Fluorescence lifetime sorting reveals tunable enzyme interactions within cytoplasmic condensates

Area of Science:

• Cell Biology
• Biophysics
• Molecular Biology

Background:

• Ribonucleoprotein (RNP) condensates are membraneless organelles crucial for cellular processes.
• Distinguishing condensate functions from the surrounding environment is challenging.
• Understanding protein-protein interactions within condensates is vital for deciphering their roles.

Purpose of the Study:

• To develop and apply a novel method for resolving and tracking protein interactions within RNP condensates.
• To investigate the dynamics of the mRNA decapping complex within P-bodies.
• To assess how cellular stress affects protein interactions in P-bodies.

Main Methods:

• Combined fluorescence lifetime imaging microscopy (FLIM) with phasor plot filtering and segmentation.
• Utilized FLIM-Förster resonance energy transfer (FRET) to measure protein-protein interactions.
• Applied condensate FLIM-FRET to analyze P-body subunit interactions in live cells.

Main Results:

• Successfully resolved RNP condensates from the dilute phase using FLIM.
• Detected core subunit interactions within P-bodies under basal conditions.
• Observed disruption of interactions between Dcp2 and Dcp1A during oxidative stress, indicating context-dependent plasticity.

Conclusions:

• FLIM-based approaches offer a robust method for studying protein dynamics in RNP condensates.
• P-body interaction networks are plastic and can be rapidly rewired in response to stimuli.
• This technique provides new insights into the functional regulation of RNP condensates.