Green fluorescent protein-based lactate and pyruvate indicators suitable for biochemical assays and live cell imaging.
Green fluorescent protein-based lactate and pyruvate indicators suitable for biochemical assays and live cell imaging.
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DOI：
10.1038/s41598-020-76440-4
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发表时间：
2020-11-11
影响因子：
4.6
通讯作者：
Tsuboi T
中科院分区：
文献类型：
--
作者：
Harada K;Chihara T;Hayasaka Y;Mita M;Takizawa M;Ishida K;Arai M;Tsuno S;Matsumoto M;Ishihara T;Ueda H;Kitaguchi T;Tsuboi T
糖酵解是将葡萄糖转化为丙酮酸的代谢途径，而发酵则可以从丙酮酸产生乳酸。在这里，我们开发了基于单一荧光蛋白（FP）的乳酸和丙酮酸指示剂，具有低EC 50，用于代谢分子的痕量检测和活细胞成像，并分别将其命名为“绿色Lindoblum”和“绿色Pegassos”。绿色Lindoblum（对乳酸的EC 50为30 µM）和绿色Pegassos（对丙酮酸的EC 50为70 µM）分别对乳酸和丙酮酸产生5.2倍和3.3倍的荧光强度变化。绿色Lindoblum测量小鼠血浆中的乳酸水平，并且绿色Pegassos与D-丝氨酸脱氢酶组合通过测量丙酮酸水平成功地估计了从小鼠原代培养的神经元和星形胶质细胞释放的D-丝氨酸水平。此外，活细胞成像分析揭示了它们在双色成像中的实用性，以及乳酸、丙酮酸和人诱导多能干细胞衍生的心肌细胞中Ca 2+之间的相互作用。因此，绿色Lindoblum和绿色Pegassos将是检测临床使用中的特定分子并监测不同细胞类型中代谢物和其他相关分子的相互作用的有用工具。
Glycolysis is the metabolic pathway that converts glucose into pyruvate, whereas fermentation can then produce lactate from pyruvate. Here, we developed single fluorescent protein (FP)-based lactate and pyruvate indicators with low EC50 for trace detection of metabolic molecules and live cell imaging and named them “Green Lindoblum” and “Green Pegassos,” respectively. Green Lindoblum (EC50 of 30 µM for lactate) and Green Pegassos (EC50 of 70 µM for pyruvate) produced a 5.2- and 3.3-fold change in fluorescence intensity in response to lactate and pyruvate, respectively. Green Lindoblum measured lactate levels in mouse plasma, and Green Pegassos in combination with D-serine dehydratase successfully estimated D-serine levels released from mouse primary cultured neurons and astrocytes by measuring pyruvate level. Furthermore, live cell imaging analysis revealed their utility for dual-colour imaging, and the interplay between lactate, pyruvate, and Ca2+ in human induced pluripotent stem cell-derived cardiomyocytes. Therefore, Green Lindoblum and Green Pegassos will be useful tools that detect specific molecules in clinical use and monitor the interplay of metabolites and other related molecules in diverse cell types.
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影响因子：
4.6
作者：
Harada K;Ito M;Wang X;Tanaka M;Wongso D;Konno A;Hirai H;Hirase H;Tsuboi T;Kitaguchi T
通讯作者：
Kitaguchi T
影响因子：
4.1
作者：
Ito, Tomokazu;Hemmi, Hisashi;Yoshimura, Tohru
通讯作者：
Yoshimura, Tohru
影响因子：
5.8
作者：
Fernandez-Fuentes, Narcis;Rai, Brajesh K.;Fiser, Andras
通讯作者：
Fiser, Andras
影响因子：
3.7
作者：
San Martín A;Ceballo S;Baeza-Lehnert F;Lerchundi R;Valdebenito R;Contreras-Baeza Y;Alegría K;Barros LF
通讯作者：
Barros LF
影响因子：
4.8
作者：
Montero, M;Lobatón, CD;Alvarez, J
通讯作者：
Alvarez, J