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

This is a first author publication from my PhD project.

For this paper, I studied the apoptotic cascade through the use of pseudo homo-FRET sensors that probe the integral activity of intrinsic, extrinsic and effector caspases simultaneously in single cells. To avoid experimenter bias and amass enough data, I worked on the design of experiments and programming of Python modules that automatically analyze acquired images. For the translation of the photophysical signal, careful consideration of its origin was taken into account in order to be able to translate from amount of photons to amount of molecules in each state and then to enzyme activity. We found that maximum activity is a proxy for activation times and their correlative values can be used to refine existing models of biological signalling networks. We also show that peak activity was reached first not by the initiator caspase, but by the effector caspase since feedbacks are key to cell death decision, and the important role of some inhibitors, such as XIAP.