Direct observation of nucleocytoplasmic transport by microinjection of GFP-tagged proteins in living cells

We established a straightforward experimental system to investigate directl y the requirements for nucleocytoplasmic transport in live cells. For this purpose, substrates were created containing nuclear localization signals (N LS) or nuclear export signals (NES) linked to a chimeric protein composed o f the glutathione S-transferase (GST)fused to the green fluorescent protein (GFP). The combination of GST/GFP-tagging allowed res to control protein e xpression in bacteria and to monitor protein purification during chromatogr aphy. Following microinjection into somatic cells, nuclear export/import of the highly fluorescent substrates could be observed directly by fluorescen ce microscopy. This system sets the stage to quantitate, in real time, the kinetics of nuclear import/export in living cells and to evaluate qualitati ve differences in various NLS/NES signals and pathways.