Automated FLIM-FRET Segmentation Within RNP Condensates.

Ribonucleoprotein (RNP) condensates are membraneless organelles that exist alongside many RNA-driven processes, such as transcription and splicing. Despite their ubiquity, the biological necessity of forming a condensed phase remains unclear, particularly because the same RNP components exist both within these organelles and in the surrounding dilute phase. Most current methods for studying biochemical interaction dynamics within condensates rely on in vitro reconstitution of minimal factors or low-throughput single-molecule studies. However, RNP condensates are complex organelles containing tens to hundreds of proteins and hundreds to thousands of different RNAs. Here, we describe a scalable, high-throughput fluorescence microscopy-based approach to analyze protein-protein interaction networks, allowing for the rigorous assessment of dynamic, process-critical interactions within RNP condensates from live cells. This method takes advantage of fluorescence lifetime imaging (FLIM) and phasor plot analysis to automate segmentation of condensate-localized fluorescence signals. Using suitable FLIM-Förster resonant energy transfer (FLIM-FRET) fluorescent pairs fused to proteins of interest, protein-protein interactions can be actively monitored throughout various conditions via changes in fluorescence lifetime. Results from this assay yield valuable insight into the organization and assembly of essential factors for different condensate-associated processes to infer the functional consequences of RNP granule partitioning. Although this protocol is tailored for studying protein interactions within condensates, the design and execution framework can be adapted to investigate protein-protein interactions across a wide variety of compartments within different biological systems. Key features • Automated segmentation of condensate-localized fluorescent signal independent of thresholding or background subtraction. • FLIM-FRET to analyze protein-protein interactions, with an emphasis on condensate interaction networks. • Monitor protein interactions from live cells over time and in response to different stimuli.