In cells transfected with GFP-ER vector, bleaching for 0.5 s caused only a 557 reduction in fluorescence within the nuclear zone, indicating that unliganded GFP-ER was extremely mobile and rapidly moved back to the bleached zone, even during the time between the bleaching process and first measurement (see Fig. The effect of ligands on the mobility of GFP-ER was studied using the FRAP technique (see Fig. The calculated data of ten individual cells from six different experiments are shown in Table I. Under these conditions, we determined the recovery rate of a mobile fraction of GFP-NRs in which fluorescence recovery could be measured ( t 1 2 ) and observed an immobile fraction in which no recovery occurred even after 3 min (original fluorescence (1007) − 7 of recovery after 3 min). They also show that ligand-binding and protein-protein interactions can affect the intracellular mobility of some NRs and thereby may contribute to their biological activity. Our findings suggest estradiol and tamoxifen exert differential effects on the intranuclear mobility of GFP-ER. Co-transfection of the co-activator, SRC-1, further slowed the mobility of liganded GFP-ER. These effects were even more pronounced with tamoxifen. In contrast, estrogen binding decreased the mobility of GFP-ER and also increased the fraction of GFP-ER that was unable to diffuse. Both GFP-TR and GFP-RAR moved rapidly in the nucleus, and ligand binding did not significantly affect their mobility. Intranuclear mobility of the GFP-NRs was studied by fluorescence recovery after photo-bleaching ± cognate ligands. Ligand addition decreased shuttling by GFP-ER, whereas heterodimerization with retinoid X receptor helped maintain TR and RAR within the nucleus. Nucleocytoplasmic shuttling was demonstrated by heterokaryon experiments and energy-dependent blockade of nuclear import and leptomycin-dependent blockade of nuclear export. These receptors were predominantly in the nucleus and, interestingly, underwent intranuclear reorganization after ligand treatment. We expressed green fluorescent protein (GFP) chimeras of estrogen, retinoic acid, and thyroid hormone receptors (ERs, RARs, and TRs, respectively) in HeLa cells to examine nucleocytoplasmic shuttling and intranuclear mobility of nuclear hormone receptors (NRs) by confocal microscopy. Glycobiology and Extracellular Matrices.
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