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

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.

Caspase cascade pathways in apoptosis of oral lichen planus.

OBJECTIVE: Apoptosis is frequently found in oral lichen planus (OLP) lesions, but the pathways leading to apoptosis are unknown. STUDY DESIGN: This study focused on analysis of caspase expression which is essential for apoptosis. Expression of caspases 2, 3, 8, 9, and 12 was studied in 70 biopsy samples from atrophic OLP to identify which cascade pathway, extrinsic or intrinsic, is of importance in apoptosis in OLP. RESULTS: Caspase-2 expression was present in every sample, and >70% of the epithelial cells were positive in 33% of the lesions. More than 70% of the epithelial cells expressed caspase-12 in 84% of the specimens. Caspase-8 expression was shown totally in 87% of the specimens. No caspase-3 expression was found in 57% of the samples, and caspase-9 expression was absent in the entire OLP specimen. CONCLUSIONS: The high frequency of intrinsic apoptotic pathway markers caspases 2 and 12 indicates intracellular stress in atrophic OLP epithelial cells.