费斯特共振能量转移
生物物理学
核糖核蛋白
化学
荧光寿命成像显微镜
蛋白质-蛋白质相互作用
蛋白质亚单位
活体细胞成像
荧光团
背景(考古学)
荧光
细胞生物学
核糖核酸
物理
生物
生物化学
细胞
古生物学
量子力学
基因
作者
Leyla E. Fahim,Joshua M. Marcus,Noah D. Powell,Zachary A. Ralston,Katherine Walgamotte,Eleonora Perego,Giuseppe Vicidomini,Alessandro Rossetta,Jason E. Lee
标识
DOI:10.1083/jcb.202311105
摘要
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.
科研通智能强力驱动
Strongly Powered by AbleSci AI