Investigation of nucleo-cytoplasmic transport using UV-guided microinjection

Active nucleo-cytoplasmic transport is mediated by dynamic signal-mediated pathways. We investigated the effects of transcription inhibitors or fluore scent lectins on nuclear import mediated by nuclear localization signals (N LSs). Therefore, a novel experimental approach that allows the controlled s equential introduction of fluorescent substances into living cells was esta blished. A microinjection system equipped with an UV-source enabled us to i dentify fluorescent-labeled cells for the subsequent introduction of additi onal fluorescent compounds, in order to study their interactions in vivo. C ells were initially labeled either by expression of autofluorescent protein s or by microinjection of fluorescent substances. Transcription inhibitors did not affect nuclear transport mediated by classical NLSs but inhibited i mport mediated by the M9-domain of hnRNPA1. Comparison of a mono- and bipar tite NLS revealed that the bipartite signal was more active in import. Sequ ential injection of differentially labeled nuclear import and export substr ates allowed monitoring of import and export simultaneously in the same liv ing cell. The introduced experimental approach will also be useful to analy ze a variety of biological processes in living mammalian cells. (C) 2001 Wi ley-Liss, Inc.