BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
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2024
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IF 2.5
Xuanming Chang,Xiaoqian Chen,Xiuze Zhang,Nian Chen,Weitao Tang,Zhuo Zhang,Sulin Zheng,Jiong Huang,Yihan Ji,Yuzheng Zhao,Yi Yang,Xie Li
Lactate plays a crucial role in energy metabolism and greatly impacts protein activities, exerting diverse physiological and pathological effects. Therefore, convenient lactate assays for tracking spatiotemporal dynamics in living cells are desirable. In this paper, we engineered and optimized a red fluorescent protein sensor for l -lactate named FiLa-Red. This indicator exhibited a maximal fluorescence change of 730 % and an apparent dissociation constant ( K d ) of approximately 460 μM. By utilizing FiLa-Red and other sensors, we monitored energy metabolism in a multiplex manner by simultaneously tracking lactate and NAD + /NADH abundance in the cytoplasm, nucleus, and mitochondria. The FiLa-Red sensor is expected to be a useful tool for performing metabolic analysis in vitro , in living cells and in vivo .
研究领域:
细胞生物学
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分子生物学
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生物技术
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代谢
✓ 已验证
产品关联
DOI
PubMed
高影响因子
Cell Metabolism
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2022
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IF 31.373
Xie Li,Yinan Zhang,Lingyan Xu,Aoxue Wang,Yejun Zou,Ting Li,Li Huang,Weicai Chen,Shuning Liu,Kun Jiang,Xiuze Zhang,Dongmei Wang,Lijuan Zhang,Zhuo Zhang,Zeyi Zhang,Xianjun Chen,Wei Jia,Aihua Zhao,Xinfeng Yan,Haimeng Zhou,Linyong Zhu,Xinran Ma,Zhenyu Ju,Weiping Jia,Congrong Wang,Joseph Loscalzo,Yi Yang,Yuzheng Zhao
Summary Despite its central importance in cellular metabolism, many details remain to be determined regarding subcellular lactate metabolism and its regulation in physiology and disease, as there is sensitive spatiotemporal resolution of lactate distribution, and dynamics remains a technical challenge. Here, we develop and characterize an ultrasensitive, highly responsive, ratiometric lactate sensor, named FiLa, enabling the monitoring of subtle lactate fluctuations in living cells and animals. Utilizing FiLa, we demonstrate that lactate is highly enriched in mammalian mitochondria and compile an atlas of subcellular lactate metabolism that reveals lactate as a key hub sensing various metabolic activities. In addition, FiLa sensors also enable direct imaging of elevated lactate levels in diabetic mice and facilitate the establishment of a simple, rapid, and sensitive lactate assay for point-of-care clinical screening. Thus, FiLa sensors provide powerful, broadly applicable tools for defining the spatiotemporal landscape of lactate metabolism in health and disease.
研究领域:
细胞生物学
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代谢
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生物医学工程
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疾病模型
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产品关联
DOI
PubMed
高影响因子
Nature Protocols
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2024
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IF 13.1
Wang Aoxue,Zou Yejun,Liu Shuning,Zhang Xiuze,Li Ting,Zhang Lijuan,Wang Ruwen,Xia Yale,Li Xie,Zhang Zhuo,Liu Tiemin,Ju Zhenyu,Wang Ru,Loscalzo Joseph,Yang Yi,Zhao Yuzheng
As a key glycolytic metabolite, lactate has a central role in diverse physiological and pathological processes. However, comprehensive multiscale analysis of lactate metabolic dynamics in vitro and in vivo has remained an unsolved problem until now owing to the lack of a high-performance tool. We recently developed a series of genetically encoded fluorescent sensors for lactate, named FiLa, which illuminate lactate metabolism in cells, subcellular organelles, animals, and human serum and urine. In this protocol, we first describe the FiLa sensor-based strategies for real-time subcellular bioenergetic flux analysis by profiling the lactate metabolic response to different nutritional and pharmacological conditions, which provides a systematic-level view of cellular metabolic function at the subcellular scale for the first time. We also report detailed procedures for imaging lactate dynamics in live mice through a cell microcapsule system or recombinant adeno-associated virus and for the rapid and simple assay of lactate in human body fluids. This comprehensive multiscale metabolic analysis strategy may also be applied to other metabolite biosensors using various analytic platforms, further expanding its usability. The protocol is suited for users with expertise in biochemistry, molecular biology and cell biology. Typically, the preparation of FiLa-expressing cells or mice takes 2 days to 4 weeks, and live-cell and in vivo imaging can be performed within 1–2 hours. For the FiLa-based assay of body fluids, the whole measuring procedure generally takes ~1 min for one sample in a manual assay or ~3 min for 96 samples in an automatic microplate assay.
研究领域:
生物化学
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分子生物学
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细胞生物学
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生物医学工程
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代谢组学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
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