Translocation Biosensors - Cellular System Integrators to Dissect CRM1-Dependent Nuclear Export by Chemicogenomics

Area of Science:

• Cellular Systems Biology
• Molecular Biology
• Pharmacology

Background:

• Regulated nucleo-cytoplasmic transport is vital for cellular functions and disease.
• Understanding nuclear transport mechanisms offers therapeutic intervention opportunities.
• Existing genetic approaches have limitations in dissecting transport dynamics.

Purpose of the Study:

• To establish and utilize high-content cellular translocation biosensors for dissecting nuclear export.
• To screen a compound library for novel nuclear export inhibitors using chemicogenomics.
• To develop robust methods for quantifying and validating export inhibition in living cells.

Main Methods:

• Engineered A431 cell lines with a glutathione S-transferase-GFP translocation biosensor.
• Utilized an optimized nuclear translocation algorithm for robust quantification on the Cellomics Arrayscan® VTI.
• Screened the 17K ChemBioNet compound collection in a 384-well high-content assay format.

Main Results:

• Successfully quantified translocation responses and identified true export inhibitors, distinguishing them from false positives.
• Selected 120 potential hit compounds for rescreening, confirming 20 effective export inhibitors (< 25 μM).
• Identified inhibitors affecting CRM1-mediated nuclear export reversibly and irreversibly via kinetic analysis.

Conclusions:

• Biosensor-based screening is effective for identifying chemicogenomic tools to study nucleo-cytoplasmic transport.
• The developed assay enables dissecting nuclear export mechanisms in living cells.
• This approach provides novel insights into regulating cellular transport for potential therapeutic applications.