Imaging of transcription factor trafficking in living cells: lessons from corticosteroid receptor dynamics.

Adrenal corticosteroids (cortisol in humans/corticosterone in rodents) readily enter the brain and exert markedly diverse effects, such as the stress response of target neural cells. These effects are regulated via two receptor systems, the mineralocorticoid receptor (MR) and the glucocorticoid receptor (GR), both are ligand-inducible transcription factors. GR and MR predominantly reside in the cytoplasm in the absence of corticosterone (CORT), but are quickly translocated into the nucleus upon binding CORT. Then these receptors form dimers to bind hormone responsive elements and regulate the expression of target genes. Given the different actions of MR and GR in the central nervous system, it is important to elucidate how the trafficking of these receptors between the cytoplasm and nucleus and their interaction are regulated by ligands or other molecules to exert transcriptional activity. However, these processes have still not been completely clarified. To address these issues, we have tried to observe more dynamic subcellular trafficking processes in living cells by employing a green fluorescent protein (GFP). In this chapter, we describe our recent studies of corticosteroid receptor dynamics in living cells focusing on three points: (1) time-lapse imaging of GFP-labeled corticosteroid receptors; (2) intranuclear dynamics of GFP-labeled corticosteroid receptors using the fluorescence recovery after photobleaching (FRAP) technique; and (3) the possibility of heterodimers formation using the fluorescence resonance energy transfer (FRET) technique. These studies demonstrate that GR and MR were quickly translocated from the cytoplasm to nucleus after CORT treatment. The time course of the nuclear translocation of GR and MR differed depending on the concentration of CORT. The FRAP study showed that liganded GR and MR in the nucleus were highly mobile, and not trapped by specific organelles. We detected GR-MR heterodimers, which were affected by changes in CORT concentrations in response to various hormonal milieu such as circadian rhythm and stress. Our findings may provide new insights into the dynamic status of corticosteroid receptors in living cells and the molecular basis of the regulation of stress by these receptors.