Image-based high-throughput mapping of TGF-β-induced phosphocomplexes at a single-cell level.

Area of Science:

• Cellular biology
• Biochemistry
• Molecular imaging

Background:

• Protein interactions and posttranslational modifications are crucial for cellular responses but challenging to monitor at scale.
• Existing methods lack the throughput for analyzing dynamic molecular events in individual cells.
• High-throughput analysis is needed to understand complex cellular signaling pathways.

Purpose of the Study:

• To develop and validate a semi-automated system for large-scale in situ proximity ligation assays (isPLA).
• To enable simultaneous digital recording of protein interactions, modifications, and subcellular morphology.
• To facilitate high-throughput, single-cell analysis of dynamic cellular events and drug responses.

Main Methods:

• Combined in situ proximity ligation assays (isPLA) in microtiter plates with automated microscopy and computer-based image analysis.
• Developed a system for digitally recording phosphorylations, protein complexes, DNA content, nuclear size, and cell positions.
• Applied the system to study TGF-β-responsive Smad2 signaling and screened phosphatase inhibitors.

Main Results:

• Successfully monitored Smad2 linker phosphorylations and complex formations across millions of individual cells over time.
• Correlated protein events with cell cycle progression and local cell crowding using DNA content and nuclear size.
• Demonstrated the system's utility in screening drug effects, specifically using phosphatase inhibitors.

Conclusions:

• The developed semi-automated isPLA system enables high-throughput, image-based single-cell analysis of protein dynamics.
• This approach integrates protein interaction/modification data with morphological features for comprehensive cellular insights.
• The system is suitable for drug screening and advancing the understanding of complex cellular signaling pathways.