Jerzy Dobrucki

Minimizing photobleaching during confocal microscopy of fluorescent probes bound to chromatin: role of anoxia and photon flux

Exposure to light can destroy the ability of a molecule to fluoresce. Such photobleaching limits the use of fluorescence and confocal microscopy in biological studies. Loss of fluorescence decreases the signal‐to‐noise ratio and so image resolution; it also prevents the acquisition of meaningful data late during repeated scanning (e.g. when collecting three‐dimensional images). The aim of this work was to investigate the role of oxygen in the photobleaching of fluorophores bound to DNA in fixed cells, and to explore whether anoxia could minimize such bleaching. Anoxia significantly reduced bleaching rates and changed the order of reaction of both propidium iodide (an intercalator) and chromomycin A3 (a minor‐groove binder) bound to DNA; it afforded the greatest protection at low photon fluxes. However, it had no effect on the bleaching of the green fluorescent protein (GFP) covalently attached to a histone and so bound to DNA, probably because the protein shielded the chromophore from oxygen. Bleaching of all three fluorophores depended on photon flux. Practical ways of minimizing bleaching were examined, and examples of three‐dimensional images of DNA marked by propidium and GFP (collected under standard and optimized conditions) are presented.

Large-conductance K+ channel openers NS1619 and NS004 as inhibitors of mitochondrial function in glioma cells

Recently, it has been reported that large-conductance Ca(2+)-activated potassium channels, also known as BK(Ca)-type potassium channels, are present in the inner mitochondrial membrane of the human glioma LN229 cell line. Hence, in the present study, we have investigated whether BK(Ca)-channel openers (BK(Ca)COs), such as the benzimidazolone derivatives NS004 (5-trifluoromethyl-1-(5-chloro-2-hydroxyphenyl)-1,3-dihydro-2H-benzimidazole-2-one) and NS1619 (1,3-dihydro-1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl)-2H-benzimidazol-2-one), affect the functioning of LN229 glioma cell mitochondria in situ. We examined the effect of BK(Ca)COs on mitochondrial membrane potential, mitochondrial respiration and plasma membrane potassium current in human glioma cell line LN229. We found that BK(Ca)COs decrease the mitochondrial membrane potential with an EC(50) value of 3.6+/-0.4 microM for NS1619 and 5.4+/-0.8 microM for NS004. This mitochondrial depolarization was accompanied by an inhibition of the mitochondrial respiratory chain. Both BK(Ca)COs induced whole-cell potassium current blocked by charybdotoxin, as measured by the patch-clamp technique. The BK(Ca)COs had no effect on membrane bilayer conductance. Moreover, the inhibition of mitochondrial function by NS004 and NS1619 was without effect on cell survival, as measured by lactate dehydrogenase release from the cells.

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. Recognition of apoptotic cells is predominantly connected with CD16+ monocytes (CD14high CD16+ and CD14dim CD16+) and requires CD36. Clearance of apoptotic polymorphonuclear leucocytes appears to be independent of the CD14 mechanism. Uptake of apoptotic Jurkat T cells by monocytes is CD14 and CD36 dependent. Liposomes containing phosphatidyl‐l‐serine reduce binding of apoptotic polymorphonuclear leucocytes. Lipopolysaccharide‐activated subpopulations of monocytes while in contact with apoptotic cells produce more anti‐inflammatory cytokine interleukin‐10 whereas the production of pro‐inflammatory cytokines, tumour necrosis factor‐α and interleukin‐1β is reduced.

Spectral-spatial ESR imaging as a method of noninvasive biological oximetry

Abstract Using a spectral range of 3.5 G, spectral-spatial ESR imaging has been applied to obtain noninvasively a quantitative spatial description of intracellular oxygen concentrations in vitro . The results of simulations and experiments with phantoms are presented to demonstrate the validity of this technique.

Studies on the role of the receptor protein motifs possibly involved in electrostatic interactions on the dopamine D1 and D2 receptor oligomerization

We investigated the influence of an epitope from the third intracellular loop (ic3) of the dopamine D2 receptor, which contains adjacent arginine residues (217RRRRKR222), and an acidic epitope from the C‐terminus of the dopamine D1 receptor (404EE405) on the receptors’ localization and their interaction. We studied receptor dimer formation using fluorescence resonance energy transfer. Receptor proteins were tagged with fluorescence proteins and expressed in HEK293 cells. The degree of D1–D2 receptor heterodimerization strongly depended on the number of Arg residues replaced by Ala in the ic3 of D2R, which may suggest that the indicated region of ic3 in D2R might be involved in interactions between two dopamine receptors. In addition, the subcellular localization of these receptors in cells expressing both receptors D1–cyan fluorescent protein, D2–yellow fluorescent protein, and various mutants was examined by confocal microscopy. Genetic manipulations of the Arg‐rich epitope induced alterations in the localization of the resulting receptor proteins, leading to the conclusion that this epitope is responsible for the cellular localization of the receptor. The lack of energy transfer between the genetic variants of yellow fluorescent protein‐tagged D2R and cyan fluorescent protein‐tagged D1R may result from differing localization of these proteins in the cell rather than from the possible role of the D2R basic domain in the mechanism of D1–D2 receptor heterodimerization. However, we find that the acidic epitope from the C‐terminus of the dopamine D1 receptor is engaged in the heterodimerization process.

Blue-light-activated phototropin2 trafficking from the cytoplasm to Golgi/post-Golgi vesicles

Summary The blue-light-induced trafficking of the UVA/blue light receptor phototropin2 is shown. Evidence is provided for the presence of two pathways, one directing phototropin2 to the Golgi and post-Golgi vesicles, and the other to degradation.

Fenofibrate enhances barrier function of endothelial continuum within the metastatic niche of prostate cancer cells

Objective: Extravasation of circulating cancer cells is an important step of the metastatic cascade and a potential target for anti-cancer strategies based on vasoprotective drugs. Reports on anti-cancer effects of fenofibrate (FF) prompted us to analyze its influence on the endothelial barrier function during prostate cancer cell diapedesis. Research design and methods: In vitro co-cultures of endothelial cells with cancer cells imitate the ‘metastatic niche’ in vivo. We qualitatively and quantitatively estimated the effect of 25 μM FF on the events which accompany prostate carcinoma cell diapedesis, with the special emphasis on endothelial cell mobilization. Results: Fenofibrate attenuated cancer cell diapedesis via augmenting endothelial cell adhesion to the substratum rather than through the effect on intercellular communication networks within the metastatic niche. The inhibition of endothelial cell motility was accompanied by the activation of PPARα-dependent and PPARα-independent reactive oxygen species signaling, Akt and focal adhesion kinase (FAK) phosphorylation, in the absence of cytotoxic effects in endothelial cells. Conclusions: Fenofibrate reduces endothelial cell susceptibility to the paracrine signals received from prostate carcinoma cells, thus inhibiting endothelial cell mobilization and reducing paracellular permeability of endothelium in the metastatic niche. Our data provide a mechanistic rationale for extending the clinical use of FF and for the combination of this well tolerated vasoactive drug with the existing multidrug regimens used in prostate cancer therapy.

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. 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.

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. 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 Staphylococcus aureus (SA) and Porphyromonas gingivalis (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.

The cellular internalization of liposome encapsulated protoporphyrin IX by HeLa cells.

The proper lipid composition of liposomes designed to carry drugs determines their surface properties ensuring their accumulation within selected tissue. The electrostatic potential and surface topology of liposomes affect the internalization by single cells. The high-resolution imaging of cancer cells and the distribution of protoporphyrin-loaded liposomes within the cytoplasm and its dependence on the liposome surface properties are presented. In the paper, HeLa cells were used to investigate the uptake of porphyrin-loaded liposomes and liposomes alone by means of confocal and differential interference contrast microscopies. The effect of liposomes surface electrostatic potential and surface topology on their intracellular distribution was evaluated. The time evolution of the intracellular distribution of liposomes labelled with Rhodamine-PE was examined on HeLa cells. These studies allow for the identification of the liposome lipid composition so the efficient delivery of the active substance to cancer cells will be achieved. The obtained results showed that neutral PC-liposomes are the most efficiently internalized by HeLa cells. Moreover, results showed that properties of liposomes affect not only the internalization efficiency of the photosensitizer but also its distribution within the cells, as revealed by colocalization measurements.