In situ localization of the estrogen receptor in living cells with the fluorescent phytoestrogen coumestrol.

Abstract

Coumestrol is a naturally occurring plant coumarin that displays high affinity for the hormone-binding site of the human estrogen receptor (hER), for which it serves as a potent non-steroidal agonist. Coumestrol emits intense blue fluorescence when bound to this protein, making it ideally suited for use as a cytological stain to detect ER in fixed and intact cells. Conditions are reported for the efficient detection of recombinant ER protein artificially expressed in cultured cells by calcium phosphate-mediated transfection. Coumestrol fluorescence co-localizes with hER protein detected by indirect immunofluorescence with an hER-specific anti-peptide antiserum, confirming the specificity of this reagent. Fluorescence from ER occupied by coumestrol shows a predominantly nuclear distribution, even when the receptor protein is cross-linked in situ by fixation with glutaraldehyde plus paraformaldehyde before coumestrol exposure. This corroborates previous conclusions, based on indirect immunofluorescence analysis, that the ER remains nuclear in the absence of hormone. Examination of the pattern of coumestrol staining in mitotic cells further indicates that hER remains associated with condensed chromatin. Such observations illustrate the potential for using coumestrol to investigate real-time effects of a variety of physiological stimuli on the subcellular distribution of hER in living cells.