A red fluorescent genetically encoded biosensor for extracellular L-lactate
A red fluorescent genetically encoded biosensor for extracellular L-lactate
复制标题
一种用于细胞外L-乳酸的红色荧光基因编码生物传感器
DOI：
10.1101/2022.08.30.505811
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发表时间：
2022
期刊：
影响因子：
--
通讯作者：
Campbell Robert E.
中科院分区：
文献类型：
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作者：
Nasu Yusuke;Kamijo Yuki;Hashizume Rina;Sato Haruaki;Hori Yukiko;Tomita Taisuke;Drobizhev Mikhail;Campbell Robert E.
L-乳酸，传统上被认为是代谢的废物，现在被认为是哺乳动物细胞间的关键能量货币。为了研究L-乳酸的细胞间穿梭，我们以前报道了eLACCO 1.1，一种用于细胞外L-乳酸的绿色荧光基因编码生物传感器。eLACCO1.1能够对培养的哺乳动物细胞和脑组织中的细胞外L-乳酸进行细胞分辨率成像。然而，eLACCO1.1在光谱上与常用的光学生物传感器和致动器重叠，限制了其在多路复用成像或与光遗传致动器组合使用中的应用。在这里，我们报告了红色荧光细胞外L-乳酸传感器，指定R-eLACCO 2。R-eLACCO 2是定向进化的终产物，在体外对L-乳酸具有高分子特异性的荧光反应。我们证明，R-eLACCO 2与优化的领导人和锚序列显示了很大的荧光变化，响应于细胞外L-乳酸对活的哺乳动物细胞的膜。R-eLACCO 2应该能够与其他荧光探针和光遗传学致动器结合，对细胞外L-乳酸进行荧光成像。
L-Lactate, traditionally recognized as a waste product of metabolism, is now appreciated as a key intercellular energy currency in mammals. To enable investigations of intercellular shuttling of L-lactate, we have previously reported eLACCO1.1, a green fluorescent genetically encoded biosensor for extracellular L-lactate. eLACCO1.1 enables cellular resolution imaging of extracellular L-lactate in cultured mammalian cells and brain tissue. However, eLACCO1.1 spectrally overlaps with commonly used optical biosensors and actuators, limiting its application for multiplexed imaging or combined use with optogenetic actuators. Here, we report a red fluorescent extracellular L-lactate biosensor, designated R-eLACCO2. R-eLACCO2 is the end-product of extensive directed evolution and exhibits a large fluorescence response to L-lactate with high molecular specificityin vitro. We demonstrate that R-eLACCO2 with optimized leader and anchor sequences shows a large fluorescence change in response to extracellular L-lactate on the membrane of live mammalian cells. R-eLACCO2 should enable multicolor imaging of extracellular L-lactate in combination with other fluorescent probes and optogenetic actuators.