Visualization of caspase-3-like activity in cells using a genetically encoded fluorescent biosensor activated by protein cleavage
Visualization of caspase-3-like activity in cells using a genetically encoded fluorescent biosensor activated by protein cleavage
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使用由蛋白质裂解激活的基因编码荧光生物传感器可视化细胞中的 caspase-3 样活性
DOI：
10.1038/ncomms3157
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发表时间：
2013-07-01
影响因子：
16.6
通讯作者：
Li, Binghui
中科院分区：
文献类型：
--
作者：
Zhang, Jiao;Wang, Xin;Li, Binghui
细胞溶质半胱天冬酶-3样蛋白酶，如半胱天冬酶-3和半胱天冬酶-7，在介导细胞凋亡的进展中具有中心作用。为了方便地监测多细胞环境中的半胱天冬酶-3样活性，我们开发了基因编码的开关荧光基指示剂，其是含有半胱天冬酶-3切割位点作为开关的环化嵌合体。当被半胱天冬酶-3样蛋白酶切割时，非荧光指示剂迅速变成荧光，从而实时检测此类半胱天冬酶的激活。我们产生了组成型表达这些嵌合体的培养细胞，所有健康细胞都是无荧光的。当这些细胞暴露于凋亡刺激时，死细胞显示出强烈的荧光，这取决于半胱天冬酶的激活。利用这些工具，我们在各种条件下实时监测每个细胞中的caspase-3样活性，并首次在改良的软琼脂试验中显示癌细胞的环境影响其对化疗药物的敏感性。这些生物传感器应该能够更好地理解半胱天冬酶-3样蛋白酶的生物相关性。
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.