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.

中文翻译：

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荧光寿命分选揭示了细胞质凝聚物内可调的酶相互作用。


核糖核蛋白 （RNP） 将 RNA 和蛋白质缩合成多个液相。富含凝聚物组分的多相特性为区分无膜凝聚物功能与周围的稀相带来了实验挑战。我们将荧光寿命成像显微镜 （FLIM） 与相量图过滤和分割相结合，以分辨稀相中的凝聚物。通过测量 FLIM-Förster 共振能量转移 （FRET），使用冷凝物特异性寿命来跟踪蛋白质-蛋白质相互作用。我们使用凝聚物 FLIM-FRET 来评估 mRNA 脱帽复合物亚基是否可以在 P 小体内形成脱帽能力相互作用。凝聚物 FLIM-FRET 揭示了在基础条件下 P 体内存在核心亚基相互作用，以及氧化应激期间脱帽酶 （Dcp2） 和关键辅因子 （Dcp1A） 之间相互作用的破坏。我们的结果表明 P 体交互网络具有上下文依赖性的可塑性，它可以在几分钟内响应刺激而重新连接。总之，我们基于 FLIM 的方法为研究人员提供了一种自动化且严格的方法，以揭示和跟踪活细胞中 RNP 凝聚物中的基本蛋白质-蛋白质相互作用动力学。