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.

journal_name

Redox Biol

journal_title

Redox biology

authors

Corbat AA,Schuermann KC,Liguzinski P,Radon Y,Bastiaens PIH,Verveer PJ,Grecco HE

doi

10.1016/j.redox.2018.07.023

subject

Has Abstract

pub_date

2018-10-01 00:00:00

pages

210-217

issn

2213-2317

pii

S2213-2317(18)30552-4

journal_volume

19

pub_type

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