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

Author:

Zhang, Jiao   Wang, Xin   Cui, Wenjing   Wang, Wenwen   Zhang, Huamei   Liu, Lu   Zhang, Zicheng   Li, Zheng   Ying, Guoguang   Zhang, Ning   Li, Binghui  

Journal:

Nature Communications

Issue Date:

2013

Abstract(summary):

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.

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