Andrea Bodnár

Lipopolysaccharide and ceramide docking to CD14 provokes ligand‐specific receptor clustering in rafts

The glycosylphosphatidylinositol‐anchored receptor CD14 plays a major role in the inflammatory response of monocytes to lipopolysaccharide. Here, we describe that ceramide, a constituent of atherogenic lipoproteins, binds to CD14 and induces clustering of CD14 to co‐receptors in rafts. In resting cells, CD14 was associated with CD55, the Fcγ‐receptors CD32 and CD64 and the pentaspan CD47. Ceramide further recruited the complement receptor 3 (CD11b/CD18) and CD36 into proximity of CD14. Lipopolysaccharide, in addition, induced co‐clustering with Toll‐like receptor 4, Fcγ‐RIIIa (CD16a) and the tetraspanin CD81 while CD47 was dissociated. The different receptor complexes may be linked to ligand‐specific cellular responses initiated by CD14.

Intensity-based energy transfer measurements in digital imaging microscopy.

Abstract Investigation of protein-protein associations is important in understanding structure and function relationships in living cells. Using Förster-type resonance energy transfer between donor and acceptor labeled monoclonal antibodies we can assess the cell surface topology of membrane proteins against which the antibodies were raised. In our current work we elaborated a quantitative image microscopic technique based on the measurement of fluorescence intensities to calculate the energy transfer efficiency on a pixel-by-pixel basis. We made use of the broad excitation and emission spectrum of cellular autofluorescence for background correction of images. In addition to the reference autofluorescence images (UV background) we recorded three fluorescent images (donor, acceptor and energy transfer signal) of donor-acceptor double labeled samples, and corrected for spectral spillage of the directly excited donor and acceptor fluorescence into the energy transfer image. After careful image registration we were able to calculate the energy transfer efficiency on a pixel-by-pixel basis. In this paper, we also present a critical comparison between results obtained with this method and other approaches (photobleaching and flow cytometric energy transfer measurements).

Colocalization and nonrandom distribution of Kv1.3 potassium channels and CD3 molecules in the plasma membrane of human T lymphocytes

Distribution and lateral organization of Kv1.3 potassium channels and CD3 molecules were studied by using electron microscopy, confocal laser scanning microscopy, and fluorescence resonance energy transfer. Immunogold labeling and electron microscopy showed that the distribution of FLAG epitope-tagged Kv1.3 channels (Kv1.3/FLAG) significantly differs from the stochastic Poisson distribution in the plasma membrane of human T lymphoma cells. Confocal laser scanning microscopy images showed that Kv1.3/FLAG channels and CD3 molecules accumulated in largely overlapping membrane areas. The numerical analysis of crosscorrelation of the spatial intensity distributions yielded a high correlation coefficient (C = 0.64). A different hierarchical level of molecular proximity between Kv1.3/FLAG and CD3 proteins was reported by a high fluorescence resonance energy transfer efficiency (E = 51%). These findings implicate that reciprocal regulation of ion-channel activity, membrane potential, and the function of receptor complexes may contribute to the proper functioning of the immunological synapse.

A biophysical approach to IL-2 and IL-15 receptor function: Localization, conformation and interactions

Interleukin-2 and interleukin-15 (IL-2, IL-15) are key participants in T and NK cell activation and function. Sharing the beta and gamma receptor subunits results in several common functions: e.g. the promotion of T cell proliferation. On the other hand, due to their distinct alpha receptor subunits, they also play opposing roles in immune processes such as activation induced cell death and immunological memory. Divergence of signaling pathways must ensue already at the plasma membrane where the cytokines interact with their receptors. Therefore understanding molecular details of receptor organization and mapping interactions with other membrane proteins that might influence receptor conformation and function, are of key importance. Biophysical/advanced microscopic methods (fluorescence resonance energy transfer (FRET), fluorescence crosscorrelation spectroscopy (FCCS), near-field scanning optical microscopy (NSOM), X-ray crystallography, surface plasmon resonance, NMR spectroscopy) have been instrumental in clarifying the details of receptor structure and organization from the atomic level to the assembly and dynamics of supramolecular clusters. In this short review some important contributions shaping our current view of IL-2 and IL-15 receptors are presented.

Modification of membrane cholesterol level affects expression and clustering of class I HLA molecules at the surface of JY human lymphoblasts

Recently we have found that class I HLA molecules, key elements of the antigen presentation system for CD8 + effector cells, show a clustered lateral distribution (homoassociation) at the surface of activated human T- and B-lymphocytes as well as virus-transformed T- and B-lymphoblasts, in contrast to a disperse distribution on resting human PBLs (Matk6 et al. (1994) J. Immunol. 152, 3353; Bene et al. (1994) Eur. J. Immunol. 24, 2115). Expression of beta2m-free HLA heavy chains and exogenous beta2m have been shown as potential regulation factors of HLA-I clustering, which in turn may affect cytotoxic activity of CD8+ effector cells. Here we report a study on the effect of plasma membrane-modification (by exogenous cholesterol and phosphatidylcholine) on the expression of free HLA heavy chains and beta2m-bound HLA-I molecules on JY human B-lymphoblasts. The modulating effect of these two treatments on the lipid fluidity of cells was demonstrated by fluorescence anisotropy of DPH lipid probe. The lateral clustering (association) of HLA-I molecules was detected by flow cytometric fluorescence resonance energy transfer (FCET) and digital imaging microscopic photobleaching energy transfer (pbFRET) methods, using flourescein-isothiocyanate (FITC) (donor)- and tetramethyl-rhodamine-isothiocyanate (TRITC) (acceptor)-labeled W6/32 or KE2 antibodies directed against intact HLA-I molecules. Cholesterol enrichment of the plasma membrane increased membrane fluidity and reduced the expression of heavy- and light-chain determinants of HLA-I molecules and free heavy chains (FHCs). This was accompanied with a higher degree of HLA-I clustering as shown by the enhanced intermolecular energy transfer efficiency. In contrast, cholesterol depletion resulted in membrane fluidization and increased expression of HLA-I epitopes. Our results suggest that both cholesterol level and lipid structure/fluidity of the plasma membrane in lymphoblastoid cells may also potentially regulate lateral organization and consequently the presentation efficiency of HLA-I molecules.

Computer program for analyzing donor photobleaching FRET image series

The photobleaching fluorescence resonance energy transfer (pbFRET) technique is a spectroscopic method to measure proximity relations between fluorescently labeled macromolecules using digital imaging microscopy. To calculate the energy transfer values one has to determine the bleaching time constants in pixel‐by‐pixel fashion from the image series recorded on the donor‐only and donor and acceptor double‐labeled samples. Because of the large number of pixels and the time‐consuming calculations, this procedure should be assisted by powerful image data processing software. There is no commercially available software that is able to fulfill these requirements.

Fluorescence Resonance Energy Transfer (FRET)

FRET involves the excitation of a donor fluorophore by incident light within its absorption spectrum. This radiative absorption elevates the donor fluorophore to a higher-energy excited state that would normally decay (return to the ground state) radiatively with a characteristic emission spectrum. If, however, another fluorophore molecule (the acceptor) exists in proximity to the donor with its energy state characterized by an absorption spectrum that overlaps the emission spectrum of the donor, then the possibility of non-radiative energy transfer between donor and acceptor exists. As an example, Figure 1 shows the overlap of the cyan fluorescent protein (CFP) emission spectrum and the yellow fluorescent protein (YFP) absorption spectrum; this pair supports a strong FRET interaction.

Antiproliferative effect of 4-thiouridylate on OCM-1 uveal melanoma cells

PURPOSE Brachytherapy is a well-established, effective treatment for uveal melanoma with a failure rate of 15%. The fatal consequence of unsuccessful treatments offers reason for improvement of the method. The authors propose using an apoptosis inducing agent locally, concomitantly with the well-established therapy, to sensitize the tumor cells. The authors propose a new nontoxic moderately active apoptosis inducing agent, 4-thio-uridylate (s4UMP), for this purpose. METHODS OCM-1 uveal melanoma cells were treated with various concentrations of s4UMP and its effect was monitored by measuring the cell viability (MTT assay). The following apoptosis detecting methods were performed to reveal the mechanism of decreased cell viability: light microscopy, DNA fragmentation assay, determination of caspase 9 activity, and FACS analysis. RESULTS The viability of uveal melanoma cells was decreased by 32%, 40%, and 9% after 24, 48, and 72 hours of treatment with 10 g/mL (30 M) s4UMP. The effect was not dose dependent; it rather followed a saturation-type inhibition and the cells at lower drug concentration recovered after 72 hours. Characteristic apoptotic cell morphology and DNA fragmentation was detected in treated cells. The caspase-9 was activated upon treatment showing maximal activity at 48 hours suggesting the induction of apoptosis. The annexin binding activity further verified the apoptogenic activity of s4UMP. CONCLUSIONS Uveal melanoma, more than other solid tumors, is resistant to most of the chemotherapeutic protocols as indicated by the high mortality rate of metastatic disease. The authors showed that s4UMP, a naturally occurring nucleotide, could induce apoptosis in uveal melanoma cells, suggesting a potential supplementary therapeutic application of the compound.

Non-random distribution of interleukin receptors on the cell surface

Spatial organization of cell surface proteins plays a key role in the process of transmembrane signalling. Receptor clustering and changes in their cell surface distribution are often determining factors in the final outcome of ligand-receptor interactions. There are several techniques for assessing the distribution of protein molecules. Fluorescence resonance energy transfer (FRET) is an excellent tool for determining distance relationships of cell surface molecules. However, it does not provide information on the distribution of molecular clusters. Different kinds of microscopies fill this gap. The evaluation of the images provided by the listed techniques is often questionable. Herein we show the applicability of Ripleys K(t) function as a tool for analyzing the cell surface receptor patterns (Y. Nakamura, et al., Nature 1994, 369, 330-333). We have implemented an effective image processing algorithm for fast localization of gold labels on biological samples. We investigated spatial organization of Interleukin-2R alpha and -15R alpha (IL-2R alpha and IL-15R alpha) on a human CD4+leukaemia T-cell line, Kit225 FT7.10 by using transmission electron microscopy (TEM). TEM analysis showed co-clustering of the two types of alpha-chains even on the few-hundred-nanometer scale. The analysis of our data may contribute to our understanding the action of the IL-2/IL-15 receptor system in T-cell function.

Distinct Spatial Relationship of the Interleukin‐9 Receptor with Interleukin‐2 Receptor and Major Histocompatibility Complex Glycoproteins in Human T Lymphoma Cells

The interleukin-9 receptor (IL-9R) consists of an α subunit and a γ(c) chain that are shared with other cytokine receptors, including interleukin-2 receptor (IL-2R), an important regulator of T cells. We previously showed that IL-2R is expressed in common clusters with major histocompatibility complex (MHC) glycoproteins in lipid rafts of human T lymphoma cells, which raised the question about what the relationship between clusters of IL-2R/MHC and IL-9R is. Confocal microscopy colocalization and fluorescence resonance energy transfer experiments capable of detecting membrane protein organization at different size scales revealed nonrandom association of IL-9R with IL-2R/MHC clusters at the surface of human T lymphoma cells. Accommodation of IL-9Rα in membrane areas segregated from the IL-2R/MHC domains was also detected. The bipartite nature of IL-9R distribution was mirrored by signal transducer and activator of transcription (STAT) activation results. Our data indicate that co-compartmentalization with MHC glycoproteins is a general property of γ(c) receptors. Distribution of receptor chains between different membrane domains may regulate their function.