School of Life Science, Faculty of Science, University of Technology Sydney, Sydney, Australia.
Cytometry A. 2022 May;101(5):387-399. doi: 10.1002/cyto.a.24527. Epub 2022 Jan 17.
Förster resonance energy transfer (FRET) is the direct energy exchange between two-component fluorescent molecules. FRET methods utilize chemically linked molecules or unlinked fluorescent molecules such as fluoresscent protein-protein interactions. FRET is therefore a powerful indicator of molecular proximity, but standardized determination of FRET efficiency is challenged when investigating natural (chemically unlinked) interactions. In this paper, we have examined the interactions of tumor necrosis factor receptor-1 (TNFR1) molecules expressed as recombinant C-terminal fusion proteins of cyan, yellow, or red fluorescent protein (-CFP, -YFP, or -RFP) to evaluate two-molecule chemically unlinked FRET by flow cytometry. We demonstrate three independent FRET pairs of TNFR1 CFP→YFP (FRET-1), YFP→RFP (FRET-2) and CFP→RFP (FRET-3), by comparing TNFR1+TNFR1 with non-interacting TNFR1+CD27 proteins, on both LSR-II and Fortessa X-20 cytometers. We describe genuine FRET activities reflecting TNFR1 homotypic interactions. The FRET events can be visualized during sample acquisition via the use of "spiked" FRET donor cells, together with TNFR1+TNFR1 co-transfected cells, as FRET channel mean fluorescence intensity (MFI) overlays. FRET events can also be indicated by comparing concatenated files of cells expressing either FRET positive events (TNFR1+TNFR1) or FRET negative events (TNFR1+CD27) to generate single-cell scatter plots showing loss of FRET donor brightness. Robust determination of FRET efficiency is then confirmed at the single-cell level by applying matrix calculations based on the measurements of FRET, using donor, acceptor, and FRET fluorescent intensities (I), detector channel emission coefficient (S), fluorescent protein extinction coefficients (ε) and the α factor. In this TNFR1-based system the mean CFP→YFP FRET-1 efficiency is 0.43 (LSR-II) and 0.41 (Fortessa X-20), the mean YFP→RFP FRET-2 efficiency is 0.30 (LSR-II) and 0.29 (Fortessa X-20), and the mean CFP→RFP FRET-3 efficiency is 0.56 (LSR-II) and 0.54 (Fortessa X-20). This study also embraces multi-dimensional clustering using t-SNE, Fit-SNE, UMAP, Tri-Map and PaCMAP to further demonstrate FRET. These approaches establish a robust system for standardized detection of chemically unlinked TNFR1 homotypic interactions with three individual FRET pairs.
Förster 共振能量转移（FRET）是两个组件荧光分子之间的直接能量交换。FRET 方法利用化学连接的分子或未连接的荧光分子，如荧光蛋白-蛋白相互作用。因此，FRET 是分子接近度的有力指标，但在研究自然（化学上未连接）相互作用时，标准化确定 FRET 效率具有挑战性。在本文中，我们研究了表达为青色、黄色或红色荧光蛋白（-CFP、-YFP 或-RFP）的重组 C 端融合蛋白的肿瘤坏死因子受体 1（TNFR1）分子的相互作用，以通过流式细胞术评估两个分子化学上未连接的 FRET。我们通过比较 TNFR1+TNFR1 与非相互作用的 TNFR1+CD27 蛋白，在 LSR-II 和 Fortessa X-20 细胞仪上，证明了三个独立的 TNFR1 CFP→YFP（FRET-1）、YFP→RFP（FRET-2）和 CFP→RFP（FRET-3）的 FRET 对。我们描述了反映 TNFR1 同源相互作用的真实 FRET 活性。通过使用“加标”FRET 供体细胞以及共转染 TNFR1+TNFR1 的细胞，可以在样品采集过程中可视化 FRET 事件，作为 FRET 通道平均荧光强度（MFI）叠加。通过比较表达 FRET 阳性事件（TNFR1+TNFR1）或 FRET 阴性事件（TNFR1+CD27）的细胞的拼接文件，也可以指示 FRET 事件，以生成显示 FRET 供体亮度损失的单细胞散点图。然后通过应用基于 FRET、供体、受体和 FRET 荧光强度（I）、探测器通道发射系数（S）、荧光蛋白消光系数（ε）和α 因子的矩阵计算，在单细胞水平上确认 FRET 效率的稳健确定。在基于 TNFR1 的系统中，平均 CFP→YFP FRET-1 效率为 0.43（LSR-II）和 0.41（Fortessa X-20），平均 YFP→RFP FRET-2 效率为 0.30（LSR-II）和 0.29（Fortessa X-20），平均 CFP→RFP FRET-3 效率为 0.56（LSR-II）和 0.54（Fortessa X-20）。本研究还采用 t-SNE、Fit-SNE、UMAP、Tri-Map 和 PaCMAP 进行多维聚类，进一步证明了 FRET。这些方法建立了一个强大的系统，用于标准化检测具有三个独立 FRET 对的化学上未连接的 TNFR1 同源相互作用。
