N-Way FRET Microscopy of Multiple Protein-Protein Interactions in Live Cells

Fluorescence Resonance Energy Transfer (FRET) microscopy has emerged as a powerful tool to visualize nanoscale protein-protein interactions while capturing their microscale organization and millisecond dynamics. Recently, FRET microscopy was extended to imaging of multiple donor-acceptor pairs, thereby enabling visualization of multiple biochemical events within a single living cell. These methods require numerous equations that must be defined on a case-by-case basis. Here, we present a universal multispectral microscopy method (N-Way FRET) to enable quantitative imaging for any number of interacting and non-interacting FRET pairs. This approach redefines linear unmixing to incorporate the excitation and emission couplings created by FRET, which cannot be accounted for in conventional linear unmixing. Experiments on a three-fluorophore system using blue, yellow and red fluorescent proteins validate the method in living cells. In addition, we propose a simple linear algebra scheme for error propagation from input data to estimate the uncertainty in the computed FRET images. We demonstrate the strength of this approach by monitoring the oligomerization of three FP-tagged HIV Gag proteins whose tight association in the viral capsid is readily observed. Replacement of one FP-Gag molecule with a lipid raft-targeted FP allowed direct observation of Gag oligomerization with no association between FP-Gag and raft-targeted FP. The N-Way FRET method provides a new toolbox for capturing multiple molecular processes with high spatial and temporal resolution in living cells.

荧光共振能量转移（Fluorescence Resonance Energy Transfer, FRET）显微镜技术已成为一款强大的研究工具，可在捕捉蛋白质纳米级相互作用的同时，可视化其微观尺度的组织形式与毫秒级动态变化过程。近年来，FRET显微镜技术已拓展至多供体-受体对成像领域，从而实现了单个活细胞内多种生化事件的可视化观测。此类方法需依托大量需逐案例定制的方程进行运算。本研究提出了一种通用多光谱显微镜方法（N-Way FRET），可实现任意数量相互作用与非相互作用FRET对的定量成像。该方法重新定义了线性解混流程，将FRET所引发的激发与发射耦合纳入考量范畴，而此类耦合在传统线性解混中无法被有效修正。本研究采用蓝、黄、红三种荧光蛋白（fluorescent proteins, FP）构建三荧光团系统开展实验，在活细胞中验证了该方法的有效性。此外，本研究提出了一种简易的线性代数框架，用于实现输入数据的误差传播，以估算计算得到的FRET图像的不确定性。研究团队通过监测三种FP标记的HIV Gag蛋白的寡聚化过程，验证了该方法的优势：此类蛋白在病毒衣壳中的紧密结合可被清晰观测到。将其中一个FP-Gag分子替换为靶向脂筏的FP后，可直接观测到Gag蛋白的寡聚化过程，且FP-Gag与脂筏靶向FP之间未出现结合现象。N-Way FRET方法为活细胞内以高时空分辨率捕捉多种分子过程提供了全新的研究工具箱。