As one of the most powerful tools to visualize PPIs in living cells, bimolecular fluorescence complementation (BiFC) has gained great advancement during recent years, including deep tissue imaging with far-red or near-infrared fluorescent proteins or super-resolution imaging with photochromic fluorescent proteins. However, little progress has been made toward simultaneous detection and visualization of multiple PPIs in the same cell, mainly due to the spectral crosstalk. In this report, we developed novel BiFC assays based on large-Stokes-shift fluorescent proteins (LSS-FPs) to detect and visualize multiple PPIs in living cells. With the large excitation/emission spectral separation, LSS-FPs can be imaged together with normal Stokes shift fluorescent proteins to realize multicolor BiFC imaging using a simple illumination scheme. We also further demonstrated BiFC rainbow combining newly developed BiFC assays with previously established mCerulean/mVenus-based BiFC assays to achieve detection and visualization of four PPI pairs in the same cell. Additionally, we prove that with the complete spectral separation of mT-Sapphire and CyOFP1, LSS-FP-based BiFC assays can be readily combined with intensity-based FRET measurement to detect ternary protein complex formation with minimal spectral crosstalk. Thus, our newly developed LSS-FP-based BiFC assays not only expand the fluorescent protein toolbox available for BiFC but also facilitate the detection and visualization of multiple protein complex interactions in living cells.

中文翻译：

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BiFC Rainbow与多种蛋白质-蛋白质相互作用的活细胞可视化

作为可视化活细胞中PPI的最强大工具之一，近年来双分子荧光互补（BiFC）取得了长足的进步，包括使用远红或近红外荧光蛋白进行深层组织成像或使用光致变色荧光进行超分辨率成像蛋白质。但是，主要由于频谱串扰，在同一单元中同时检测和可视化多个PPI方面进展甚微。在本报告中，我们开发了基于大斯托克斯位移荧光蛋白（LSS-FPs）的新颖BiFC测定法，以检测和可视化活细胞中的多个PPI。借助大的激发/发射光谱分离，可以将LSS-FP与正常的Stokes位移荧光蛋白成像，从而使用简单的照明方案实现多色BiFC成像。我们还进一步证明了BiFC Rainbow将新开发的BiFC测定法与先前建立的基于mCerulean / mVenus的BiFC测定法相结合，以实现同一细胞中四个PPI对的检测和可视化。此外，我们证明了通过mT-蓝宝石和CyOFP1的完全光谱分离，基于LSS-FP的BiFC分析可以轻松地与基于强度的FRET测量结合使用，以最小的光谱串扰检测三元蛋白质复合物的形成。因此，我们新开发的基于LSS-FP的BiFC分析不仅扩展了BiFC可用的荧光蛋白工具箱，而且还促进了活细胞中多种蛋白复合物相互作用的检测和可视化。