Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina.
Departments of Bioinformatics and Cell Biology, University of Texas Southwestern Medical Center, Dallas, Texas.
Biophys J. 2023 Sep 19;122(18):3646-3655. doi: 10.1016/j.bpj.2023.04.020. Epub 2023 Apr 20.
Imaging two or more fluorescent biosensors in the same living cell can reveal the spatiotemporal coordination of protein activities. However, using multiple Förster resonance energy transfer (FRET) biosensors together is challenging due to toxicity and the need for orthogonal fluorophores. Here we generate a biosensor component that binds selectively to the activated conformation of three different proteins. This enabled multiplexed FRET with fewer fluorophores, and reduced toxicity. We generated this MultiBinder (MB) reagent for the GTPases RhoA, Rac1, and Cdc42 by combining portions of the downstream effector proteins Pak1 and Rhotekin. Using FRET between mCherry on the MB and YPet or mAmetrine on two target proteins, the activities of any pair of GTPases could be distinguished. The MB was used to image Rac1 and RhoA together with a third, dye-based biosensor for Cdc42. Quantifying effects of biosensor combinations on the frequency, duration, and velocity of cell protrusions and retractions demonstrated reduced toxicity. Multiplexed imaging revealed signaling hierarchies between the three proteins at the cell edge where they regulate motility.
在同一个活细胞中同时成像两个或更多荧光生物传感器可以揭示蛋白质活性的时空协调。然而，由于毒性和对正交荧光团的需求，同时使用多个Förster 共振能量转移（FRET）生物传感器具有挑战性。在这里，我们生成了一个生物传感器组件，该组件选择性地结合到三种不同蛋白质的激活构象上。这使得可以使用更少的荧光团进行多路复用 FRET，并且降低了毒性。我们通过组合下游效应蛋白 Pak1 和 Rhotekin 的部分来生成针对 GTPases RhoA、Rac1 和 Cdc42 的多结合物（MB）试剂。使用 MB 上的 mCherry 与两个靶蛋白上的 YPet 或 mAmetrine 之间的 FRET，可以区分任何一对 GTPases 的活性。该 MB 用于与基于染料的第三个 Cdc42 生物传感器一起成像 Rac1 和 RhoA。定量分析生物传感器组合对细胞突起和缩回的频率、持续时间和速度的影响表明毒性降低。多路复用成像揭示了它们在调节运动的细胞边缘处三种蛋白质之间的信号层次结构。
