Lactate biosensors for spectrally and spatially multiplexed fluorescence imaging

Abstract

L-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.

Fig. 1. Development of eLACCO2.1 and R-iLACCO1.

a Schematic representation of TTHA0766-based eLACCO and its mechanism of response to l-lactate. b Schematic representation of LldR-based R-iLACCO and its mechanism of response to l-lactate. c Schematic of directed evolution workflow. Specific sites (i.e., the linkers) or the entire gene of template l-lactate biosensor were randomly mutated and the resulting mutant library was used to transform E. coli. Bright colonies were picked and cultured, and then proteins were extracted to examine ΔF/F upon addition of 10 mM l-lactate. A mixture of the variants with the highest ΔF/F was used as the template for the next round. d ΔF/F rank plot representing all crude proteins tested during the directed evolution of eLACCO. For each round, tested variants are ranked from lowest to highest ΔF/F value from left to right. e Excitation and emission spectra of purified eLACCO2.1 in the presence (10 mM) and absence of l-lactate. f Overall representation of the eLACCO1 crystal structure (PDB ID: 7E9Y [https://www.rcsb.org/structure/7E9Y]) with the position of mutations indicated. l-Lactate and Ca²⁺ (black) are shown in a sphere representation. In the primary structure of eLACCO2.1 (bottom), linker regions are shown in black and the two “gate post” residues in cpGFP are highlighted in dark orange (His195) and purple (Phe437). g ΔF/F rank plot representing all crude proteins tested during the directed evolution of R-iLACCO. h Excitation and emission spectra of purified R-iLACCO1 in the presence (10 mM) and absence of l-lactate. i Overall representation of the R-iLACCO1 model structure with the position of mutations indicated. In the primary structure of R-iLACCO1 (bottom), linker regions are shown in black and the two “gate post” residues in cpmApple are highlighted in dark orange (Val112) and purple (Trp351). Source data are provided as a Source Data file.

Fig. 2. Optimization of leader/anchor combination for cell-surface expression.

a Overview of biosensor optimization. b Localization of eLACCO1.1 with CD59 leader sequence and different anchors in rat primary neurons. Scale bars, 200 μm. Right panels show magnified images in the presence of l-lactate. Scale bars, 50 μm. c Localization index and ΔF/F of eLACCO1.1 with CD59 leader sequence and different anchors in rat primary neurons. Mean ± s.e.m., n = 3 field of views (FOVs) over three wells per construct. d Localization of eLACCO1.1 with different leader sequences and NGR GPI anchor in rat primary neurons in the presence of l-lactate. Scale bars, 200 μm. e Localization index and neurite brightness of eLACCO1.1 with different leader sequences and NGR GPI anchor in rat primary neurons. Mean ± s.e.m., n = 9 FOVs over three wells per construct. Source data are provided as a Source Data file.

Fig. 3. Characterization of eLACCO2.1 in live mammalian cells and acute brain slices.

a Localization of eLACCO2.1 and control biosensor deLACCO1 with the optimized leader sequence and anchor, and eLACCO1.1 with CD59-derived leader and anchor domain in HEK293T cells. mRFP1 (ref. ) with the Igκ leader sequence and PDGFR transmembrane domain was used as a cell surface marker. Scale bars, 10 μm. b Expression on cell surface of HeLa cells before and after 10 mM l-lactate stimulation. Scale bars, 10 μm. c ΔF/F on HeLa cells. d In situ l-lactate titration. Mean ± s.e.m. e In situ Ca²⁺ titration in the presence (10 mM) of l-lactate. Mean ± s.e.m. f Time course of the fluorescence response. Mean ± s.d. Two-tailed Student’s t test. *P = 0.0043, **P < 0.0001. g Photobleaching curves (left) and τ_bleach (right). Mean ± s.e.m. One-way ANOVA followed by Tukey’s multiple comparison. *P < 0.0001. h Expression of eLACCO variants expressed on the surface of rat primary neurons and astrocytes. Scale bars, 10 μm. i l-Lactate (10 mM) response of eLACCO variants expressed on cell surface of rat primary neurons and astrocytes. Scale bars, 10 μm. j ΔF/F of eLACCO biosensors under hSyn promoter on rat primary neurons. n = 3 FOVs over three wells per construct. k ΔF/F of eLACCO biosensors under gfaABC1D promoter expressed on rat cortical astrocytes. l Schematic illustration of AAV injection into the somatosensory cortex. Image shows expression of hSyn-HA-eLACCO2.1-NGR. m Two-photon imaging of extracellular l-lactate around neurons expressing in hSyn-HA-eLACCO2.1-NGR, in response to a zero-glucose challenge in acute brain slices. Pseudo coloured images, summary trace (mean ± s.e.m.), and summary data are shown. n = 5 slices, **P = 0.0093, two-tailed unpaired Student’s t test compared with vehicle control experiment. Scale bar, 50 μm. n Two-photon imaging of extracellular l-lactate around neurons in response to electrical afferent stimulation (theta burst) in acute brain slices. Pseudo coloured images, a representative trace, and summary data are shown. n = 10 slices, *P = 0.0191, two-tailed paired Student’s t test comparing peak to baseline. Scale bar, 100 μm. Source data are provided as a Source Data file.

Fig. 4. Characterization of R-iLACCO variants in live mammalian cells.

a Fluorescence images in HeLa cells. Scale bars, 10 μm. b ΔF/F in HeLa cells in (a). Parentheses represent number of cells investigated. c In situ l-lactate titration. n = 10, 18, and 18 cells for R-iLACCO1, R-iLACCO1.1, and R-iLACCO1.2, respectively (mean ± s.e.m.). d Time course of the fluorescence response in HeLa cells. n = 19, 21, and 21 cells for R-iLACCO1, R-iLACCO1.1, and R-iLACCO1.2, respectively (mean ± s.d.). One-way ANOVA followed by Tukey’s multiple comparison. *P < 0.0001. e Fluorescence traces (left) and ΔF/F (right) in response to blue-light illumination of HeLa cells. n = 17, 14, 11, and 14 cells for R-iLACCO1, R-iLACCO1.1, R-iLACCO1.2, and R-diLACCO1, respectively. Fluorescence traces represent mean ± s.e.m. f Photobleaching curves (left, mean ± s.e.m.) and integrated fluorescence (right) in HeLa cells. Inset shows the photobleaching curves in the first 10 s. n = 18, 20, 18, 19, and 17 cells for mApple, R-iLACCO1, R-iLACCO1.1, R-iLACCO1.2, and R-diLACCO1, respectively. One-way ANOVA followed by Tukey’s multiple comparison. *P < 0.0001. g Representative images (top) and fluorescence traces (middle) in HEK293T upon 5 mM d-glucose treatment after starvation. Box plots (bottom) show ΔF/F or ΔR/R for R-iLACCO variants and other biosensors. Parentheses represent number of cells investigated. Scale bars, 10 μm. h Representative images (top) and fluorescence traces (middle) in HeLa cells upon 1 μM AR-C155858 treatment. Box plots (bottom) show ΔF/F or ΔR/R for R-iLACCO variants and other biosensors. Parentheses represent number of cells investigated. Scale bars, 10 μm. i Representative images (top) and fluorescence traces (middle) in primary neurons upon 1 μM AR-C155858 treatment. Box plots (bottom) show ΔF/F or ΔR/R for R-iLACCO variants and other biosensors. Parentheses represent number of neurons investigated. Scale bars, 20 μm. j Representative images (top) and fluorescence traces (middle) in primary astrocytes upon 1 μM AR-C155858 treatment. Box plots (bottom) show ΔF/F or ΔR/R for R-iLACCO variants and other biosensors. Parentheses represent number of astrocytes investigated. Scale bars, 20 μm. Source data are provided as a Source Data file.

Fig. 5. In vivo l-lactate imaging in mice.

a Schematic illustration of in vivo mouse imaging upon i.c.v. l-lactate injection. b Fluorescence response traces (left) of the visual cortex in CaMKII-HA-eLACCO2.1-NGR expressing mice upon i.c.v. injection of l-lactate. Right panel shows the area under the curve (AUC) of the traces for each stimulation condition. Saline (n = 8 traces from three mice), 300 nmol l-lactate (n = 7 traces from three mice), and 1 μmol l-lactate (n = 10 traces from three mice). Mean ± s.e.m. One-way ANOVA with the Dunnett’s post hoc tests. *P = 0.0066. c Fluorescence response traces (left) upon i.c.v. injection of 1 μmol l-lactate. Right panel shows the AUC of the traces for each construct. eLACCO2.1 (n = 10 traces from three mice) and deLACCO1 (n = 7 traces from three mice). Mean ± s.e.m. Two-tailed Student’s t test. *P = 0.0493. d Timeline of animal surgery and recording. The right panels show representative images of the coordinate of integrated GRIN lens implantation (upper) and eLACCO2.1 in the field of view (bottom). Scar bars, 50 μm (upper) and 100 μm (bottom). e Fluorescence responses in freely moving mice in response to the i.p. injection of saline or insulin. Each recording was normalized to 10 min baseline recording. n = 5 and four mice for eLACCO2.1 and deLACCO1, respectively. Mean ± s.e.m. Two-tailed paired Student’s t test. *P = 0.02533. f Schematic illustration of in vivo one-photon imaging of the mouse stimulated by air pff. g Histological verification of R-iLACCO1.1 expression in the somatosensory cortex. Bottom image shows a magnified image in the box of the upper image. Similar results were obtained for more than five independent experiments. Scale bars, 1 mm (upper) and 200 μm (bottom). h Traces of air puff-evoked intracellular l-lactate response (mean ± s.e.m.). R-iLACCO1.1 (n = 8 traces from 6 mice of which one mouse was subjected to shorter post stimulation of 120 s) and R-diLACCO1 (n = 8 traces from 6 mice). i AUC of the traces for R-iLACCO1.1 (n = 6 mice) and R-diLACCO1 (n = 6 mice). Mean ± s.e.m. Two-tailed Student’s t test. *P < 0.0001. Source data are provided as a Source Data file.

Fig. 6. Spectrally and spatially multiplexed l-lactate imaging.

a Schematic illustration of l-lactate imaging in extracellular space and cytosol. b Representative images of cell surface-targeted eLACCO2.1 and cytosolic R-iLACCO1.2 expressed in T98 cells before and after 5 mM glucose treatment. Similar results were observed in more than ten cells. Scale bar, 10 μm. c Fluorescence traces for imaging of extracellular and cytosolic l-lactate in T98 cells upon glucose treatment. Mean ± s.e.m. d Schematic illustration of l-lactate imaging in mitochondrial matrix and cytosol. COXIV N-terminal yeast cytochrome c oxidase subunit IV (MLSLRQSIRFFKRSGI). e Representative images of cytosolic iLACCO1 and mitochondrial matrix-targeted R-iLACCO1.2 expressed in T98 cells before and after 5 mM glucose treatment. Similar results were observed in more than ten cells. White arrowhead indicates a mitochondrial fusion. Scale bars, 10 μm and 2 μm (magnified image). f Fluorescence traces for imaging of l-lactate in cytosol and mitochondrial matrix in T98 cells upon glucose treatment. Mean ± s.e.m. g Schematic illustration of l-lactate imaging in endoplasmic reticulum (ER) and cytosol. Calr N-terminal human calreticulin (MLLPVLLLGLLGAAAD). h Representative images of cytosolic iLACCO1 and ER-targeted R-iLACCO1.2 expressed in T98 cells before and after 5 mM glucose treatment. Similar results were observed in more than ten cells. White arrowhead indicates an ER fusion. Scale bars, 10 μm and 3 μm (magnified image). i Fluorescence traces for imaging of l-lactate in cytosol and ER in T98 cells upon glucose treatment. Mean ± s.e.m. Source data are provided as a Source Data file.