Genetically encoded biosensors for visualizing live-cell biochemical activity at super-resolution.
Genetically encoded biosensors for visualizing live-cell biochemical activity at super-resolution.
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DOI：
10.1038/nmeth.4221
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发表时间：
2017-04
期刊：
影响因子：
48
通讯作者：
Zhang J
中科院分区：
文献类型：
--
作者：
Mo GC;Ross B;Hertel F;Manna P;Yang X;Greenwald E;Booth C;Plummer AM;Tenner B;Chen Z;Wang Y;Kennedy EJ;Cole PA;Fleming KG;Palmer A;Jimenez R;Xiao J;Dedecker P;Zhang J
区隔化的生化活动对所有细胞过程都是必不可少的，但目前还没有一种通用的方法可以在与它们的区隔化相适应的分辨率下可视化活细胞中的动态蛋白质活动。在这里，我们介绍了一类新的荧光生物传感器，检测生物化学活动在活细胞的分辨率高达三倍优于衍射极限。这些生物传感器利用特异性的、结合诱导的蛋白质荧光动力学变化，将激酶活性或蛋白质-蛋白质相互作用转化为荧光波动的变化，这些变化可以通过随机光学波动成像来量化。蛋白激酶A （PKA）生物传感器使我们能够解析活细胞质膜上微小的PKA活性微域，并揭示聚集锚定蛋白在组织这些活性微域中的作用。总之，这些发现表明，细胞的生化活动在空间上被组织成一个活动结构，这些新的生物传感器可以揭示其结构和功能特征。
Compartmentalized biochemical activities are essential to all cellular processes, but there is no generalizable method to visualize dynamic protein activities in living cells at a resolution commensurate with their compartmentalization. Here we introduce a new class of fluorescent biosensors that detect biochemical activities in living cells at a resolution up to three-fold better than the diffraction limit. Utilizing specific, binding-induced changes in protein fluorescence dynamics, these biosensors translate kinase activities or protein-protein interactions into changes in fluorescence fluctuations, which are quantifiable through stochastic optical fluctuation imaging. A Protein Kinase A (PKA) biosensor allowed us to resolve minute PKA activity microdomains on the plasma membrane of living cells and uncover the role of clustered anchoring proteins in organizing these activity microdomains. Together, these findings suggest that biochemical activities of the cell are spatially organized into an activity architecture, whose structural and functional characteristics can be revealed by these new biosensors.