Translocation biosensors to study signal-specific nucleo-cytoplasmic transport, protease activity and protein-protein interactions.
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 nucleo-cytoplasmic transport pathways but also to be employed in numerous screening applications for the early stage evaluation of potential drug candidates.
Knauer
Shirley K
SK
Georg-Speyer-Haus, Paul-Ehrlich-Str. 42-44, D-60596 Frankfurt, Germany.
Moodt
Sabrina
S
Berg
Thorsten
T
Liebel
Urban
U
Pepperkok
Rainer
R
Stauber
Roland H
RH
D001692:Biological Transport; D015374:Biosensing Techniques; D002467:Cell Nucleus / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism*; D010447:Peptide Hydrolases / Q000378:metabolism*; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism*; D015398:Signal Transduction
D011506:Proteins; D010447:Peptide Hydrolases