2-COLOR GREEN FLUORESCENT PROTEIN TIME-LAPSE IMAGING

The Aequorea victoria green fluorescent protein (GFP) is widely recogn ized as a powerful tool in cell biology, serving as a vital reporter f or monitoring localization and dynamics of intracellular proteins and organelles over time. GFP variants with shifted spectral characteristi cs have been described and offer enormous potential Sor double-labelin g experiments and protein-protein interaction studies. However, most G FP variant combinations are not suitable for double-label, time-lapse imaging experiments because of either extremely rapid photobleaching o f blue-shifted GFP variants or crossover of their excitation and emiss ion spectra, which must then be computer corrected. Here, we describe the successful use of two photostable spectral GFP variants, W7 and 10 C, in dual-color, time-lapse imaging of fusion proteins in living cell s using either wide-field or confocal microscopy. W7 and 10C were high ly photostable during repetitive long-term imaging and were cleanly se parated by their different excitation spectra alone with negligible cr ossover of fluorescence. We present time-lapse image sequences of COS- 7 cells co-expressing both a marker of the Golgi complex (galactosyl t ransferase) fused to W7 and a marker of the nuclear envelope (lamin-B receptor) fused to 10C. To our knowledge, these image sequences provid e the first simultaneous visualization of Golgi and nuclear envelope m embranes in living cells.