A cell based, high throughput assay for quantitative analysis of Hedgehog pathway activation using a Smoothened...

Area of Science:

• Cell Biology
• Molecular Biology
• Developmental Biology

Background:

• The Hedgehog (Hh) signalling pathway is crucial for development and disease.
• Understanding Smoothened (Smo) regulation by Patched (Ptc) and Hh ligand is limited.
• Lack of tools for real-time subcellular monitoring of Smo activation hinders research.

Purpose of the Study:

• To develop a high-throughput cell culture assay for monitoring Drosophila Smo activation.
• To screen small molecule inhibitors for their effect on Smo activation.
• To investigate the cell biological basis of Hh pathway activation.

Main Methods:

• Developed a fluorescent sensor assay for Drosophila Smo activation.
• Screened a small molecule inhibitor library.
• Performed biochemical validation and single-cell level monitoring of Smo activation.

Main Results:

• Identified inhibitors targeting MAPK signalling, polo, and aurora kinases that increase Smo sensor fluorescence.
• Biochemical validation confirmed screen results and revealed distinct Smo activation modes for inhibitors like dobrafenib, tak-733, and volasertib.
• Single-cell analysis showed varied cellular responses to Hh stimulation, suggesting a mechanism for graded pathway activation.

Conclusions:

• The developed fluorescent sensor assay is a valuable tool for studying Hh pathway activation.
• The findings provide new molecular targets and insights into the regulation of Smo.
• The assay enables dissection of the cell biological mechanisms underlying Hh signalling.