Language

English

Publication Date

1-6-2025

Journal

Journal of Cell Biology

DOI

10.1083/jcb.202311105

PMID

39400294

PMCID

PMC11472878

PubMedCentral® Posted Date

10-14-2024

PubMedCentral® Full Text Version

Post-print

Abstract

Ribonucleoprotein (RNP) condensates partition RNA and protein into multiple liquid phases. The multiphasic feature of condensate-enriched components creates experimental challenges for distinguishing membraneless condensate functions from the surrounding dilute phase. We combined fluorescence lifetime imaging microscopy (FLIM) with phasor plot filtering and segmentation to resolve condensates from the dilute phase. Condensate-specific lifetimes were used to track protein-protein interactions by measuring FLIM-Förster resonance energy transfer (FRET). We used condensate FLIM-FRET to evaluate whether mRNA decapping complex subunits can form decapping-competent interactions within P-bodies. Condensate FLIM-FRET revealed the presence of core subunit interactions within P-bodies under basal conditions and the disruption of interactions between the decapping enzyme (Dcp2) and a critical cofactor (Dcp1A) during oxidative stress. Our results show a context-dependent plasticity of the P-body interaction network, which can be rewired within minutes in response to stimuli. Together, our FLIM-based approaches provide investigators with an automated and rigorous method to uncover and track essential protein-protein interaction dynamics within RNP condensates in live cells.

Keywords

Humans, Fluorescence Resonance Energy Transfer, Ribonucleoproteins, Microscopy, Fluorescence, Cytoplasm, Biomolecular Condensates, HeLa Cells, Oxidative Stress, Endoribonucleases, Protein Binding

Published Open-Access

yes

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