Localization of the glucocorticoid receptor in discrete clusters in the cell nucleus

Abstract

The cell nucleus is highly organized. Many nuclear functions are localized in discrete domains, suggesting that compartmentalization is an important aspect of the regulation and coordination of nuclear functions. We investigated the subnuclear distribution of the glucocorticoid receptor, a hormone-dependent transcription factor. By immunofluorescent labeling and confocal microscopy we found that after stimulation with the agonist dexamethasone the glucocorticoid receptor is concentrated in 1,000-2,000 clusters in the nucleoplasm. This distribution was observed in several cell types and with three different antibodies against the glucocorticoid receptor. A similar subnuclear distribution of glucocorticoid receptors was found after treatment of cells with the antagonist RU486, suggesting that the association of the glucocorticoid receptor in clusters does not require transformation of the receptor to a state that is able to activate transcription. By dual labeling we found that most dexamethasone-induced receptor clusters do not colocalize with sites of pre-mRNA synthesis. We also show that RNA polymerase II is localized in a large number of clusters in the nucleus. Glucocorticoid receptor clusters did not significantly colocalize with these RNA polymerase II clusters or with domains containing the splicing factor SC-35. Taken together, these results suggest that most clustered glucocorticoid receptor molecules are not directly involved in activation of transcription.

A Gold Nanoparticle Bio-Optical Transponder to Dynamically Monitor Intracellular pH

“…where i denotes intensity values for a given pixel and a and b denote the intensity averages over the whole image per channel, A and B) for each image overlap position. 76,77 Here the observation of maxima at the 0 pixelshift position indicates colocalization of the pH-BOT probe with the EE, LE, and LY trackers (Figure 5A−F). Slight lateral favoring of maxima away from 0 pixel-shift for EE and LE trackers indicates a slight degree of partial colocalization, which is most likely due to the slightly larger and more heterogeneous morphologies of EEs and LEs, as compared to LYs.…”

“…78 This is in contrast to an observation of minima at 0 shift, a sign of delocalization, which is not seen for any of the tracker labels compared with the DL700. 76 This indicates that the pH-BOT probe is found in all components of the endolysosomal pathway at 2 and 6 h after transfection. Traditional use of r p alone to determine the degree of colocalization in a fluorescent microscope image is limited by a necessity to have similar intensity values for each fluorescence channel, where an r p value of 1 would lead to complete one-forone intensity matching at each pixel, 0 would be the absence of any colocalization, and −1 would mean opposite intensity matching.…”

“…Slight lateral favoring of maxima away from 0 pixel-shift for EE and LE trackers indicates a slight degree of partial colocalization, which is most likely due to the slightly larger and more heterogeneous morphologies of EEs and LEs, as compared to LYs . This is in contrast to an observation of minima at 0 shift, a sign of delocalization, which is not seen for any of the tracker labels compared with the DL700 . This indicates that the pH-BOT probe is found in all components of the endolysosomal pathway at 2 and 6 h after transfection.…”

“…Figure A–F display the cross-correlation functions (CCF) of colocalization for the RFP fluorescence channel versus the DL700 fluorescence channel averaged over five image fields for each time point and tracker, to give a clear picture of the overall colocalization of the pH-BOT with each vesicular component of the endolysosomal pathway, before and after complete release of the DNA from the AuNP. The van Steensel CCF is defined by the best Gaussian fit of the plot created by laterally shifting the image pixels in one fluorescence intensity channel “A” (DL700) versus another potentially colocalizing fluorescence intensity channel “B” (RFP) and comparing the effect of the pixel shift on the Pearson’s correlation coefficient ( r p = where i denotes intensity values for a given pixel and a and b denote the intensity averages over the whole image per channel, A and B) for each image overlap position. , Here the observation of maxima at the 0 pixel-shift position indicates colocalization of the pH-BOT probe with the EE, LE, and LY trackers (Figure A–F). Slight lateral favoring of maxima away from 0 pixel-shift for EE and LE trackers indicates a slight degree of partial colocalization, which is most likely due to the slightly larger and more heterogeneous morphologies of EEs and LEs, as compared to LYs .…”

Corticosterone and Glucocorticoid Receptor in the Cortex of Rats during Aging—The Effects of Long-Term Food Restriction

“…They act on target cells by specific receptors, the high-affinity mineralocorticoid receptor (MR), and the lower-affinity glucocorticoid receptor (GR). Both receptors primarily remain in the cytoplasm as part of multiprotein complexes (containing chaperone proteins such as Heat shock protein 90 (Hsp90), and immunophilins), whereas upon ligand binding receptors translocate into the nucleus to act as transcription factors [22,23]. In addition, the availability of glucocorticoids in the tissues is regulated by a locally expressed specific enzyme, 11β-hydroxysteroid dehydrogenase (11β-HSD) [24,25].…”