Yellow fluorescent protein-tagged and cyan fluorescent protein-tagged imaging analysis of glucocorticoid receptor and importins in single living cells.
Glucocorticoid receptor (GR) acts as a ligand-dependent transcription factor after nuclear transport from the cytoplasm in the liganded state. Importins are docking proteins for karyopherin-mediated binding of substrate in a nuclear import pathway. To investigate the spatial and temporal relation between GR and importins, we analyzed the subcellular distribution of GR and importins in response to ligand in single living cells using fusion proteins labeled with different spectral variants of green fluorescent protein. Upon activation with ligand treatment, fluorescent protein-tagged (FP-) GR was translocated from the cytoplasm to the nucleus, showing a similar time course as FP-importin-alpha in the coexpressed cells with the fusion proteins. In contrast to FP-importin-alpha, the distribution of FP-importin-beta was little changed upon ligand treatment in the coexpressed cells with FP-GR and FP-importin-beta. Analysis using fluorescence resonance energy transfer proved that GR directly interacted with importin-alpha in the whole area of the cytoplasm upon ligand treatment and detached importin-alpha shortly after nuclear import. However, direct interaction between GR and importin-beta was not detected. These studies showed visual evidence of the nuclear importing of GR in association with importin-alpha in single living cells.
Tanaka
Masayuki
M
Department of Anatomy and Neurobiology, Kyoto Prefectural University of Medicine, Kawaramachi Hirokoji, Kamigyo-ku, Kyoto 602-8566, Japan.
Nishi
Mayumi
M
Morimoto
Masafumi
M
Sugimoto
Tohru
T
Kawata
Mitsuhiro
M
D000595:Amino Acid Sequence; D000818:Animals; D000893:Anti-Inflammatory Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000235:genetics; D019556:COS Cells; D002467:Cell Nucleus / Q000737:chemistry / Q000378:metabolism; D003345:Corticosterone / Q000494:pharmacology; D003593:Cytoplasm / Q000737:chemistry / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D015870:Gene Expression; D049452:Green Fluorescent Proteins; D007202:Indicators and Reagents / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics; D018613:Microscopy, Confocal / Q000379:methods; D008856:Microscopy, Fluorescence / Q000379:methods*; D008969:Molecular Sequence Data; D011965:Receptors, Glucocorticoid / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics; D014162:Transfection; D028901:alpha Karyopherins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D028961:beta Karyopherins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism
D000893:Anti-Inflammatory Agents; D001426:Bacterial Proteins; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D011965:Receptors, Glucocorticoid; D011993:Recombinant Fusion Proteins; D028901:alpha Karyopherins; D028961:beta Karyopherins; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins; D003345:Corticosterone