Confocal Scanning Laser Microscopy of Steroid Receptors in Brain

Steensel, Bas van; Binnendijk, E.P. van; Driel, Roel van

doi:10.1016/b978-0-12-185292-4.50016-4

Cited by 5 publications

(4 citation statements)

References 23 publications

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“…Immunofluorescent labelling combined with 3-D confocal microscopy, shows that in nuclei of HeLa cells RNA polymerase II and the transcription factors BRG1, Oct1, E2F-1, GR, TFIIF and TFIIH are distributed in punctate patterns throughout the nucleoplasm excluding the nucleoli. These distributions are similar to those observed for the glucocorticoid receptor (GR) and the mineralocorticoid receptor in other cell types and in hypocampus neurons (Van Steensel et al, 1994 and that of nascent RNA (Jackson et al, 1993;Wansink et al, 1993). However, careful analysis of images of dual labelled cells showed consistently that there is remarkably little relationship between the spatial distributions of the transcription factors on the one hand and RNA polymerase II and nascent RNA on the other hand.…”

Section: Discussionsupporting

confidence: 67%

Nuclear distribution of transcription factors in relation to sites of transcription and RNA polymerase II

Grande

Kraan

Jong

et al. 1997

Journal of Cell Science

192

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We have investigated the spatial relationship between sites containing newly synthesized RNA and domains containing proteins involved in transcription, such as RNA polymerase II and the transcription factors TFIIH, Oct1, BRG1, E2F-1 and glucocorticoid receptors, using dual immunofluorescence labelling followed by confocal microscopy on cultured cells. As expected, a high degree of colocalisation between the RNA polymerase II and sites containing newly synthesised RNA was observed. Like the newly synthesised RNA and the RNA polymerase II, we found that all the transcription factors that we studied are distributed more or less homogeneously throughout the nucleoplasm, occupying numerous small domains. In addition to these small domains, TFIIH was found concentrated in coiled bodies and Oct1 in a single large domain of about 1.5 microm in 30% of the cells in an asynchronous HeLa cell culture. Remarkably, we found little or no relationship between the spatial distribution of the glucocorticoid receptor, Oct1 and E2F-1 on the one hand and RNA polymerase II and transcription sites on the other hand. In contrast, a significant but incomplete overlap was observed between the spatial distributions of transcription sites and BRG1 and TFIIH. These results indicate that many of the transcription factor-rich nuclear domains are not actively involved in transcription. They may represent incomplete transcription initiation complexes, inhibitory complexes, or storage sites.

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Section: Discussionsupporting

confidence: 67%

Nuclear distribution of transcription factors in relation to sites of transcription and RNA polymerase II

Grande

Kraan

Jong

et al. 1997

Journal of Cell Science

192

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“…Fejes-Toth et al (15) reported that agonistactivated MR accumulates in discrete clusters in the nucleus, and that this phenomenon occurs only with transcriptionally active MR. In contrast, van Steensel et al (44,45) demonstrated the spatial distribution of GR and MR in clusters in specific nuclear domains using an immunofluorescence technique with confocal microscopy. They indicated that there was no correlation between the GR clusters and the distribution of newly synthesized pre-mRNA, suggesting that the clusters are not directly involved in active transcription.…”

Section: Relationship With Transcriptional Sitesmentioning

confidence: 95%

Dynamic Changes in Subcellular Localization of Mineralocorticoid Receptor in Living Cells: In Comparison with Glucocorticoid Receptor using Dual-Color Labeling with Green Fluorescent Protein Spectral Variants

Nishi

Ogawa

Itō

et al. 2001

Molecular Endocrinology

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Mineralocorticoid receptor (MR) and glucocorticoid receptor (GR) are ligand-dependent transcription factors. Although it is generally accepted that GR is translocated into the nucleus from the cytoplasm only after ligand binding, the subcellular localization of MR is still quite controversial. We examined the intracellular trafficking of MR in living neurons and nonneural cells using a fusion protein of green fluorescent protein (GFP) and rat MR (GFP-MR). Corticosterone (CORT) induced a rapid nuclear accumulation of GFP-MR, whereas in the absence of ligand, GFP-MR was distributed in both cytoplasm and nucleus in the majority of transfected cells. Given the differential action of MR and GR in the central nervous system, it is important to elucidate how the trafficking of these receptors between cytoplasm and nucleus is regulated by ligand. To examine the simultaneous trafficking of MR and GR within single living cells, we use different spectral variants of GFP, yellow fluorescent protein (YFP) and cyan fluorescent protein (CFP), linked to MR and GR, respectively. In COS-1 cells, expressing no endogenous corticosteroid receptors, the YFP-MR chimera was accumulated in the nucleus faster than the CFP-GR chimera in the presence of 10(-9) M CORT, while there was no significant difference in the nuclear accumulation rates in the presence of 10(-6) M CORT. On the other hand, in primary cultured hippocampal neurons expressing endogenous receptors, the nuclear accumulation rates of the YFP-MR chimera and CFP-GR chimera were nearly the same in the presence of both concentrations of CORT. These results suggest that CORT-induced nuclear translocation of MR and GR exhibits differential patterns depending on ligand concentrations or cell types.

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“…Animals were anaesthesized with Nembutal and brain tissue was fixed by cardiac perfusion with 4% formaldehyde in phosphate-buffered saline; preparation of 30 µm brain slices and dual-labeling immunocytochemistry was carried out as described previously (van Steensel et al, 1994). The MR was detected with rabbit polyclonal antibody MINREC4 (Ahima et al, 1991), followed by biotinylated donkey anti-rabbit antibody and streptavidin-FITC (Jackson ImmunoResearch Laboratories, West Grove, PA); the GR was detected with mouse monoclonal antibody 7 (Okret et al, 1984), followed by TRITC-conjugated donkey anti-mouse antibody (Jackson ImmunoResearch Laboratories).…”

Section: Immunocytochemistrymentioning

confidence: 99%

Partial colocalization of glucocorticoid and mineralocorticoid receptors in discrete compartments in nuclei of rat hippocampus neurons

Steensel

Binnendijk

Hornsby

et al. 1996

Journal of Cell Science

324

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The glucocorticoid receptor and the mineralocorticoid receptor are hormone-dependent transcription factors. They regulate the excitability of rat hippocampus CA1 neurons in a coordinated fashion. We studied the spatial distribution of these transcription factors in nuclei of CA1 neurons by dual labeling immunocytochemistry and confocal microscopy, combined with novel image restoration and image analysis techniques. We found that both receptors are concentrated in about one thousand clusters within the nucleus. Some clusters contain either mineralocorticoid receptors or glucocorticoid receptors, but a significant number of clusters contains both receptors. These results indicate that the two receptor types are targeted to specific compartments in the nucleus. The coordinated action of the glucocorticoid and mineralocorticoid receptor on gene expression may be established in a specific set of nuclear domains that contain both receptors.

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Confocal Scanning Laser Microscopy of Steroid Receptors in Brain

Cited by 5 publications

References 23 publications

Nuclear distribution of transcription factors in relation to sites of transcription and RNA polymerase II

Nuclear distribution of transcription factors in relation to sites of transcription and RNA polymerase II

Dynamic Changes in Subcellular Localization of Mineralocorticoid Receptor in Living Cells: In Comparison with Glucocorticoid Receptor using Dual-Color Labeling with Green Fluorescent Protein Spectral Variants

Partial colocalization of glucocorticoid and mineralocorticoid receptors in discrete compartments in nuclei of rat hippocampus neurons

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