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

Abstract

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.

A fate worse than death: apoptosis as an oncogenic process

“…Along these lines, fluorescent reporters have recently been developed that accurately detect caspase activity even at sub lethal levels 71,115 . While apoptosis has been extensively imaged in vitro very few studies have extended these analyses to an in vivo setting [116][117][118] .…”

Bax/Bak-dependent, Drp1-independent Targeting of X-linked Inhibitor of Apoptosis Protein (XIAP) into Inner Mitochondrial Compartments Counteracts Smac/DIABLO-dependent Effector Caspase Activation

“…6, C-E) (19). Importantly, 6 h of exposure to CCCP had no impact on caspase activity in XIAPoverexpressing cells, indicating that XIAP-induced mitochondrial permeabilization does not per se result in caspase activation.…”

“…For stable lentiviral gene transfer, pCDH-puro-CMV carrying the super-folding GFPtagged caspase-3/-7 sensor (GC3A1) (19) was calcium-phosphate-transfected into 293T cells together with pCMVdeltaR8.91 and pMD2.G. MCF7 cells were infected with virus particle-containing supernatants and selected and maintained with puromycin (1 g/ml), following selection of a stable single clone line.…”

Persistent luminescence nanoprobe for biosensing and lifetime imaging of cell apoptosis via time-resolved fluorescence resonance energy transfer

“…Indeed, many elegant fluorescent probes including graphene oxideepeptide conjugate [35], aggregation light-up tetraphenylethene fluorogen [36], and a genetically encoded fluorescent protein [37], have been developed for detection of caspase activity in live cells [38,39]. However, these proposed methods were usually susceptible to the short-lived auto-fluorescence interference from cells and tissues.…”