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Quantitation of Glucocorticoid Receptor DNA-Binding Dynamics by Single-Molecule Microscopy and FRAP

Please always quote using this URN: urn:nbn:de:bvb:20-opus-117085
  • Recent advances in live cell imaging have provided a wealth of data on the dynamics of transcription factors. However, a consistent quantitative description of these dynamics, explaining how transcription factors find their target sequences in the vast amount of DNA inside the nucleus, is still lacking. In the present study, we have combined two quantitative imaging methods, single-molecule microscopy and fluorescence recovery after photobleaching, to determine the mobility pattern of the glucocorticoid receptor (GR) and the mineralocorticoidRecent advances in live cell imaging have provided a wealth of data on the dynamics of transcription factors. However, a consistent quantitative description of these dynamics, explaining how transcription factors find their target sequences in the vast amount of DNA inside the nucleus, is still lacking. In the present study, we have combined two quantitative imaging methods, single-molecule microscopy and fluorescence recovery after photobleaching, to determine the mobility pattern of the glucocorticoid receptor (GR) and the mineralocorticoid receptor (MR), two ligand-activated transcription factors. For dexamethasone-activated GR, both techniques showed that approximately half of the population is freely diffusing, while the remaining population is bound to DNA. Of this DNA-bound population about half the GRs appeared to be bound for short periods of time (similar to 0.7 s) and the other half for longer time periods (similar to 2.3 s). A similar pattern of mobility was seen for the MR activated by aldosterone. Inactive receptors (mutant or antagonist-bound receptors) show a decreased DNA binding frequency and duration, but also a higher mobility for the diffusing population. Likely, very brief (<= 1 ms) interactions with DNA induced by the agonists underlie this difference in diffusion behavior. Surprisingly, different agonists also induce different mobilities of both receptors, presumably due to differences in ligand-induced conformational changes and receptor complex formation. In summary, our data provide a consistent quantitative model of the dynamics of GR and MR, indicating three types of interactions with DNA, which fit into a model in which frequent low-affinity DNA binding facilitates the search for high-affinity target sequences.show moreshow less

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Metadaten
Author: Femke L. Groeneweg, Martin E. van Royen, Susanne Fenz, Veer I. P. Keizer, Bart Geverts, Jurrien Prins, E. Ron de Kloet, Adriaan B. Houtsmuller, Thomas S. Schmidt, Marcel J. M. Schaaf
URN:urn:nbn:de:bvb:20-opus-117085
Document Type:Journal article
Faculties:Fakultät für Biologie / Theodor-Boveri-Institut für Biowissenschaften
Language:English
Parent Title (English):PLOS ONE
Year of Completion:2014
Schriftenreihe:PLoS ONE 9(3): e90532. doi:10.1371/journal.pone.0090532
Volume:9
Issue:3
Pagenumber:e90532
DOI:https://doi.org/10.1371/journal.pone.0090532
Pubmed Id:http://www.ncbi.nlm.nih.gov/pubmed?term=24632838
Dewey Decimal Classification:5 Naturwissenschaften und Mathematik / 57 Biowissenschaften; Biologie / 570 Biowissenschaften; Biologie
Tag:NF-KAPPA-B; human mineralocorticoid receptor; image correlation spectroscopy; in-vivo; living cells; mobility; nuclear-pore complexes; protein; reveals; transcription
Release Date:2015/08/14
Licence (German):License LogoCC BY: Creative-Commons-Lizenz: Namensnennung