Localization, trafficking, and temperature-dependence of the Aequorea green fluorescent protein in cultured vertebrate cells.
The localization, trafficking, and fluorescence of Aequorea green fluorescent protein (GFP) in cultured vertebrate cells transiently transfected with GFP cDNA were studied. Fluorescence of GFP in UV light was found to be strongest when cells were incubated at 30 degrees C but was barely visible at an incubation temperature of 37 degrees C. COS-1 cells, primary chicken embryonic retina cells, and carp epithelial cells were fluorescently labeled under these conditions. GFP was distributed uniformly throughout the cytoplasm and nucleus independent of cell type examined. When GFP was fused to PML protooncogene product, fluorescence was detected in a unique nuclear organelle pattern indistinguishable from that of PML protein, showing the potential use of GFP as a fluorescent tag. To analyze both function and intracellular trafficking of proteins fused to GFP, a GFP-human glucocorticoid receptor fusion construct was prepared. The GFP-human glucocorticoid receptor efficiently transactivated the mouse mammary tumor virus promoter in response to dexamethasone at 30 degrees C but not at 37 degrees C, indicating that temperature is important, even for function of the GFP fusion protein. The dexamethasone-induced translocation of GFP-human glucocorticoid receptor from cytoplasm to nucleus was complete within 15 min; the translocation could be monitored in a single living cell in real time.
Graduate School of Biological Sciences, Nara Institute of Science and Technology, Japan.
H
H
Ogawa
S
S
Inouye
F I
FI
Tsuji
K
K
Yasuda
K
K
Umesono
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001692:Biological Transport; D015415:Biomarkers; D002451:Cell Compartmentation; D002478:Cells, Cultured; D003907:Dexamethasone / Q000494:pharmacology; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D000071156:Promyelocytic Leukemia Protein; D011965:Receptors, Glucocorticoid / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013696:Temperature; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D025521:Tumor Suppressor Proteins; D014714:Vertebrates
D015415:Biomarkers; D008164:Luminescent Proteins; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; C513293:Pml protein, mouse; D000071156:Promyelocytic Leukemia Protein; D011965:Receptors, Glucocorticoid; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins; D049452:Green Fluorescent Proteins; D003907:Dexamethasone