Automated FLIM-FRET Segmentation Within RNP Condensates

Area of Science:

• Cell Biology
• Molecular Biology
• Biophysics

Background:

• Ribonucleoprotein (RNP) condensates are essential membraneless organelles involved in RNA processing.
• Understanding the dynamic protein interactions within these complex structures is crucial but challenging.
• Current methods lack the throughput and scalability to analyze RNP condensate dynamics in live cells.

Purpose of the Study:

• To develop a scalable, high-throughput fluorescence microscopy approach for analyzing protein-protein interaction networks within RNP condensates.
• To rigorously assess dynamic, process-critical interactions within RNP condensates in live cells.
• To infer the functional consequences of RNP granule partitioning.

Main Methods:

• Utilized fluorescence lifetime imaging (FLIM) and phasor plot analysis for automated segmentation of condensate signals.
• Employed FLIM-Förster resonant energy transfer (FLIM-FRET) with fluorescent protein pairs to monitor protein-protein interactions.
• Applied the method to analyze protein interactions in live cells under various conditions and stimuli.

Main Results:

• Developed a method for automated segmentation of condensate-localized fluorescent signals, independent of thresholding.
• Successfully monitored protein-protein interactions within RNP condensates using FLIM-FRET in live cells.
• Generated insights into the organization and assembly of factors critical for condensate-associated processes.

Conclusions:

• The developed FLIM-based approach offers a powerful tool for studying dynamic protein interactions within RNP condensates.
• This method provides valuable insights into the functional consequences of RNP granule organization.
• The framework is adaptable for investigating protein interactions in diverse biological compartments and systems.