Laconic, a FRET lactate sensor based on the transcriptional regulator LldR.

<p>(A) Crystallographic structure of LldR from <i>Corynebacterium glutamicum </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057712#pone.0057712-Gao1" target="_blank">[19]</a>, and 3D-structure of LldR from <i>Escherichia coli</i> predicted using LldR from <i>C. glutamicum</i> and FadR from <i>E. coli</i> as templates (M4T Server 3.0 from the Fiser Laboratory <a href="http://www.fiserlab.org/servers_table.htm" target="_blank">http://www.fiserlab.org/servers_table.htm</a>). (B) General design: the transcriptional regulator LldR is sandwiched between fluorescent proteins mTFP and Venus, with artificial peptides separating the proteins (blue and orange linkers). (C) Effect of 10 mM lactate on the fluorescence ratio of 8 variants of the lactate sensor based on LldR from either <i>E. coli</i> or <i>C. glutamicum</i>. The most responsive of the constructs, indicated by the arrow, was used in the rest of the study.</p

Inhibition of the sodium-dependent HCO3- transporter SLC4A4, produces a cystic fibrosis-like airway disease phenotype

International audienceBicarbonate secretion is a fundamental process involved in maintaining acid-base homeostasis. Disruption of bicarbonate entry into airway lumen, as has been observed in cystic fibrosis, produces several defects in lung function due to thick mucus accumulation. Bicarbonate is critical for correct mucin deployment and there is increasing interest in understanding its role in airway physiology, particularly in the initiation of lung disease in children affected by cystic fibrosis, in the absence of detectable bacterial infection. The current model of anion secretion in mammalian airways consists of CFTR and TMEM16A as apical anion exit channels, with limited capacity for bicarbonate transport compared to chloride. However, both channels can couple to SLC26A4 anion exchanger to maximise bicarbonate secretion. Nevertheless, current models lack any details about the identity of the basolateral protein(s) responsible for bicarbonate uptake into airway epithelial cells. We report herein that the electrogenic, sodium-dependent, bicarbonate cotransporter, SLC4A4, is expressed in the basolateral membrane of human and mouse airways, and that it’s pharmacological inhibition or genetic silencing reduces bicarbonate secretion. In fully differentiated primary human airway cells cultures, SLC4A4 inhibition induced an acidification of the airways surface liquid and markedly reduced the capacity of cells to recover from an acid load. Studies in the Slc4a4 -null mice revealed a previously unreported lung phenotype, characterized by mucus accumulation and reduced mucociliary clearance. Collectively, our results demonstrate that the reduction of SLC4A4 function induced a CF-like phenotype, even when chloride secretion remained intact, highlighting the important role SLC4A4 plays in bicarbonate secretion and mammalian airway function

A Genetically Encoded FRET Lactate Sensor and Its Use To Detect the Warburg Effect in Single Cancer Cells

<div><p>Lactate is shuttled between and inside cells, playing metabolic and signaling roles in healthy tissues. Lactate is also a harbinger of altered metabolism and participates in the pathogenesis of inflammation, hypoxia/ischemia, neurodegeneration and cancer. Many tumor cells show high rates of lactate production in the presence of oxygen, a phenomenon known as the Warburg effect, which has diagnostic and possibly therapeutic implications. In this article we introduce Laconic, a genetically-encoded Forster Resonance Energy Transfer (FRET)-based lactate sensor designed on the bacterial transcription factor LldR. Laconic quantified lactate from 1 µM to 10 mM and was not affected by glucose, pyruvate, acetate, betahydroxybutyrate, glutamate, citrate, α-ketoglutarate, succinate, malate or oxalacetate at concentrations found in mammalian cytosol. Expressed in astrocytes, HEK cells and T98G glioma cells, the sensor allowed dynamic estimation of lactate levels in single cells. Used in combination with a blocker of the monocarboxylate transporter MCT, the sensor was capable of discriminating whether a cell is a net lactate producer or a net lactate consumer. Application of the MCT-block protocol showed that the basal rate of lactate production is 3–5 fold higher in T98G glioma cells than in normal astrocytes. In contrast, the rate of lactate accumulation in response to mitochondrial inhibition with sodium azide was 10 times lower in glioma than in astrocytes, consistent with defective tumor metabolism. A ratio between the rate of lactate production and the rate of azide-induced lactate accumulation, which can be estimated reversibly and in single cells, was identified as a highly sensitive parameter of the Warburg effect, with values of 4.1 ± 0.5 for T98G glioma cells and 0.07 ± 0.007 for astrocytes. In summary, this article describes a genetically-encoded sensor for lactate and its use to measure lactate concentration, lactate flux, and the Warburg effect in single mammalian cells.</p> </div

Imaging of cytosolic lactate.

<p>(A) HEK293 cells expressing Laconic imaged at 440 excitation/535 emission). Scale bar is 20 µm. (B) Fluorescence ratio was measured at 0.01, 0.1, 1 and 10 mM extracellular lactate in HEK293 cells treated with metabolic inhibitors and permeabilized to H⁺ as described in Material and Methods. (C) The time course of mTFP and Venus fluorescences were measured in the presence of 2 mM glucose/1 mM lactate or 10 mM pyruvate, as indicated. A cell with low expression of the sensor requiring strong illumination was chosen to demonstrate the insensitivity of the fluorescence ratio to photobleaching. The fluorescence ratio was converted into lactate concentration using the ratio in pyruvate (zero lactate) as described in the text.</p

Metabolic characterization of tumor cells.

<p>(A) An astrocyte expressing Laconic was sequentially exposed to 5 mM azide, 50 µM phloretin and 500 µM pCMBS. The straight lines represent initial slopes of lactate accumulation fitted by linear regression within the same range of ratio values. (B) A T98G glioma cell expressing Laconic was sequentially exposed to 5 mM azide, 50 µM phloretin and 500 µM pCMBS. The straight lines represent initial slopes of lactate accumulation fitted by linear regression within the same range of ratio values. (C) Summary of the initial slopes (Δ ratio/min) obtained in three experiments of the type shown in A and B. (D) Correlation plot between the rates of lactate accumulation (Δ ratio/min) in azide and in pCMBS. Symbols represent single astrocytes (white), HEK293 cells (gray) or T98G cells (black). (E) The Warburg Index was estimated as the ratio between the rates of lactate production with pCMBS and lactate accumulation with azide, and used to color the silhouette of each cell according to the 16-color look up table. The inset shows an isolated cell that was located about 100 µm from the cluster. The bar graph summarizes data from 3 experiments in each cell type. Scale bars are 20 µm. *, p < 0.05 between every cell type.</p

<i>In vitro</i> characterization of Laconic.

<p>(A) Emission spectra in the absence and presence of 10 mM lactate. (B) The ratio between mTFP and Venus fluorescence (at 430 nm excitation) was measured at 0, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 5 and 10 mM lactate. The continuous line corresponds to the best fit of a double rectangular hyperbola to the data, with apparent dissociation constant (K_D) values of 8 ± 2 µM and 830 ± 160 µM, and respective maximum ΔR values of 8 ± 0.4% and 11 ± 0.4%. (C) Lactate dose-response curves were measured at the indicated pH values. The continuous line corresponds to the best fit of a double rectangular hyperbola to the data at pH .4. (D) Response of the sensor to 5 mM of lactate, pyruvate, acetate, glutamate, β-hydroxy-butyrate and glucose, 1 mM of α-ketoglutarate, succinate, malate or oxalacetate, or increasing concentrations of citrate (0.01, 0.1 and 1 mM). In panels E to I, lactate dose-response curves were measured in the presence of 1 mM acetate, glutamate, β-hydroxy-butyrate or glucose (E), in 1 mM of α-ketoglutarate, succinate, malate or oxalacetate (F), in 0.25, 1 mM or 10 mM pyruvate (G), in 0.01, 0.1 mM or 1 mM citrate (H), and in 0.2 µM NADH, 100 µM NAD+, or 0.2 µM NADH plus 100 µM NAD+ (I). The continuous lines correspond to the best fit of a double rectangular hyperbola to control data.</p