Cell Biophysics Group of Hung. Acad. Sci.
Heads of the group:
Professor Emeritus, member of EMBO, member of the Hungarian Academy of Sciences
Co-Principle Investigator, Senior Research Fellow
Dr. Zsolt Fazekas, Research fellow
Ferenc Papp, Junior research fellow
Rita Szabó, Laboratory assistant
Gábor Mocsár, Phd student
Éva Nagy, Phd student
Enikő Nizsalóczki, Phd student
Nikoletta Szalóki, Phd student
Julianna Volkó, Phd student
Balázs Bravics, Phd student
Attila Horváth, Diploma student
Tibor Szekeres, Diploma student
Tamás Domoszlai, Diploma student
Gergely Molnár, Diploma student
István Csomós, Diploma student
Experimental observations accumulated in the past decades led to the “membrane microdomain” concept describing organization of membrane components into non-random, well-defined patterns at different hierarchical levels with diameters from 10 nm to several microns. The primary target of external stimuli is the plasma membrane and non-random co-distribution of membrane proteins plays an important role in signal transduction across the cell membrane. Our research efforts are mainly focused on mapping protein patterns formed by molecules playing key roles in T-cell-mediated immunity (e.g. IL-2/15R, -9R, MHC I and II, Kv1.3 K+ channel, etc.). Factors (lipid environment, membrane potential, etc.) controlling the cell surface distribution of these proteins as well as functional consequences of their association patterns are also under investigation.
Interleukin-2 and -15 (IL-2, -15) are key participants in T and NK cell homeostasis and function. Their multisubunit receptors share the β and γc chains, which results in several common functions (e.g. promotion of T cell proliferation). On the other hand, presumably due to their distinct α chains, they also play opposing roles in immune processes such as AICD and immunological memory. Divergence of signaling pathways must ensue already at the plasma membrane where the cytokines interact with their receptors. We have used fluorescence resonance energy transfer (FRET), near-field scanning optical microscopy (NSOM) and fluorescence cross-correlation spectroscopy (FCCS) to map the pair-wise interactions of the IL-2/15R subunits in CD4+ T lymphoma cells. Our data are compatible with a tetrameric structure of the IL-2/15R complex, where cytokine binding rearranges the receptor subunits to form the appropriate high-affinity receptor trimer (αβγc), whereas the “unused” α-chain rotates away).
These receptor complexes are partitioned into membrane microdomains (lipid rafts) and form supramolecular clusters with other proteins (MHC I and II, ICAM-1). Clusters of IL-2/15R with MHC and ICAM-1 were detected in CD4+ T cells derived from draining lymph nodes and peripheral blood of colorectal carcinoma patients. Interestingly, in draining lymph nodes interactions weakened as compared to the control, implying that different stimuli/tissue environment can change association patterns of the same cell type.
In collaboration with the Dept. of Biophysics of Macromolecules (DKFZ, Heidelberg) we applied FRET and FCCS and molecular dynamic modeling to describe the conformation of the activator protein-1 transcription factor. AP-1, which is induced at the last stage of the IL-2/15 signaling cascade, is a heterodimer containing one of each of the Fos and Jun subfamilies of basic-region leucine-zipper proteins. Our studies imply that the C terminal ends of the Jun-Fos heterodimer are in the close vicinity of each other. Measurements performed with Fos molecules of different lengths suggested that, in addition to N terminal transactivation domains, the C termini may also contribute to DNA binding and may have transactivating function. Current research projects
Proper function of T cells requires the highly organized intercellular communication with antigen presenting or target cells, which is achieved through the “immunological synapse” (IS), a contact region having high complexity. The membrane potential (MP) of T cells is a key determinant of the transmission of the mitogenic signal upon activation. Recently we have proven that Kv1.3, the determinant voltage gated K+ channel controlling MP in T cells, is recruited to the IS. We have also found that T cells engaged in the IS respond to depolarizations with characteristic, large amplitude oscillations. This finding brings up the possibility of the electrophysiological regulation of the function and assembly of the IS. In collaboration with the Electrophysiology Group we characterize the conditions required for triggering MP oscillations in conjugated T cells. Recent evidence suggests the paracrine signaling of IL-2R in homotypic interaction of activating T cells. We investigate the mechanism of this interaction, which may by similar to the transpresentation of IL-15 between dendritic and T cells during activation. The process may play an important role in acute T cell lymphomas where IL-2R expression is increased. The nuclear receptors retinoic acid receptor (RAR) and retinoid X receptor (RXR) regulate the expression of their target genes in a ligand dependent manner. According to the classical “molecular switch” model, the DNA-associated receptor binds a corepressor in the absence of ligand. Upon agonist binding the corepressor is released and a coactivator is bound, promoting gene transcription. In collaboration with the Nuclear Receptor Group we analyze the molecular events following receptor activation by applying biophysical techniques (FCS, FRET) to describe the mobility and pair-wise interactions of receptors and their cofactors.
Figure 1: Possible cytokine dependent arrangements of the IL-2/IL-15 receptor complex together with MHC I and MHC II
Figure 2: MD modeling structures of the Jun-Fos complex compatible with FRET results.
Brock R, Vámosi G, Vereb G, Jovin TM: Rapid characterization of green fluorescent protein fusion proteins on the molecular and cellular level by fluorescence correlation microscopy. Proc Natl Acad Sci U S A 96(18): 10123-8, 1999.
Vereb G, Matkó J, Vámosi G, Ibrahim SM, Magyar E, Varga S, Szöllősi J, Jenei A, Gáspár R Jr, Waldmann TA, Damjanovich S: Cholesterol-dependent clustering of IL-2Ralpha and its colocalization with HLA and CD48 on T lymphoma cells suggest their functional association with lipid rafts. Proc Natl Acad Sci U S A 97(11): 6013-8, 2000.
Panyi G, Bagdány M, Bodnár A, Vámosi G, Szentesi G, Jenei A, Mátyus L, Varga S, Waldmann TA, Gáspár R, Damjanovich S: Colocalization and nonrandom distribution of Kv1.3 potassium channels and CD3 molecules in the plasma membrane of human T lymphocytes. Proc Natl Acad Sci U S A 100(5): 2592-7, 2003.
Panyi G, Vámosi G, Bodnár A, Gáspár R, Damjanovich S: Looking through ion channels: recharged concepts in T cell signaling. Trends in Immunology, 25(11):565-9, 2004.
Vámosi G, Bodnár A, Vereb G, Jenei A, Goldman CK, Dubois S, Langowski J, Tóth K, Mátyus L, Szöllősi J, Waldmann TA, Damjanovich S: Interleukin-2 and -15 receptor alpha subunits are co-expressed in a supramolecular receptor cluster in lipid rafts of T-cells. Proc Natl Acad Sci U S A. 101(30):11082-7, 2004.
Vámosi G, Baudendistel N, von der Lieth CW, Szalóki N, Mocsár G, Müller G, Brázda P, Waldeck W, Damjanovich S, Langowski J, Tóth K: Conformation of the c-Fos / c-Jun complex in vivo: a combined FRET, FCCS and MD-modeling study. Biophys J. 94(7):2859-68, 2008
Bodnár A, Nizsalóczki E, Mocsár G, Szalóki N, Waldmann TA, Damjanovich S, Vámosi G: A biophysical approach to IL-2 and IL-15 receptor function: localization, conformation and interactions. Immunol Lett. 116(2):117-25, 2008.
Fazekas Z, Petrás M, Fábián A, Pályi-Krekk Z, Nagy P, Damjanovich S, Vereb G, Szöllősi J: Two-sided fluorescence resonance energy transfer for assessing molecular interactions of up to three distinct species in confocal microscopy. Cytometry A. 73(3):209-19, 2008.
de Bakker BI, Bodnár A, van Dijk EMHP, Vámosi G, Damjanovich S, Waldmann TA, van Hulst NF, Jenei A, Garcia-Parajo MF: Nanometer scale organization of the alpha subunits of interleukin-2 and -15 receptors (IL2Ra-IL15Ra) on Kit 225 FT7.10 human T lymphoma cells. J. Cell Sci.;121(Pt 5):627-33, 2008.
Panyi G., Vámosi G, Bacsó Z, Bagdány M, Bodnár A, Varga Z, Gáspár R, Mátyus L, Damjanovich S: Kv1.3 potassium channels are localized in the immunological synapse formed between cytotoxic and target cells. Proc Natl Acad Sci U S A 101(5): 1285-90, 2004.