Department of Physics, FCEN, University of Buenos Aires and IFIBA, CONICET, Buenos Aires, Argentina.
Department of Systemic Cell Biology, Max Planck Institute of Molecular Physiology, Dortmund, Germany.
Redox Biol. 2018 Oct;19:210-217. doi: 10.1016/j.redox.2018.07.023. Epub 2018 Aug 9.
In order to overcome intercellular variability and thereby effectively assess signal propagation in biological networks it is imperative to simultaneously quantify multiple biological observables in single living cells. While fluorescent biosensors have been the tool of choice to monitor the dynamics of protein interaction and enzymatic activity, co-measuring more than two of them has proven challenging. In this work, we designed three spectrally separated anisotropy-based Förster Resonant Energy Transfer (FRET) biosensors to overcome this difficulty. We demonstrate this principle by monitoring the activation of extrinsic, intrinsic and effector caspases upon apoptotic stimulus. Together with modelling and simulations we show that time of maximum activity for each caspase can be derived from the anisotropy of the corresponding biosensor. Such measurements correlate relative activation times and refine existing models of biological signalling networks, providing valuable insight into signal propagation.
为了克服细胞间的可变性，从而有效地评估生物网络中的信号传递，有必要在单个活细胞中同时定量多个生物学观察值。虽然荧光生物传感器一直是监测蛋白质相互作用和酶活性动力学的首选工具，但同时测量两个以上的生物传感器已经被证明具有挑战性。在这项工作中，我们设计了三种光谱分离的基于各向异性的Förster 共振能量转移（FRET）生物传感器来克服这一困难。我们通过监测凋亡刺激后外源、内源和效应半胱天冬酶的激活来证明这一原理。通过建模和模拟，我们表明每个半胱天冬酶的最大活性时间可以从相应生物传感器的各向异性中得出。这种测量方法可以关联相对激活时间，并改进现有的生物信号网络模型，为信号传递提供有价值的见解。
