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

被引：209

作者：

Zhang, Jiao ^{[1
,2
]}

Wang, Xin ^{[2
]}

Cui, Wenjing ^{[1
,2
]}

Wang, Wenwen ^{[1
,2
]}

Zhang, Huamei ^{[1
]}

Liu, Lu ^{[1
]}

Zhang, Zicheng ^{[1
]}

Li, Zheng ^{[3
]}

Ying, Guoguang ^{[1
]}

Zhang, Ning ^{[1
]}

Li, Binghui ^{[1
]}

机构：

[1] Tianjin Med Univ, Canc Inst & Hosp, Key Lab Breast Canc Prevent & Therapy, Canc Cell Biol Lab, Tianjin 300060, Peoples R China

[2] Tianjin Med Univ, Canc Inst & Hosp, Key Lab Breast Canc Prevent & Therapy, Dept Breast Tumor 1, Tianjin 300060, Peoples R China

[3] Tianjin Med Univ, Canc Inst & Hosp, Key Lab Breast Canc Prevent & Therapy, Dept Biochem & Mol Biol, Tianjin 300060, Peoples R China

来源：

NATURE COMMUNICATIONS | 2013年 / 4卷

基金：

中国国家自然科学基金;

关键词：

RESONANCE ENERGY-TRANSFER; ALPHA-INDUCED APOPTOSIS; PHYSIOLOGICAL CONDITIONS; COMPLEMENTATION SYSTEM; CANCER-CELLS; DNAE INTEIN; CASPASES; RED; REPORTER; DEATH;

D O I：

10.1038/ncomms3157

中图分类号：

O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];

学科分类号：

07 ; 0710 ; 09 ;

摘要：

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.

引用

页数：13

共 39 条

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