Fluorescence lifetime sorting reveals tunable enzyme interactions within cytoplasmic condensates.

IF 7.4 1区 生物学 Q1 CELL BIOLOGY Journal of Cell Biology Pub Date : 2024-10-14 DOI:10.1083/jcb.202311105
Leyla E Fahim,Joshua M Marcus,Noah D Powell,Zachary A Ralston,Katherine Walgamotte,Eleonora Perego,Giuseppe Vicidomini,Alessandro Rossetta,Jason E Lee
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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.
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荧光寿命分选揭示了细胞质凝聚体内可调的酶相互作用。
核糖核蛋白(RNP)凝聚体将 RNA 和蛋白质分隔成多个液相。冷凝物富集成分的多相特征给区分无膜冷凝物功能和周围稀释相带来了实验挑战。我们将荧光寿命成像显微镜(FLIM)与相位图过滤和分割相结合,从稀释相中分辨出冷凝物。通过测量 FLIM-Förster 共振能量转移 (FRET),凝集物的特异性寿命被用于跟踪蛋白质与蛋白质之间的相互作用。我们利用凝集物 FLIM-FRET 来评估 mRNA 解旋复合物亚基是否能在 P 体内形成解旋作用。冷凝物 FLIM-FRET 揭示了在基础条件下 P 体内存在的核心亚基相互作用,以及在氧化应激过程中解旋酶(Dcp2)与关键辅助因子(Dcp1A)之间相互作用的破坏。我们的研究结果表明,P-body 相互作用网络具有上下文相关的可塑性,它可以在几分钟内根据刺激重新布线。总之,我们基于 FLIM 的方法为研究人员提供了一种自动化的严谨方法,用于发现和跟踪活细胞中 RNP 凝聚物内重要的蛋白质-蛋白质相互作用动态。
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来源期刊
Journal of Cell Biology
Journal of Cell Biology 生物-细胞生物学
CiteScore
12.60
自引率
2.60%
发文量
213
审稿时长
1 months
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.
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