Miroslaw Zarebski - Academia.edu (original) (raw)

Papers by Miroslaw Zarebski

A. Transmission light (upper two rows) and fluorescence (lower two rows) micrographs (x ... more A. Transmission light (upper two rows) and fluorescence (lower two rows) micrographs (x 40) of control (upper panel) and S. aureus-infected cells (bottom panel) maintained in culture for 2h (A) and 1 day (B), 4- (C), 5- (D), and 6-days (E). Propidium iodide-positive cells represent infected host cells with leaky plasma membranes. B. BODIPY495/503 staining of lipid droplets of control and S. aureus infected hMDM cultures on five consecutive days post-phagocytosis. Cells were permeabilized with 0.2 % Triton X-100 and stained as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001409#s4" target="_blank">Materials and Methods</a> section. All scale bars = 10 µm. C. Transmission electron micrographs of control (panel A) and infected cells (panels B and C) one day post-phagocytosis. Black arrowheads point to lipid droplets. Magnification: x10,000 (A and C) and ×7,500 (B). The photographs presented are representative of a minimum of 20 fields observed. D. Cytotoxicity (%) of S. aureus infection. Plasma membrane permeabilization or cell lysis induced in macrophage cultures by S. aureus infection at different MOI was determined as LDH activity levels. Cytotoxicity was calculated according to the formula: % cytotoxicity = [(experimental value–low control)/(high control–low control)] × 100, where a low control is the LDH activity in the conditioned medium of the control non-infected culture, while the LDH activity in the whole cell culture with cells lysed with detergent (2% Triton X-100) constitutes a high control. An experimental value was the activity in the conditioned medium from the culture infected with S. aureus. According to this calculation the control non-infected culture was assumed to show 0% cytotoxicity. All assays were performed in triplicate.</p

The photographs presented are representative of a minimum of 20 fields observed. hMDMs w... more The photographs presented are representative of a minimum of 20 fields observed. hMDMs were allowed to engulf bacteria for 2h at a MOI of 25, and infected macrophages were fixed with Karnovsky's fixative either immediately following phagocytosis (2 h), or on consecutive days post-phagocytosis, before being processed by standard electron microscopic techniques. At any given point post-phagocytosis dividing bacterial cells were visible (arrowheads). Magnified views of selected bacteria framed on main micrographs are shown in the bottom row labeled from A' to H', respectively. A. Control, non-infected hMDM has a morphological appearance typical for that of professional phagocyte, with vesicles localized around the nucleus (N) and only few lipid droplets (arrows). Magnification: x14,000. B. Immediately post-phagocytosis (2h), the bacteria were located predominantly in very tight membranous compartments (arrows). Magnification: x50,000. C. Already 1 day post-phagocytosis the majority of S. aureus cells were found in clearly defined membrane-enclosed vacuoles (arrows). A partially degraded bacterium in the vacuole is framed. Magnification: x20,000. D and E. At day 2 the cells of S. aureus can only be found in well defined vacuoles (arrows), occasionally spacious vacuoles (asterisk) containing several bacteria. Magnification: x32,500. F. On day 3 S. aureus persists in vacuoles which often reveal signs of partial membrane discontinuity (arrows). Magnification: x32,500. G and H. On day 4, the majority of bacteria were found in vacuoles with profoundly damaged or fully disintegrated membranes (arrow). Magnification: x54,000 (G) and x32,000 (H).</p

Oligonucleotides used in this study.</p

A. CFU of S. aureus in cell lysates and culture medium on six consecu... more A. CFU of S. aureus in cell lysates and culture medium on six consecutive days post-infection. Macrophages were allowed to engulf S. aureus at a MOI of 25 for 2 h, washed, and extracellular bacteria killed by gentamycin. Macrophages were cultured in media without antibiotics. At consecutive days post-phagocytosis media was aspired and hMDMs were lysed. Both conditioned media and cell lysates were plated onto TSA for CFU enumeration. The data shown details of one representative experiment (means±SD) of the 76 we performed in triplicate. B. CFU of S. aureus and LDH activity levels (A490 nm) in the conditioned culture medium on six consecutive days post infection (left panel); and transmission (upper row) and fluorescent (propidium iodide staining, lower row) light micrographs (x40) of S. aureus-infected cells maintained for the indicated time interval post infection. C. Confocal fluorescence microscopy images of hMDMs after 1, 3, and 5 days post-infection at MOI of 25. Cells were fixed, treated with RNase and stained with acridine orange (see Experimental Procedures section). Internalized bacteria are observed as green spots. All scale bars = 10 µm. D. Confocal fluorescence microscopy images of viable bacteria in hMDMs on the 5th day post-phagocytosis. Infected hMDMs were collected, permeabilized with 0.2 % Triton X-100 and double- labeled with propidium iodide and Syto9 (LIVE/DEAD BacLight Kit). Viable S. aureus cells are stained green while red signals represent dead bacteria and the host cell's nuclear DNA stained mainly with propidium. Scale bar = 7.5 µm. E. Specific PCR amplification of the S. aureus 16S rRNA gene (left panel) from cell lysates and the MLVF pattern (right panel) of bacteria infecting cells on consecutive days post-phagocytosis (numbers above lanes). Lane 0, sample collected immediately upon completion of phagocytosis (2h). Lane N, the MLVF pattern of S. aureus Newman before infection.</p

Bacterial strains.</p

Intracellular persistence of S. aureus was determined as described in... more Intracellular persistence of S. aureus was determined as described in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001409#pone-0001409-g003" target="_blank">Fig. 3A</a>. Bars represent mean CFU values ±SD and are representative of at least three separate experiments, performed in triplicate, using hMDMs derived from different donors. An electron microphotograph of infected hMDMs was obtained 1 day post-phagocytosis and shows partially (white arrows) and totally degraded (black arrows) bacterial cells inside tight vacuoles. At this time point some apparently intact bacterial cells are also present (black arrowheads). Magnification: x32,500. The photograph presented is a representative of a minimum of 20 fields observed.</p

Control hMDMs, and cells 4 days post infection with S. aureus strain ... more Control hMDMs, and cells 4 days post infection with S. aureus strain Newman, were stimulated to phagocytose either live bacteria at a MOI of 1∶25 (1.25×107 CFU) (panel A) or latex beads (panel B). Generation of reactive oxygen species (ROS) determined as the level of the mean fluorescence intensity (MFI) was measured at various time intervals. The data shown is representative of at least three separate experiments, performed in triplicate, using hMDMs derived from different donors.</p

Quantitative Analysis of Spatial Association between Discrete Events Represented by Small Foci in... more Quantitative Analysis of Spatial Association between Discrete Events Represented by Small Foci in Multicolor 3D Confocal Microscopy Krzysztof Berniak1, Tytus Bernas2, Hong Zhao3, Paulina Rybak4, Miroslaw Zarebski4, Zbigniew Darzynkiewicz5, Jerzy Dobrucki4 1Jagiellonian University, Cracow, Poland, Poland, 2Jagiellonian University in Krakow, 3New York Medical College, 4Jagiellonian University, Cracow, Poland, 5Brander Cancer Research Institute, New York, Medical College

We here describe a technique termed STRIDE (SensiTive Recognition of Individual DNA Ends), which ... more 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. Sensitivity of STRIDE was tested using 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 TUNEL assay or methods based on monitoring of recruitment of repair proteins or histone modifications at the damage site (e.g. γH2AX). Even individual genome site-specific DNA double-strand cuts induced by CRISPR/Cas9, as well as individual single-strand DNA scissions induced by the nickase version of Cas9, can be detected by STRIDE and precisely localized within the cell nucleus. We further show that STRIDE can detect ...

Biological chemistry, Apr 25, 2018

FK506-binding proteins (FKBPs) belong to a distinct class of immunophilins that interact with imm... more 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. The Drosophila melanogaster peptidyl-prolyl cis-trans isomerase FK506-binding protein of 39 kDa (FKBP39) is thought to act as a transcriptional modulator of gene expression in 20-hydroxyecdysone and juvenile hormone signal transduction. The aim of this study was to analyze the molecular determinants responsible for the subcellular distribution of an FKBP39-yellow fluorescent protein (YFP) fusion construct (YFP-FKBP39). We found that YFP-FKBP39 was predominantly nucleolar. To identify the nuclear localization signal (NLS), a series of YFP-tagged FKBP39 deletion mutants were prepared and examined in vivo. The identified NLS signal is located in a basic domain. Detailed muta...

Journal of virology, Feb 15, 2017

First steps of human coronavirus NL63 (HCoV-NL63) infection were previously described. The virus ... more First steps of human coronavirus NL63 (HCoV-NL63) infection were previously described. The virus binds to target cells by heparan sulfate proteoglycans, and interacts with the ACE2 protein. Subsequent events, including virus internalization and trafficking, remain to be elucidated. In this study, we mapped the process of HCoV-NL63 entry into LLC-Mk2 cell line and ex vivo 3D tracheobronchial tissue.Using a variety of techniques we have shown that HCoV-NL63 virions require endocytosis for successful entry to the LLC-MK2 cells, and interaction between the virus and the ACE2 molecule triggers recruitment of clathrin. Subsequent vesicle scission by dynamin results in virus internalization, and the newly formed vesicle passes the actin cortex, what requires active cytoskeleton rearrangement. Finally, acidification of the endosomal microenvironment is required for successful fusion and release of viral genome into the cytoplasm. Also for 3D tracheobronchial tissue cultures we observed that...

Journal of Leukocyte Biology, 2017

Heat shock proteins (HSPs) are typical intracellular chaperones which also appear on the cell sur... more 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. In this report, we provide the first evidence, to our knowledge, of the geldanamycin (Ge)-inhibitable HSP90 on the surface of live monocyte-derived Mϕs (hMDMs). Using cytometry and specific Abs, we showed both HSP90 isoforms (α and β) on the surface of human monocytes and hMDMs. The cell-surface HSP90 pool was also labeled with cell-impermeable Ge derivatives. Confocal analysis of hMDMs revealed that HSP90-inhibitor complexes were rapidly clustered on the cell surface and recycled through the endosomal compartment. This finding suggests that the N-terminal (ATPase) domain of HSP90 is exposed and accessible from the extracellular space. To study the role of cell-surface HSP90 in pattern recognition, we used pathogen (PAMPs)- or apoptotic cell-associated molecular patterns (ACAMPs). We showed that blocking the cell-surface HSP90 pool leads to a dramatic decrease in TNF production by monocytes and hMDMs exposed to soluble (TLRs-specific ligands) and particulate [bacteria (SA) and (PG)] PAMPs. Surprisingly, in hMDMs the functional cell-surface HSP90 was not necessary for the engulfment of either apoptotic neutrophils or bacteria. The presented data suggest that the cell-surface HSP90 is a "signaling complex chaperone," with activity that is essential for cytokine response but not for target engulfment by Mϕ.

Oncotarget, 2016

Phosphorylation of histone H2AX on serine 139 (γH2AX) is an early step in cellular response to a ... more 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. We studied DNA damage induced in A549 human lung adenocarcinoma cells by topoisomerase type I and II inhibitors (0.2 µM camptothecin, 10 µM etoposide or 0.2 µM mitoxantrone for 1 h), and using 3D high resolution quantitative confocal microscopy, assessed the number, size and the integrated intensity of immunofluorescence signals of individual γH2AX foci induced by these drugs. Also, investigated was spatial association between γH2AX foci and foci of 53BP1, the protein involved in DSB repair, both in relation to DNA replication sites (factories) as revealed by labeling nascent DNA with EdU. Extensive 3D and correlation data analysis demonstrated that γH2AX foci exhibit a wide range of sizes and levels of H2AX phosphorylation, and correlate differently with 53BP1 and DNA replication. This is the first report showing lack of a link between low level phosphorylation γH2AX sites and double-strand DNA breaks in cells exposed to topoisomerase I or II inhibitors. The data are discussed in terms of mechanisms that may be involved in formation of γH2AX sites of different sizes and intensities.

Journal of leukocyte biology, Jan 27, 2015

The sphingolipid analog fingolimod is known to induce apoptosis of tumor cells and lymphocytes. I... more 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. Fingolimod also induces typical apoptotic features, including rapid externalization of phosphatidylserine and activation of caspase-8. Fingolimod-induced neutrophil death is independent of sphingosine-1-phosphate receptors and positively regulated by protein phosphatase A. Externalization of phosphatidylserine and heat shock protein 27 can be partially inhibited by inhibitors of caspase-8 [Z-Ile-Glu(O-Me)-Thr-Asp(O-Me)-fluoromethyl ketone], receptor-interacting protein kinase 1 (necrostatin-1), recepto...

PLOS ONE, 2015

Drosophila melanogaster germ cell-expressed protein (GCE) belongs to the family of bHLH-PAS trans... more 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. Recently, it has been shown that the functions of MET and GCE are only partially redundant and tissue specific. The ability of bHLH-PAS proteins to fulfill their function depends on proper intracellular trafficking, determined by specific sequences, i.e. the nuclear localization signal (NLS) and the nuclear export signal (NES). Nevertheless, until now no data has been published on the GCE intracellular shuttling and localization signals. We performed confocal microscopy analysis of the subcellular distribution of GCE fused with yellow fluorescent protein (YFP) and YFP-GCE derivatives which allowed us to characterize the details of the subcellular traffic of this protein. We demonstrate that GCE possess specific pattern of localization signals, only partially consistent with presented previously for MET. The presence of a strong NLS in the Cterminal part of GCE, seems to be unique and important feature of this protein. The intracellular localization of GCE appears to be determined by the NLSs localized in PAS-B domain and C-terminal fragment of GCE, and NESs localized in PAS-A, PAS-B domains and C-terminal fragment of GCE. NLSs activity can be modified by juvenile hormone (JH) and other partners, likely 14-3-3 proteins.

PLOS ONE, 2015

Human coronavirus (HCoV) NL63 was first described in 2004 and is associated with respiratory trac... more 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. The coronaviral nucleocapsid protein is abundantly present in infected cells. It is a multi-domain, multi-functional protein important for viral replication and a number of cellular processes. The aim of the present study was to characterize the HCoV-NL63 nucleocapsid protein. Biochemical analyses revealed that the protein shares characteristics with homologous proteins encoded in other coronaviral genomes, with the N-terminal domain responsible for nucleic acid binding and the C-terminal domain involved in protein oligomerization. Surprisingly, analysis of the subcellular localization of the N protein of HCoV-NL63 revealed that, differently than homologous proteins from other coronaviral species except for SARS-CoV, it is not present in the nucleus of infected or transfected cells. Furthermore, no significant alteration in cell cycle progression in cells expressing the protein was observed. This is in stark contrast with results obtained for other coronaviruses, except for the SARS-CoV.

Journal of Virology, 2014

Human coronavirus NL63 (HCoV-NL63) is an alphacoronavirus that was first identified in 2004 in th... more 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. However, comparative analysis showed that directed expression or selective scission of the ACE2 protein had no measurable effect on virus adhesion. In contrast, binding of HCoV-NL63 to heparan sulfates was required for viral attachment and i...

European Biophysics Journal, 2014

PLoS ONE, 2008

Although considered to be an extracellular pathogen, Staphylococcus aureus is able to invade a va... more Although considered to be an extracellular pathogen, Staphylococcus aureus is able to invade a variety of mammalian, nonprofessional phagocytes and can also survive engulfment by professional phagocytes such as neutrophils and monocytes. In both of these cell types S. aureus promptly escapes from the endosomes/phagosomes and proliferates within the cytoplasm, which quickly leads to host cell death. In this report we show that S. aureus interacted with human monocyte-derived macrophages in a very different way to those of other mammalian cells. Upon phagocytosis by macrophages, S. aureus persisted intracellularly in vacuoles for 3-4 days before escaping into the cytoplasm and causing host cell lysis. Until the point of host cell lysis the infected macrophages showed no signs of apoptosis or necrosis and were functional. They were able to eliminate intracellular staphylococci if prestimulated with interferon-c at concentrations equivalent to human therapeutic doses. S. aureus survival was dependent on the alternative sigma factor B as well as the global regulator agr, but not SarA. Furthermore, isogenic mutants deficient in a-toxin, the metalloprotease aureolysin, protein A, and sortase A were efficiently killed by macrophages upon phagocytosis, although with different kinetics. In particular a-toxin was a key effector molecule that was essential for S. aureus intracellular survival in macrophages. Together, our data indicate that the ability of S. aureus to survive phagocytosis by macrophages is determined by multiple virulence factors in a way that differs considerably from its interactions with other cell types. S. aureus persists inside macrophages for several days without affecting the viability of these mobile cells which may serve as vehicles for the dissemination of infection.

Oligonucleotides used in this study.</p

Bacterial strains.</p

Biological chemistry, Apr 25, 2018

Journal of virology, Feb 15, 2017

Journal of Leukocyte Biology, 2017

Oncotarget, 2016

Journal of leukocyte biology, Jan 27, 2015

PLOS ONE, 2015

Journal of Virology, 2014

European Biophysics Journal, 2014

PLoS ONE, 2008