Co-imaging extrinsic, intrinsic and effector caspase activity by fluorescence anisotropy microscopy

Abstract

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.

Resveratrol Enhances Apoptotic and Oxidant Effects of Paclitaxel through TRPM2 Channel Activation in DBTRG Glioblastoma Cells

“…Several proteins act in extrinsic apoptosis; for instance, caspase 8 shears and activates downstream apoptosis-inducing proteins including caspase 3 [ 84 ]. Caspase 3 acts through both extrinsic and intrinsic apoptosis pathways [ 85 ]. In the current study, caspase 3 but not caspase 9 activity was increased by PAX treatment.…”

CASPAM: a triple modality biosensor for multiplexed imaging of caspase network activity

“…Homo-FRET measured by fluorescence polarization anisotropy (FPA) microscopy has been used as a tool to detect molecular interactions, conformational changes and assembly state [12, 21, 14]. Briefly, the emission of FP is highly anisotropic upon excitation with linearly polarized light, due to the rather larger size and therefore their large rotational diffusion time as compared to the fluorescence lifetime.…”

“…Different detection channels were configured to image each individual biosensor. Due to the lack of significant spectral overlap between fluorophores [12] linear unmixing adjustments were dismissed. As shown in Figure 2B , none of the cells transfected with the plasmids encoding the individual biosensors showed fluorescence signal in a detection channel other than that specified for each fluorophore-pair.…”

“…Genetically encoded hetero-FRET-based biosensors (consisting of two spectrally separated fluorescent proteins (FP): a donor and a red-shifted acceptor) have uncovered relevant spatiotemporal aspects of protein interaction networks [10, 11]. Despite this, due to the broad spectra of FP, co-imaging in the visible range is limited to two sensors (4 FP) [1, 12]. In this sense, single fluorophore (or spectrally similar fluorophore) homo-FRET based biosensors considerably shrink the spectral window per sensor by means of fluorescence polarization anisotropy (FPA) [13, 14, 15], thus enabling the insertion of up to three reporters in a single cell [12].…”

“…Despite this, due to the broad spectra of FP, co-imaging in the visible range is limited to two sensors (4 FP) [1, 12]. In this sense, single fluorophore (or spectrally similar fluorophore) homo-FRET based biosensors considerably shrink the spectral window per sensor by means of fluorescence polarization anisotropy (FPA) [13, 14, 15], thus enabling the insertion of up to three reporters in a single cell [12].…”