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Investigation of nucleo‐cytoplasmic transport using UV‐guided microinjection
Author(s) -
Stauber Roland H.,
Krätzer Friedrich,
Schneider Grit,
Hirschmann Nicole,
Hauber Joachim,
Rosorius Olaf
Publication year - 2000
Publication title -
journal of cellular biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.028
H-Index - 165
eISSN - 1097-4644
pISSN - 0730-2312
DOI - 10.1002/1097-4644(20010301)80:3<388::aid-jcb130>3.0.co;2-v
Subject(s) - microinjection , nuclear localization sequence , nls , nuclear transport , fluorescence , microbiology and biotechnology , cytoplasm , transcription factor , chemistry , transcription (linguistics) , nuclear export signal , biophysics , biochemistry , biology , cell nucleus , gene , physics , linguistics , philosophy , quantum mechanics
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. J. Cell. Biochem. 80:388–396, 2001. © 2001 Wiley‐Liss, Inc.