Visualization of caspase-3-like activity in cells using a genetically encoded fluorescent biosensor activated by protein cleavage

• DOI: 10.1038/ncomms3157
• Publication Date: 2013-07-16T09:58:52Z
• Authors: Jiao Zhang; Xin Wang; Wenjing Cui; Wenwen Wang; Huamei Zhang; Lu Liu; Zicheng Zhang; Zheng Li; Guoguang Ying; Ning Zhang; Binghui Li
• Keywords: 0303 health sciences; Tumor Necrosis Factor-alpha; Genetic Vectors; Lentivirus; Apoptosis; Biosensing Techniques; Fluorescence; Molecular Imaging; 3. Good health; Enzyme Activation; 03 medical and health sciences; Microscopy, Fluorescence; Caspases; Molecular Probes; Humans; HeLa Cells

Cytosolic caspase-3-like proteases, such as caspase-3 and caspase-7, have a central role in mediating the progress of apoptosis. Here to conveniently monitor caspase-3-like activity in the multicellular environment, we have developed genetically encoded switch-on fluorescence-base indicators that are cyclized chimeras containing a caspase-3 cleavage site as a switch. When cleaved by caspase-3-like proteases, the non-fluorescent indicator rapidly becomes fluorescent, and thus detects in real-time the activation of such caspases. We generate cultured cells constitutively expressing these chimeras, and all the healthy cells are non-fluorescent. When these cells are exposed to apoptotic stimuli, dead cells show strong fluorescence depending on caspase activation. With these tools, we monitor in real-time caspase-3-like activity in each cell under various conditions, and show for the first time that the environment of cancer cells affects their sensitivity to chemotherapeutic drugs in a modified soft agar assay. These biosensors should enable better understanding of the biological relevance of caspase-3-like proteases.