Mapping transcription factors dynamics and interactions by advanced fluorescence microscopy techniques - eScholarship

Field: Biochemistry, Genetics and Molecular Biology (Life Sciences)

ABSTRACT spatial resolution is especially advantageous for bacterial cells because of their small sizes. In the past few years the spatial organization and dynamics of a variety of bacterial cellular structures and protein macromachineries have been revealed with unprecedented details. As the field matures, it is now time to focus on the functional aspect of the observed spatial organizations and dynamics. Are they essential in carrying out a specific cellular function? Do they play a regulatory role in controlling the on and off of a certain cellular process? In this work I will present a few examples from our laboratory that examine the spatial and functional organization of macromolecules involved in bacterial cell division. PG-003 Mapping transcription factors dynamics and interactions by advanced fluorescence microscopy techniques Martin Stortz 1 , Luciana Bruno 2 , Paolo Annibale 3 , Enrico Gratton 3 , Adali Pecci 2 , Valeria Levi 4,5 IFIByNE-Conicet, 2 IFIBA-Conicet & Dept. of Physics, University of Buenos Aires, 3 LFD-University of California, 4 Dept. of Biological Chemistry, University of Buenos Aires, 5 IQUIBICEN-Conicet Transcription factors (TF) exert their function by interacting with other proteins and binding to DNA. The nucleus is a compartmentalized space, and the spatial organization of TFs and their partners represents other step of gene expression regulation. We used the glucocorticoid receptor (GR) as a model of TF’s mechanism of action. GR is a ligand-activated TF with a relevant role in physiology and a great variety of effects. It can be recruited to specific response elements on DNA or interact with other TFs. Also, the activity of GR is modu- lated by different co-regulators, e.g. TIF2/GRIP1. GR and TIF2 do not distribute homogeneously within the nucleus but accumulate in distinctive clusters. The functional role of this particular intranuclear organization remains unknown. We used advanced fluorescence microscopy techniques to study the dynamics of GR and TIF2 in the nucleus of living cells with high spatial and time resolution. GR and TIF2 fused to fluorescent tags were transiently expressed in newborn hamster kidney (BHK) cells and visualized by a confocal microscope. Fluorescence correlation spectroscopy (FCS) experiments were carried on to measure the intranuclear mobil- ity of both proteins. The method is based on the analysis of fluorescence intensity fluctuations due to the movement of fluorescent molecules in and out the confocal volume. The data could be fitted with a model that considers a free diffusion of TIF2 and GR in the nucleus and their binding to fixed targets. We also stud- ied the dynamics of different GR mutants in the presence of different ligands and our results suggest that the binding depends on DNA. Both GR and TIF2 autocorrelation curves reveal an increase in the bound popula- tion upon GR activation by its agonist dexamethasone (Dex). A cross-correlation analysis showed that, as expected, Dex-stimulus increases the population of GR-TIF2 complexes. Without hormone, GR shows a homogeneous distribution and TIF2 forms large clusters in the nucleus. Upon Dex-binding, GR accumulates in the nucleus, is rapidly recruited to TIF2 foci and there is an important re-distribution of both proteins, that co-localize in the same pattern of small intranuclear clusters. The dynamics of GR and TIF2 molecules at these clusters were studied by performing orbital-scanning measurements, tracking the clusters position in silico and analyzing the intensity fluctuations of the clusters along time. A positive cross-correlation between both channels indicates that Dex-bound GR and TIF2 interact at these foci and dissociate from them forming TIF2- GR hetero-complexes. In conclusion, advanced fluorescence microscopy methods allowed obtaining a dynamical map of GR distribution and function in the nucleus of mammalian living cells. PG-004 Assembly of membrane pores as a mechanism for amyloid cytotoxicity by the bacterial prionoid RepA-WH1 ~ ez-Ramirez 1 , Mercedes Jimenez 1 , Germ an Rivas 1 , Rafael Giraldo 1 Cristina Fern andez 1 , Rafael N u n gicas, CSIC Centro de Investigaciones Biol o Amyloid fibril formation is associated with human neurodegenerative diseases. Prefibrillar oligomers formed during the fibril assembly process, rather than mature fibrils are known to be central to disease