Knauer, Shirley; Moodt, Sabrina; Berg, Thorsten; Liebel, Urban; Pepperkok, Rainer; Stauber, Roland H.:

Translocation Biosensors to Study Signal-Specific Nucleo-Cytoplasmic Transport, Protease Activity and Protein–Protein Interactions

In: Traffic, Jg. 6 (2005) ; Nr. 7, S. 594-606
Zeitschriftenaufsatz / Fach: Biologie
Abstract:
Regulated nucleo-cytoplasmic transport is crucial for
cellular homeostasis and relies on protein interaction
networks. In addition, the spatial division into the
nucleus and the cytoplasm marks two intracellular
compartments that can easily be distinguished by
microscopy. Consequently, combining the rules for
regulated nucleo-cytoplasmic transport with autofluorescent
proteins, we developed novel cellular biosensors
composed of glutathione S-transferase, mutants
of green fluorescent protein and rational combinations
of nuclear import and export signals. Addition of regulatory
sequences resulted in three classes of biosensors
applicable for the identification of signal-specific
nuclear export and import inhibitors, small molecules
that interfere with protease activity and compounds
that prevent specific protein–protein interactions in living
cells. As a unique feature, our system exploits
nuclear accumulation of the cytoplasmic biosensors
as the reliable readout for all assays. Efficacy of the
biosensors was systematically investigated and also
demonstrated by using a fully automated platform for
high throughput screening (HTS) microscopy and
assay analysis. The introduced modular biosensors
not only have the potential to further dissect nucleocytoplasmic
transport pathways but also to be
employed in numerous screening applications for the
early stage evaluation of potential drug candidates.