In vivo investigation of estrogenic activity by fluorescence techniques

Hormone binding nuclear receptors are transcription factors that activate gene transcription by binding to specific DNA sequences in the gene promoter region and then recruiting the necessary coactivator proteins for transcriptional activation. The knowledge of the function of these receptors, e.g. how they activate or repress transcription are deduced mainly from in vitro biochemical experiments. A general model has been proposed that these receptors and the recruited coactivator proteins form relatively stable complexes with gene promoters. Only recently, in the last four years, additional in vivo data measured with fluorescence recovery after photobleaching (FRAP) have shown that many hormone receptors and coactivators are very mobile inside the nucleus and their association to chromatin is very dynamic, much faster than what has been anticipated from earlier biochemical studies. Although the mobility is reported to be high for the hormone binding receptors, there are consistent reports about reduced mobility in the presence of agonists and antagonists. However, little is known about the significance of the altered mobility. Open questions are, whether the change of mobility is due to increasing interactions with other proteins, e.g. by forming larger protein complexes or if it is due to interactions with chromatin and other relative immobile domains of the nucleus. In this work, fluorescence correlation spectroscopy (FCS) has been employed to monitor the mobility of both a hormone binding receptor, the estrogen receptor (ER), and coactivator proteins belonging to the steroid receptor coactivator (SRC)-family inside the nucleus, to investigate how their mobility is influenced by different ligands. The main advantages of FCS compared to FRAP are (i) its sensitivity which enables measurements of proteins of low abundance in live cells and (ii) its higher temporal resolution which allows us to resolve different diffusing species in the millisecond range. This work has been divided into four major parts. The first contains the construction and characterization of activity of fluorescently labeled molecules. The second part describes the optimization of the experimental conditions for measuring the mobility of yellow fluorescent protein (YFP) in living cells. The third part reports on the effects of YFP-labeled ER inside the nucleus in the presence of different ligands. Finally the fourth part investigates the effects of different ligands on the interaction between receptor and coactivator in living cells. The main results of this thesis are the following: The finding of the different mobility patterns of YFP-ER in the presence of agonists and antagonists indicate that the different ligands induce multiple discrete diffusive states; some are re-occuring for different ligands while others are characteristic for particular ligands. The potency of the different ligands to induce a lower mobility of the YFP-ER was estimated, and the relative order of the potencies to induce the lower mobility corresponds well to the relative order of their affinities for ER. ER and coactivator proteins were shown to interact in the absence of ligands and there are also clear indications of the co-existence of multiple ER-SRC complexes in the presence of agonists. The full antagonist abolishes these interactions completely in the case of the full-length coactivator and only partly when a truncated coactivator, containing only the receptor interaction domain, was used.