Real-time monitoring of caspase cascade activation in living cells.
We introduce a simple, versatile and robust one-step technique that enables real-time imaging of multiple intracellular caspase activities in living cells without the need for complicated synthetic protocols. Conventional fluorogenic probes or recently reported activatable probes have been designed to target various proteases but are limited to extracellular molecules. Only a few have been applied to image intracellular proteases in living cells because most of these probes have limited cell-permeability. Our platform does not need complicated synthetic processes; instead it involves a straightforward peptide synthesis and a simple mixing step with a commercial transfection agent. The transfection agent efficiently delivered the highly quenched fluorogenic probes, comprised of distinctive pairs of dyes and quenchers, to the initiator caspase-8 and the effector caspase-3 in MDA-MB-435 cells, allowing dual-imaging of the activities of both caspases during the apoptotic process induced by TNF-related apoptosis induced ligand (TRAIL). With the combination of multiple fluorogenic probes, this simple platform can be applied to multiplexed imaging of selected intracellular proteases to study apoptotic processes in pathologies or for cell-based high throughput screening systems for drug discovery.
Center for Molecular Imaging and Translational Medicine, School of Public Health, Xiamen University, Xiamen, 361005, China.
Lei
L
Zhu
Xinglu
X
Huang
Ki Young
KY
Choi
Ying
Y
Ma
Fan
F
Zhang
Gang
G
Liu
Seulki
S
Lee
Xiaoyuan
X
Chen
D017209:Apoptosis; D053148:Caspase 3 / Q000378:metabolism*; D053181:Caspase 8 / Q000378:metabolism*; D045744:Cell Line, Tumor; D005456:Fluorescent Dyes / Q000008:administration & dosage / Q000737:chemistry; D006801:Humans; D008163:Luminescent Measurements; D009842:Oligopeptides / Q000008:administration & dosage / Q000737:chemistry; D053221:TNF-Related Apoptosis-Inducing Ligand
D005456:Fluorescent Dyes; D009842:Oligopeptides; D053221:TNF-Related Apoptosis-Inducing Ligand; D053148:Caspase 3; D053181:Caspase 8