CASPAM: A Triple-Modality Biosensor for Multiplexed Imaging of Caspase Network Activity

Area of Science:

• Molecular Biology
• Cellular Signaling
• Biophysics

Background:

• Monitoring multiple biological network nodes simultaneously is crucial for understanding signal propagation and correlations.
• Existing methods face challenges in tracking more than two enzymatic activities on short timescales.
• Homo-FRET-based biosensors offer potential for multiplexed imaging due to their narrow spectral width.

Purpose of the Study:

• To introduce a novel single-plasmid, triple-modality reporter for monitoring key nodes in the apoptotic network.
• To enable simultaneous imaging of up to three enzymatic activities using fluorescence polarization anisotropy microscopy.
• To facilitate the study of interactions within the intrinsic, extrinsic, and effector pathways of apoptosis.

Main Methods:

• Development of the Caspase Activity Sensor by Polarization Anisotropy Multiplexing (CASPAM).
• Utilizing fluorescence polarization anisotropy microscopy for imaging.
• Characterization of biosensor performance and expression levels.

Main Results:

• CASPAM successfully enables simultaneous monitoring of up to three enzymatic activities.
• Equimolar expression simplifies experimental procedures and reduces observable variations.
• The biosensor facilitates robust data-driven modeling of molecular pathways.

Conclusions:

• CASPAM is an essential tool for analyzing molecular pathways requiring simultaneous monitoring of multiple nodes.
• The biosensor simplifies complex experiments and enhances the reliability of data for pathway analysis.
• This technology advances the study of cellular signaling, particularly in the context of apoptosis.