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Visualization of caspase-3-like activity in cells using a genetically encoded fluorescent biosensor activated by protein cleavage.
|Title||Visualization of caspase-3-like activity in cells using a genetically encoded fluorescent biosensor activated by protein cleavage.|
|Publication Type||Journal Article|
|Year of Publication||2013|
|Authors||Zhang J, Wang X, Cui W, Wang W, Zhang H, Liu L, Zhang Z, Li Z, Ying G, Zhang N, Li B|
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.
|Alternate Journal||Nat Commun|