GEM-IL: A highly responsive fluorescent lactate indicator

Abstract

Lactate metabolism has been shown to have increasingly important implications in cellular functions as well as in the development and pathophysiology of disease. The various roles as a signaling molecule and metabolite have led to interest in establishing a new method to detect lactate changes in live cells. Here we report our development of a genetically encoded metabolic indicator specifically for probing lactate (GEM-IL) based on superfolder fluorescent proteins and mutagenesis. With improvements in its design, specificity, and sensitivity, GEM-IL allows new applications compared with the previous lactate indicators, Laconic and Green Lindoblum. We demonstrate the functionality of GEM-IL to detect differences in lactate changes in human oncogenic neural progenitor cells and mouse primary ventricular myocytes. The development and application of GEM-IL show promise for enhancing our understanding of lactate dynamics and roles.

Keywords: cancer; cardiac metabolism; cellular metabolism; genetically encoded fluorescent indicators; lactate; metabolite indicator.

Graphical abstract

Figure 1

Development of a genetically encoded metabolic indicator for lactate (GEM-IL) (A) Overview of the lactate indicator GEM-IL binding upon introduction of lactate. (B) Relative fluorescence change of the cyan, green, and yellow fluorescent protein-based GEM-IL constructs after 10 mM lactate addition in NIH3T3 cells with the superfolder CFP (sfCFP), full-length cyan fluorescent protein (CFP), Laconic, and Green Lindoblum signal changes as points of comparison. Error bars represent standard deviations (n = 23, 16, 12, 18, 29, 26, and 30 samples, respectively). One-way ANOVA with multiple comparison test (Laconic versus cyan, ∗p = 0.0368; Laconic versus Green Lindoblum, ∗∗∗p = 2.76 × 10⁻¹¹; Green Lindoblum versus cyan, ∗∗∗p = 0.0008; cyan versus sfCFP, ∗∗∗p = 1.58 × 10⁻¹³; cyan versus CFP, ∗∗∗p = 2.25 × 10⁻¹⁴). (C) Representative fluorescence change traces of cyan fluorescent protein-based GEM-IL (C-GEM-IL) in NIH3T3 cells. (D) Construct maps of GEM-IL prototype, C-GEM-IL 1.0, and C-GEM-IL 1.1. (E) Fluorescence change of G-GEM-IL with various linker types after addition of 10 mM lactate (i.e., [Lactate]i = ∼545 μM according to [B]) to the imaging solution. Error bars represent standard deviations (n = 12, 8, 9, 10, 7, 9, 6, and 15, respectively). One-way ANOVA with multiple comparison tests (∗p < 0.05, ∗∗p < 0.01) was used. (F) Cyan fluorescence and bright-field images of C-GEM-IL full-length and C-GEM-IL ΔDBD in NIH3T3 cells. Arrowheads indicate the puncta seen when overexpressing C-GEM-IL. Scale bar, 10 μm.

Figure 2

Fluorophore point mutation improves response to lactate (A) Amino acid sequence alignment showing the inclusion of the I146N mutation (red outline) in C-GEM-IL (gray, a match; yellow, a mismatch). (B) C-GEM-IL 2.0 construct map. (C) Fluorescence intensity changes of the wild-type (WT) and I146N mutant in NIH3T3 cells. Error bars represent standard deviations (n = 31 and 43 traces, using unpaired t test, ∗∗p < 0.01). (D) Emission spectra of recombinant C-GEM-IL 2.0 proteins purified from bacterial expression system. (E) Lactate binding curve of the purified recombinant C-GEM-IL 2.0 protein. The non-linear curve fit for total binding used to calculate dissociation constant is shown (red). (F) Substrate binding tests of the recombinant C-GEM-IL 2.0 protein with each substrate at 10 mM. Error bars represent standard deviations (pH 7.0, n = 3, using one-way ANOVA with multiple comparisons tests to control column [blank], ∗p < 0.05; n.s., no other significant differences among the groups).

Figure 3

Mutation in lactate binding domain improves response and specificity to lactate (A) Model of the LldR structure with the predicted lactate binding site (purple) and E103 (red). (B) Effects of targeted amino acid substitutions E103D and H151M in the lactate binding domain on lactate-induced fluorescence of C-GEM-IL 2.0 in NIH3T3 cells. Error bars represent standard deviations (n = 63 and 37). One-way ANOVA using Dunnett’s multiple comparison tests was used with wild -type (WT; ∗∗∗p < 0.001). (C) C-GEM-IL 3.0 construct map. (D) Overview of C-GEM-IL binding upon introduction of lactate and the effect of AR-C155858 (MCT1 inhibitor). (E) Fluorescence intensity changes of C-GEM-IL 3.0 and its response to 10 mM lactate addition with (black) and without treatment with AR-C155858 (100 nM, blue). Student's t test was used (∗∗∗p < 0.001). (F) Schematic representation of C-GEM-IL lactate sensing upon introduction of glucose after NIH3T3 cells are glucose -starved. (G) Average fluorescence intensity changes of C-GEM-IL 3.0 and its response to glucose (10 mM) in the absence and presence of 100 nM AR-C155858 (red and blue, respectively) and a non-metabolizable glucose analog, 2DG (10 mM; green). Before glucose or 2DG was added, the cells were kept in glucose-free Tyrode's solution. Error bars represent standard deviations. One-way ANOVA with multiple comparisons test (∗∗∗p < 0.001). (H) Image of SDS-PAGE gel of purified recombinant C-GEM-IL 3.0 proteins (arrow). (I) Emission spectrum of the purified recombinant proteins of C-GEM-IL 3.0. (J) Lactate binding curve of the purified recombinant C-GEM-IL 3.0 proteins. The non-linear curve fit for total binding used to calculate the dissociation constant is shown (red). (K) Substrate binding test of the recombinant C-GEM-IL 3.0 with each substrate at 10 mM. Error bars represent standard deviations (n = 3, using one-way ANOVA with multiple comparisons test, ∗∗∗p < 0.001; n.s., no other significant differences among the groups).

Figure 4

Comparison of C-GEM-IL, Laconic, and Green Lindoblum (A) Representative cyan/green fluorescence and bright-field images of NIH3T3 cells expressing Laconic, Green Lindoblum, and C-GEM-IL 3.0 (scale bar, 5 μm). White arrowheads shown highlighting puntae formation (B) Fluorescence change of Laconic (mTFP/Venus), Green Lindoblum, and C-GEM-IL 2.0 and 3.0 (cyan) in response to 10 mM glucose perfusions to glucose-starved NIH3T3 cells. The experimental design is identical to the one conducted in Figure 3F (n = 32, 30, 17, and 45 traces, respectively, using one-way ANOVA with multiple comparisons test, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.001). (C) Anti-GFP Western blot results interrogating the expression of the various iterations of Laconic and C-GEM-IL expressed in NIH3T3 cells. Arrowheads show Laconic (black) and C-GEM-IL proteins (red). ∗Truncated Laconic proteins. β-tubulin, a housekeeping molecule, was also tested as a reference. (D) Fluorescence change of various iterations of Laconic and Green Lindoblum in response to the exposure of NIH3T3 cells to 10 mM lactate. Red dashed line represents the average fluorescence change observed with C-GEM-IL 3.0. Error bars represent standard deviations (n = 22, 51, 33, 22, and 26).

Figure 5

Applications of C-GEM indicators (A) Quantification of the average fluorescence changes of C-GEM-IL 3.0, Laconic, and Green Lindoblum in NIH3T3 cells upon exposure to 1 mM octyl-R-2HG. Error bars represent standard deviations (n = 27, 14, 17, and 17 samples in three independent experiments, one-way ANOVA with multiple comparisons, ∗∗∗∗p < 0.0001). (B) Cumulative trace overlay of c-Myc-infected and non-infected human neural progenitors upon addition of 10 mM lactate to imaging solution. Error bars represent standard deviations (n = 46 and 31 traces in two independent experiments). (C) C-GEM-IL 3.0 fluorescence changes in c-Myc-infected human neuronal progenitors. Error bars represent standard deviations (n = 46 and 31 traces in two independent experiments, using unpaired two-tailed t test with Welch's correction, ∗∗∗p < 0.001). (D) Cumulative trace overlay of c-Myc-infected and non-infected NIH3T3 cells upon addition of 10 mM lactate. Error bars represent standard deviations. (E) Fluorescence changes in c-Myc-infected NIH3T3 cells and controls. Error bars represent standard deviations (n = 31 and 15 samples, using unpaired two-tailed Student's t test with Welch's correction, ∗∗∗p < 0.001). (F) Simplified diagram showing the insertion of the C-GEM-IL 2.0 or 3.0 indicator into mouse Rosa26 locus and the effect of crossing with a Cre line. (G) Cumulative traces showing the extracellular lactate-induced fluorescence increase in adult ventricular myocytes isolated from C-GEM-IL 2.0 and 3.0 mice crossed with cardiac Cre drivers. Error bars represent standard deviations. (H) Quantification of the fluorescence changes upon introduction of 10 mM lactate in the imaging solution to the cardiomyocytes isolated from C-GEM-IL 2.0- and 3.0-expressing mice. Error bars represent standard deviations (n = 36 and 34 traces, respectively, using unpaired two-tailed t test with Welch's correction, ∗∗∗p < 0.001).