Design of FRET sensor molecules for detection of local activation of specific caspases. (A) Schematic outline of FRET sensor molecules. ECFP and EYFP are connected with different spacer sequences containing two tandemly arranged tetra-amino acid cleavage sequences preferentially cleaved by different caspases (as specified) or a non-cleavable control tetra-amino acid LEVA sequence. The microtubule binding Tau protein is fused to the C-terminus of EYFP to target the sensors to microtubules. (B) Distribution of the caspase-3 (DEVD) sensor to microtubules of mitotic (left) and interphase (right) HeLa cells. (C) Quantitative acceptor bleaching experiments in differentiated neuroblastoma SH-SY5Y cells expressing different caspase sensors, showing approximately 50% increase in ECFP fluorescence after bleaching of EYFP. (D) U2OS cells expressing FRET sensors for caspase-6 (VEID) treated with 10 μM staurosporine. The apoptotic cell (star) showed maintained localization of EYFP fluorescence to microtubules (right cell), in contrast to ECFP fluorescence, after loss of FRET.