Investigation of nucleo-cytoplasmic transport using UV-guided microinjection.

Abstract:

: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.

journal_name

J Cell Biochem

authors

Stauber RH,Krätzer F,Schneider G,Hirschmann N,Hauber J,Rosorius O

doi

10.1002/1097-4644(20010301)80:3<388::aid-jcb130>3.

subject

Has Abstract

pub_date

2001-01-01 00:00:00

pages

388-96

issue

3

eissn

0730-2312

issn

1097-4644

pii

10.1002/1097-4644(20010301)80:3<388::AID-JCB130>3.

journal_volume

80

pub_type

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