LOCALIZATION, TRAFFICKING, AND TEMPERATURE-DEPENDENCE OF THE AEQUOREAGREEN FLUORESCENT PROTEIN IN CULTURED VERTEBRATE CELLS

The localization, trafficking, and fluorescence of Aequorea green fluo rescent protein (GFP) in cultured vertebrate cells transiently transfe cted with GFP cDNA were studied, Fluorescence of GFP in UV light was f ound 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 ce lls, primary chicken embryonic retina cells, and carp epithelial cells were fluorescently labeled under these conditions. GFP was distribute d uniformly throughout the cytoplasm and nucleus independent of cell t ype examined. When GFP was fused to PML protooncogene product, fluores cence was detected in a unique nuclear organelle pattern indistinguish able from that of PML protein, showing the potential use of GFP as a f luorescent 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 efficien tly transactivated the mouse mammary tumor virus promoter in response to dexamethasone at 30 degrees C but not at 37 degrees C, indicating t hat temperature is important, even for function of the GFP fusion prot ein. The dexamethasone-induced translocation of GFP-human glucocortico id receptor from cytoplasm to nucleus was complete within 15 min; the translocation could be monitored in a single living cell in real time.