Real-time imaging of glucocorticoid receptor dynamics in living neurons and glial cells in comparison with non-neural cells.
To investigate the intracellular trafficking of glucocorticoid receptor (GR) in response to various conditions in a single living cell, a green fluorescent protein (GFP) and rat GR chimera construct (GFP-GR) was prepared. We transiently transfected GFP-GR into primary cultured rat hippocampal neurons, cortical glial cells, and non-neural cells, e.g. COS-1 cells and CV-1 cells, and compared the dynamic changes in subcellular localization of GFP-GR in these cells. When GFP-GR was expressed in the cells, GFP-GR efficiently transactivated the mouse mammary tumour virus promoter in response to dexamethasone (DEX). The cytoplasm-to-nuclear translocation of GFP-GR induced with 10(-7) m DEX, a specific agonist of GR, at 37 degrees C was completed within 30 min in all cell types used, and the rate of nuclear translocation was dependent on the ligand dose. The translocation of GFP-GR into the nucleus from the cytoplasm was induced in a ligand-specific manner, similar to that of the native GR. The disruption of microtubules by colchicine or nocodazole showed no significant effect on the DEX-induced GFP-GR translocation from the cytoplasmic region to the nuclear region. The cells were not deteriorated during time-lapse imaging analysis for 1 h at 37 degrees C. The present findings suggest that the subcellular localization of GFP-GR is dynamically changed in response to extracellular and intracellular conditions, and that there are no conspicuous variations in the manner of trafficking of GR among different types of cells in vitro.
Nishi
M
M
Department of Anatomy and Neurobiology, Kyoto Prefectural University of Medicine, Kyoto, Japan. nmayumi@koto.kpu-m.ac.jp
Takenaka
N
N
Morita
N
N
Ito
T
T
Ozawa
H
H
Kawata
M
M
D000818:Animals; D001692:Biological Transport / Q000502:physiology; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D002678:Chimera; D003199:Computer Systems; D003593:Cytoplasm / Q000378:metabolism; D003599:Cytoskeleton / Q000502:physiology; D049452:Green Fluorescent Proteins; D007202:Indicators and Reagents; D008024:Ligands; D008164:Luminescent Proteins / Q000235:genetics; D051379:Mice; D009457:Neuroglia / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism*; D051381:Rats / Q000196:embryology; D017207:Rats, Sprague-Dawley; D011965:Receptors, Glucocorticoid / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology; D014162:Transfection
D007202:Indicators and Reagents; D008024:Ligands; D008164:Luminescent Proteins; D011965:Receptors, Glucocorticoid; D049452:Green Fluorescent Proteins