NTnC-like genetically encoded calcium indicator with a positive and enhanced response and fast kinetics.
NTnC-like genetically encoded calcium indicator with a positive and enhanced response and fast kinetics.
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DOI：
10.1038/s41598-018-33613-6
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发表时间：
2018-10-15
影响因子：
4.6
通讯作者：
Subach FV
中科院分区：
文献类型：
--
作者：
Barykina NV;Doronin DA;Subach OM;Sotskov VP;Plusnin VV;Ivleva OA;Gruzdeva AM;Kunitsyna TA;Ivashkina OI;Lazutkin AA;Malyshev AY;Smirnov IV;Varizhuk AM;Pozmogova GE;Piatkevich KD;Anokhin KV;Enikolopov G;Subach FV
NTnC基因编码的钙指示剂具有有利的设计，因为与广泛使用的来自GCaMP家族的指示剂相比，其尺寸更小，具有GFP样的N-和C-末端，并且钙结合位点的数量减少两倍。然而，NTnC具有倒置和适度的钙响应以及低时间分辨率。通过用EYFP取代NTnC中的mNeonGreen荧光部分，我们设计了一种NTnC样指示剂，称为YTnC，其具有积极的和显著改善的钙响应和更快的动力学。YTnC在体外具有比NTnC高3倍的钙响应和低13.6倍的亮度。根据体外进行的停流实验，YTnC的钙解离动力学比NTnC快4倍。在HeLa细胞中，YTnC表现出比NTnC低3.3倍的亮度和4.9倍的钙瞬变响应。神经元培养物的自发活动诱导YTnC的ΔF/F响应比先前显示的NTnC大3.6倍。在斑贴神经元上，YTnC的ΔF/F比GCaMP 6s低2.6倍。YTnC使用单光子和双光子显微镜成功地可视化了麻醉小鼠皮质和清醒小鼠海马中神经元的钙瞬变。此外，YTnC在培养的HeLa和神经元细胞的线粒体和内质网中优于GCaMP 6s。
The NTnC genetically encoded calcium indicator has an advantageous design because of its smaller size, GFP-like N- and C-terminal ends and two-fold reduced number of calcium binding sites compared with widely used indicators from the GCaMP family. However, NTnC has an inverted and modest calcium response and a low temporal resolution. By replacing the mNeonGreen fluorescent part in NTnC with EYFP, we engineered an NTnC-like indicator, referred to as YTnC, that had a positive and substantially improved calcium response and faster kinetics. YTnC had a 3-fold higher calcium response and 13.6-fold lower brightness than NTnC in vitro. According to stopped-flow experiments performed in vitro, YTnC had 4-fold faster calcium-dissociation kinetics than NTnC. In HeLa cells, YTnC exhibited a 3.3-fold lower brightness and 4.9-fold increased response to calcium transients than NTnC. The spontaneous activity of neuronal cultures induced a 3.6-fold larger ΔF/F response of YTnC than previously shown for NTnC. On patched neurons, YTnC had a 2.6-fold lower ΔF/F than GCaMP6s. YTnC successfully visualized calcium transients in neurons in the cortex of anesthetized mice and the hippocampus of awake mice using single- and two-photon microscopy. Moreover, YTnC outperformed GCaMP6s in the mitochondria and endoplasmic reticulum of cultured HeLa and neuronal cells.
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影响因子：
48
作者：
Subach, Fedor V.;Piatkevich, Kiryl D.;Verkhusha, Vladislav V.
通讯作者：
Verkhusha, Vladislav V.
改善绿色和红色荧光蛋白的亮度和光稳定性，用于活细胞成像和FRET报告。
影响因子：
4.6
作者：
Bajar BT;Wang ES;Lam AJ;Kim BB;Jacobs CL;Howe ES;Davidson MW;Lin MZ;Chu J
通讯作者：
Chu J
影响因子：
4.6
作者：
Barykina, Natalia V.;Subach, Oksana M.;Enikolopov, Grigori N.
通讯作者：
Enikolopov, Grigori N.
影响因子：
4.8
作者：
Heim, N;Griesbeck, O
通讯作者：
Griesbeck, O
影响因子：
25
作者：
Shemesh OA;Tanese D;Zampini V;Linghu C;Piatkevich K;Ronzitti E;Papagiakoumou E;Boyden ES;Emiliani V
通讯作者：
Emiliani V