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 fluorescent lectins on nuclear import mediated by nuclear localization signals (NLSs). Therefore, a novel experimental approach that allows the controlled sequential introduction of fluorescent substances into living cells was established. A microinjection system equipped with an UV-source enabled us to identify fluorescent-labeled cells for the subsequent introduction of additional fluorescent compounds, in order to study their interactions in vivo. Cells were initially labeled either by expression of autofluorescent proteins or by microinjection of fluorescent substances. Transcription inhibitors did not affect nuclear transport mediated by classical NLSs but inhibited import mediated by the M9-domain of hnRNPA1. Comparison of a mono- and bipartite NLS revealed that the bipartite signal was more active in import. Sequential injection of differentially labeled nuclear import and export substrates allowed monitoring of import and export simultaneously in the same living cell. The introduced experimental approach will also be useful to analyze a variety of biological processes in living mammalian cells.
Stauber
R H
RH
Institute for Clinical and Molecular Virology, University of Erlangen-Nürnberg, Schlossgarten 4, D-91054 Erlangen, Germany. rdstaube@viro.med.uni-erlangen.de
Krätzer
F
F
Schneider
G
G
Hirschmann
N
N
Hauber
J
J
Rosorius
O
O
D000595:Amino Acid Sequence; D001483:Base Sequence; D002467:Cell Nucleus / Q000201:enzymology / Q000378:metabolism*; D003593:Cytoplasm / Q000201:enzymology / Q000378:metabolism*; D005982:Glutathione Transferase / Q000378:metabolism; D008845:Microinjections; D008969:Molecular Sequence Data; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014158:Transcription, Genetic; D014466:Ultraviolet Rays
D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase