High performance genetically-encoded green fluorescent biosensors for intracellular L-lactate

Le, Giang N. T.; Hario, Saaya; Takahashi, Kei; Li, Selene; Drobizhev, Mikhail; Nasu, Yusuke; Campbell, Robert E.

doi:10.1101/2022.10.19.512892

Cited by 5 publications

(6 citation statements)

References 46 publications

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“…Interestingly, L-lactate titration revealed that R-iLACCO1 and its affinity variants have Hill coefficients of ~1 (Supplementary Table 2), whereas a green fluorescent biosensor iLACCO1 based on the same LldR and cpGFP displays a Hill coefficient of 0.6 (ref. 10). These results suggest that the L-lactate-dependent fluorescence response of iLACCO1 is negatively cooperative, but that of R-iLACCO1 variants is not cooperative.…”

Section: Discussionmentioning

confidence: 75%

“…19), into the L-lactate binding domain (LBD) of the Escherichia coli LldR at position 109 (Supplementary Fig. 6a) 10 . Relative to .…”

Section: Resultsmentioning

confidence: 99%

“…19), into the l -Lactate binding domain (LBD) of the Escherichia coli LldR at position 109 ( Supplementary Fig. 6a ) 10 . Relative to Ca 2+ -requiring TTHA0766, LldR is a preferred binding domain for an intracellular l -Lactate biosensor because LldR has no Ca 2+ binding site and has been demonstrated to be functional in the cytosol where Ca 2+ concentration is low (∼ 0.1–1 μM) 10, 11 .…”

Section: Resultsmentioning

confidence: 99%

“…6a ) 10 . Relative to Ca 2+ -requiring TTHA0766, LldR is a preferred binding domain for an intracellular l -Lactate biosensor because LldR has no Ca 2+ binding site and has been demonstrated to be functional in the cytosol where Ca 2+ concentration is low (∼ 0.1–1 μM) 10, 11 . The resulting variant, designated R-iLACCO0.1, exhibited dim fluorescence and only a slight increase in fluorescence intensity (Δ F / F = 0.7, crude protein extract) upon treatment with l -Lactate ( Supplementary Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Investigations of the emerging roles of L-lactate in cells and tissues would be facilitated by a set of genetically encoded fluorescent biosensors that could enable high resolution spatially and temporally resolved imaging of L-lactate in the extracellular and intracellular environment. As steps towards this end, we previously developed two classes of genetically encoded green fluorescent biosensors: eLACCO1.1 for extracellular L-lactate 9 and iLACCO1 for intracellular L-lactate 10 . In addition to our LACCO series, others have developed cyan or green fluorescent genetically encoded biosensors for intracellular L-lactate [11][12][13][14][15][16][17] .…”

Section: Introductionmentioning

confidence: 99%

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Improved genetically encoded fluorescent biosensors for monitoring of intra- and extracellular L-lactate

Nasu

Aggarwal

et al. 2022

Preprint

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L-Lactate is increasingly appreciated as a key metabolite and signaling molecule in mammals. To enable investigations of both the inter- and intra-cellular dynamics of L-lactate, we develop a second-generation green fluorescent extracellular L-lactate biosensor, designated eLACCO2.1, and a red fluorescent intracellular L-lactate biosensor, designated R-iLACCO1. Compared to the first-generation eLACCO1.1 (ΔF/F = 1.5 in cultured neurons), eLACCO2.1 exhibits better membrane localization and fluorescence response (ΔF/F = 8.1 in cultured neurons) with faster response kinetics to extracellular L-lactate on the surface of live mammalian cells. R-iLACCO1 and its affinity variants exhibit large fluorescence responses to changes in L-lactate concentration in vitro (ΔF/F = 15 to 22) and in live mammalian cells (ΔF/F = 5.5 to 11). We demonstrate that these biosensors enable cellular-resolution imaging of extracellular and intracellular L-lactate in cultured mammalian cells.

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Section: Discussionmentioning

confidence: 75%

“…19), into the L-lactate binding domain (LBD) of the Escherichia coli LldR at position 109 (Supplementary Fig. 6a) 10 . Relative to .…”

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Improved genetically encoded fluorescent biosensors for monitoring of intra- and extracellular L-lactate

Nasu

Aggarwal

et al. 2022

Preprint

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Lactate biosensors for spectrally and spatially multiplexed fluorescence imaging

Nasu,

Aggarwal,

et al. 2023

Nat Commun

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Abstractl-Lactate is increasingly appreciated as a key metabolite and signaling molecule in mammals. However, investigations of the inter- and intra-cellular dynamics of l-lactate are currently hampered by the limited selection and performance of l-lactate-specific genetically encoded biosensors. Here we now report a spectrally and functionally orthogonal pair of high-performance genetically encoded biosensors: a green fluorescent extracellular l-lactate biosensor, designated eLACCO2.1, and a red fluorescent intracellular l-lactate biosensor, designated R-iLACCO1. eLACCO2.1 exhibits excellent membrane localization and robust fluorescence response. To the best of our knowledge, R-iLACCO1 and its affinity variants exhibit larger fluorescence responses than any previously reported intracellular l-lactate biosensor. We demonstrate spectrally and spatially multiplexed imaging of l-lactate dynamics by coexpression of eLACCO2.1 and R-iLACCO1 in cultured cells, and in vivo imaging of extracellular and intracellular l-lactate dynamics in mice.

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An expanded palette of ATP sensors for subcellular and multiplexed imaging

Sahan

Youngblood

Das

et al. 2023

Preprint

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Genetically encoded fluorescent biosensors that detect changes in ATP levels in live cells have enabled the discovery of novel roles for ATP in cellular processes and signaling. Many of these available ATP biosensors have a limited dynamic range, or have ATP affinities that are not suitable for sensing the physiological concentrations of ATP in mammalian cells. To address these limitations, we developed a FRET-based ATP biosensor with enhanced dynamic range and signal to noise ratio, eATeam. Using eATeam, we uncovered distinct spatiotemporal dynamics of ATP changes upon inhibition of cellular energy production. We also developed dimerization-dependent GFP and RFP-based ATP biosensors with enhanced dynamic ranges compared to the current standard in the field. Using the single-color ATP biosensors, we visualized the complex interplay between AMPK activity, ATP, lactate, and calcium by multiplexed imaging in single cells. This palette of ATP sensors expands the toolbox for interrogating subcellular ATP regulation and metabolic signaling in living cells.

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High performance genetically-encoded green fluorescent biosensors for intracellular L-lactate

Cited by 5 publications

References 46 publications

Improved genetically encoded fluorescent biosensors for monitoring of intra- and extracellular L-lactate

Improved genetically encoded fluorescent biosensors for monitoring of intra- and extracellular L-lactate

Lactate biosensors for spectrally and spatially multiplexed fluorescence imaging

An expanded palette of ATP sensors for subcellular and multiplexed imaging

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