AbstractsBiology & Animal Science

SUMMARY We characterized and compared the nuclear dynamics of RXR and RAR during activation in single cells on the sub-second scale using live-cell imaging methods. By applying FRAP and fluorescence correlation spectroscopy (FCS), techniques with different temporal resolution, a highly dynamic behaviour could be shown, which is best described by a two-state model of receptor mobility. In the unliganded state most NRs belonged to the fast population. Upon agonist treatment, the ratio of the slow population increased to as a result of an immediate redistribution. Coactivator binding appears to be indispensable for redistribution and has a major contribution to chromatin association. A comparison of the FCS results gained from the RXR and from the RAR studies revealed differences on the behaviour of these two molecules: RXR appeared to be more dynamic. It changed its mobility at a larger scale upon ligand activation. The redistribution detected by FCS was confirmed and refined by single plane illumination microscopy (SPIM-FCS). In addition, it was revealed that the distribution of populations was rather homogenous, as no nuclear architecture related pattern was recognized either before or after activation. Investigation of activation dependent changes showed that the occupancy of RXR’s genomic binding regions increased, but no significant change in the number of sites was revealed by ChIP-Seq. The relationship of the RXR and other nuclear receptors during activation is an interesting question of the field. How the available binding sites, the ligands and the dimer partners determine and regulate the formation of different RXR heterodimers might now be answered. Another interesting topic is the relationship of the chromatin, the transcription factors and the actual active sites of transcriptions. Newer applications partially related to these methods are already available; such as SPIM-FCCS (SPIM - Fluorescence Cross- Correlation Spectroscopy) for the nuclear map of protein-protein interactions, or the GROseq (Global Run-On sequencing) for the detection of active RNA-production on a global scale. When the aim is to describe a mechanism, the key might still be the combined use of methods with different ways of targeting. ÖSSZEFOGLALÁS Az RXR és az RAR sejtmagon belüli mobilitását vizsgáltuk aktiválás során élő-sejtes konfokális mikroszkópiás módszerekkel. A FRAP és az FCS technikák kombinált alkalmazásával széles időskálán tudtuk tanulmányozni a magreceptorok rendkívül dinamikus viselkedését. A rendszert legjobban a két-komponensű szabad diffúziós modell írja le. Ligand távollétében a legtöbb receptor a gyorsabb populációhoz tartozik. Agonista kezelést követően, a populációk eloszlása gyorsan megváltozik; megnő a lassú receptorok aránya. A koaktivátorkötés nélkülözhetetlen ehhez a változáshoz. Az RXR és az RAR receptorok FCS-mérése jelentős dinamikai különbségekre mutatott rá: RXR dinamikusabb molekulaként viselkedett. Az aktiválás során szélesebb spektrumon változott a mobilitása. SPIM-FCS méréseink alapján ezek a…