Leyla E Fahim,Joshua M Marcus,Noah D Powell,Zachary A Ralston,Katherine Walgamotte,Eleonora Perego,Giuseppe Vicidomini,Alessandro Rossetta,Jason E Lee
{"title":"Fluorescence lifetime sorting reveals tunable enzyme interactions within cytoplasmic condensates.","authors":"Leyla E Fahim,Joshua M Marcus,Noah D Powell,Zachary A Ralston,Katherine Walgamotte,Eleonora Perego,Giuseppe Vicidomini,Alessandro Rossetta,Jason E Lee","doi":"10.1083/jcb.202311105","DOIUrl":null,"url":null,"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.","PeriodicalId":15211,"journal":{"name":"Journal of Cell Biology","volume":null,"pages":null},"PeriodicalIF":7.4000,"publicationDate":"2024-10-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Journal of Cell Biology","FirstCategoryId":"99","ListUrlMain":"https://doi.org/10.1083/jcb.202311105","RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"CELL BIOLOGY","Score":null,"Total":0}
引用次数: 0
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.
期刊介绍:
The Journal of Cell Biology (JCB) is a comprehensive journal dedicated to publishing original discoveries across all realms of cell biology. We invite papers presenting novel cellular or molecular advancements in various domains of basic cell biology, along with applied cell biology research in diverse systems such as immunology, neurobiology, metabolism, virology, developmental biology, and plant biology. We enthusiastically welcome submissions showcasing significant findings of interest to cell biologists, irrespective of the experimental approach.