diseases associated with synaptic dysfunction but also for type 2 diabetes and pancreas transplantation. The ability of bHLH-PAS proteins to fulfil their function depends on their intracellular trafficking, which is regulated by specific sequences, the nuclear localization signal (NLS) and the nuclear export signal (NES).

Molecular determinants of Drosophila immunophilin FKBP39 nuclear localization.

FK506-binding proteins (FKBPs) belong to a distinct class of immunophilins that interact with immunosuppressants. They use their peptidyl-prolyl isomerase (PPIase) activity to catalyze the cis-trans conversion of prolyl bonds in proteins during protein-folding events. FKBPs also act as a unique group of chaperones.

Entry of Human Coronavirus NL63 into the Cell.

The first steps of human coronavirus NL63 (HCoV-NL63) infection were previously described. The virus binds to target cells by use of heparan sulfate proteoglycans and interacts with the ACE2 protein. Subsequent events, including virus internalization and trafficking, remain to be elucidated.

Involvement of cell surface 90 kDa heat shock protein (HSP90) in pattern recognition by human monocyte-derived macrophages.

Heat shock proteins (HSPs) are typical intracellular chaperones which also appear on the cell surface and in extracellular milieu. HSP90, which chaperones many proteins involved in signal transduction, is also a regular component of LPS-signaling complexes on Mϕ. As LPS is a prototypical PAMP, we speculated that HSP90 is engaged in pattern recognition by professional phagocytes.

Low level phosphorylation of histone H2AX on serine 139 (γH2AX) is not associated with DNA double-strand breaks.

Phosphorylation of histone H2AX on serine 139 (γH2AX) is an early step in cellular response to a DNA double-strand break (DSB). γH2AX foci are generally regarded as markers of DSBs. A growing body of evidence demonstrates, however, that while induction of DSBs always brings about phosphorylation of histone H2AX, the reverse is not true - the presence of γH2AX foci should not be considered an unequivocal marker of DNA double-strand breaks.

Rapid externalization of 27-kDa heat shock protein (HSP27) and atypical cell death in neutrophils treated with the sphingolipid analog drug FTY720.

The sphingolipid analog fingolimod is known to induce apoptosis of tumor cells and lymphocytes. Its effect on neutrophils has not been investigated so far. Here, we describe a fingolimod-induced atypical cell death mechanism in human neutrophils, characterized by rapid translocation of heat shock protein 27 to the cell surface, extensive cell swelling and vacuolization, atypical chromatin staining and nuclear morphology, and phosphorylation of mixed lineage kinase domain-like protein.

Mapping of the Sequences Directing Localization of the Drosophila Germ Cell-Expressed Protein (GCE).

Drosophila melanogaster germ cell-expressed protein (GCE) belongs to the family of bHLH-PAS transcription factors that are the regulators of gene expression networks that determine many physiological and developmental processes. GCE is a homolog of D. melanogaster methoprene tolerant protein (MET), a key mediator of anti-metamorphic signaling in insects and the putative juvenile hormone receptor.

The nucleocapsid protein of human coronavirus NL63.

Human coronavirus (HCoV) NL63 was first described in 2004 and is associated with respiratory tract disease of varying severity. At the genetic and structural level, HCoV-NL63 is similar to other members of the Coronavirinae subfamily, especially human coronavirus 229E (HCoV-229E). Detailed analysis, however, reveals several unique features of the pathogen.

Human coronavirus NL63 utilizes heparan sulfate proteoglycans for attachment to target cells.

Unlabelled: Human coronavirus NL63 (HCoV-NL63) is an alphacoronavirus that was first identified in 2004 in the nasopharyngeal aspirate from a 7-month-old patient with a respiratory tract infection. Previous studies showed that HCoV-NL63 and the genetically distant severe acute respiratory syndrome (SARS)-CoV employ the same receptor for host cell entry, angiotensin-converting enzyme 2 (ACE2), but it is largely unclear whether ACE2 interactions are sufficient to allow HCoV-NL63 binding to cells. The present study showed that directed expression of angiotensin-converting enzyme 2 (ACE2) on cells previously resistant to HCoV-NL63 renders them susceptible, showing that ACE2 protein acts as a functional receptor and that its expression is required for infection.

Spatial heterogeneity of dynamics of H1 linker histone.

Linker histone H1 participates in maintaining higher order chromatin structures. It is a dynamic protein that binds to DNA and exchanges rapidly with a mobile pool. Therefore, the dynamics of H1 were probed in the nuclei of intact, live cells, using an array of microscopy techniques: fluorescence recovery after photobleaching (FRAP), raster image correlation spectroscopy (RICS), fluorescence correlation spectroscopy (FCS), pair correlation functions (pCF) and fluorescence anisotropy.

Photosensitized damage inflicted on plasma membranes of live cells by an extracellular generator of singlet oxygen--a linear dependence of a lethal dose on light intensity.

We describe a study of the influence of a dose rate, i.e. light intensity or photon flux, on the efficiency of induction of a loss of integrity of plasma membranes of live cells in culture.

Analysis of spatial correlations between patterns of DNA damage response and DNA replication in nuclei of cells subjected to replication stress or oxidative damage.

Sites of DNA replication (EdU incorporation) and DNA damage signaling (γH2AX) induced by camptothecin (Cpt) or hydrogen peroxide (H2O2) form characteristic patterns of foci in cell nuclei. The overlap between these patterns is a function of the number of DNA double strand breaks (DSBs) formed in replication sites. The goal of this study was to optimize a method of quantitative assessment of a degree of correlation between these two patterns.

Relationship between DNA damage response, initiated by camptothecin or oxidative stress, and DNA replication, analyzed by quantitative 3D image analysis.

A method of quantitative analysis of spatial (3D) relationship between discrete nuclear events detected by confocal microscopy is described and applied in analysis of a dependence between sites of DNA damage signaling (γH2AX foci) and DNA replication (EdU incorporation) in cells subjected to treatments with camptothecin (Cpt) or hydrogen peroxide (H2O2). Cpt induces γH2AX foci, likely reporting formation of DNA double-strand breaks (DSBs), almost exclusively at sites of DNA replication. This finding is consistent with the known mechanism of induction of DSBs by DNA topoisomerase I (topo1) inhibitors at the sites of collisions of the moving replication forks with topo1-DNA "cleavable complexes" stabilized by Cpt.

Daunomycin, an antitumor DNA intercalator, influences histone-DNA interactions.

Although daunomycin and adriamycin are considered effective antitumor drugs and have been used in the clinic for over 40 years, their mechanism of action is still a matter of debate. We investigated the influence of daunomycin on interaction between linker or core histones and DNA in live HeLa cells in vitro, using image and flow cytometry. Exposure to daunomycin at clinically relevant concentrations (25-250 nM) caused dissociation of wild-type H1.

Inducing local DNA damage by visible light to study chromatin repair.

Dynamics of DNA repair and recruitment of repair factors to damaged DNA can be studied by live cell microscopy. DNA damage is usually inflicted by a laser beam illuminating a DNA-interacting photosensitizer in a small area of the nucleus. We demonstrate that a focused beam of visible low intensity light alone can inflict local DNA damage and permit studies of DNA repair, thus avoiding potential artifacts caused by exogenous photosensitizers.

Cell fixation in zinc salt solution is compatible with DNA damage response detection by phospho-specific antibodies.

By virtue of superior preservation of proteins and nucleic acids the zinc salt-based fixatives (ZBF) has been proposed as an alternative to precipitants and cross-linking fixatives in histopathology. It was recently reported that ZBF is compatible with analysis of cell surface immunophenotype and detection of intracellular epitopes by flow cytometry. The aim of this study was to explore whether ZBF is also compatible with the detection of DNA damage response assessed by phospho-specific antibodies (Abs) detecting phosphorylation of the key proteins of that pathway.

Interaction of human peripheral blood monocytes with apoptotic polymorphonuclear cells.

Macrophages have the potential to recognize apoptotic neutrophils and phagocytose them while the same function for monocytes is uncertain. In fact, early findings indicated that monocytes started to phagocytose neutrophils on the third day of differentiation to macrophages. Here we show, using flow cytometry and confocal microscopy, that peripheral blood monocytes phagocytose apoptotic but not freshly isolated granulocytes.

Recruitment of heterochromatin protein 1 to DNA repair sites.

Heterochromatin protein 1 (HP1) was originally identified as a constitutive component of heterochromatin. However it is recognized now that it plays an important role in a number of dynamic processes in the cell nucleus, including transcriptional repression and regulation of euchromatic genes. Recent reports demonstrate that HP1 may be involved in the DNA damage response.

Staphylococcal cysteine protease staphopain B (SspB) induces rapid engulfment of human neutrophils and monocytes by macrophages.

Abstract Circulating neutrophils and monocytes constitute the first line of antibacterial defence, which is responsible for the phagocytosis and killing of microorganisms. Previously, we have described that the staphylococcal cysteine proteinase staphopain B (SspB) cleaves CD11b on peripheral blood phagocytes, inducing the rapid development of features of atypical cell death in protease-treated cells. Here, we report that exposure of phagocytes to SspB critically impairs their antibacterial functions.