Subcellular and subnuclear distributions of estrogen receptor α in living cells using green fluorescent protein and immunohistochemistry

Abstract

Although the subcellular localization of estrogen receptor α (ERα) is generally recognized as in the nucleus, some studies have shown a cytoplasmic distribution. We used a time-lapse imaging technique to examine intracellular and intranuclear localizations of ERα using a green fluorescent protein (GFP)-ERα chimera protein in living COS-1 cells of different external milieu in a cultured condition. Transfected cells were treated with the ERα agonist, estradiol and the partial antagonist, tamoxifen, to investigate the ligand specific changes of distribution of GFP-ERα in a single cell. GFP-ERα was accumulated in the nucleus even in the absence of ligand exposure. Cells showed diffuse fine-grained fluorescence of GFP-ERα in the nucleus without ligands. These results were confirmed by using anti-ERα antibody immunocytochemistry. GFP-ERα gradually and clearly formed small clusters or more punctate patterns in the nuclei of cells after ligand incubation for 60 min, while some of the transfected cells showed a finely diffuse pattern. The manner of nuclear redistribution did not depend on the type of ligand. The present study showed that GFP-ERα is restricted to the nucleus and accumulates with estradiol and tamoxifen, suggesting that accumulation of ERα in the nucleus could be necessary for ERα to function with ligands.