Highly responsive single-fluorophore indicator to explore lactate dynamics in high calcium environments

Abstract

Lactate is an energy substrate and intercellular signaling molecule with multiple bodily functions. Lactate has physiological roles in neurogenesis, axon integrity, memory consolidation, immune response, exercise, adipose tissue lipolysis, etc, and is involved in inflammation, cancer and neurodegeneration. The FRET lactate indicator Laconic has been instrumental in the discovery of mechanisms involved in neurometabolic coupling, and has advanced the understanding of lactate transport, glycolysis and mitochondrial physiology. However, the low fluorescent response and the complex saturation kinetics of Laconic limit its use for high-throughput screening and quantitation. Using the bacterial periplasmic binding protein TTHA0766 from Thermus thermophilus, we have now developed the first single-fluorophore indicator for lactate. The sensor exhibited an intensiometric fluorescence increase of ΔF/F0 3.0 and a single binding site with a KD of 293 micromolar. The fluorescence is not affected by other monocarboxylates or pH. However, it is sensitive to Ca2+ in the nanomolar range. Targeting of the sensor to the endoplasmic reticulum revealed that this organelle presents a high permeability for lactate. The functionality of the sensor in living tissue is demonstrated in the brain of Drosophila melanogaster larvae. This indicator, which we have termed CanlonicSF, is well suited to explore lactate dynamics in environments with micromolar Ca2+ or higher, such as the endoplasmic reticulum and the extracellular space.

Monitoring glycolytic dynamics in single cells using a fluorescent biosensor for fructose 1,6-bisphosphate

“…Moreover, the ratiometric signal and high dynamic range of HYlight combined with the large changes in FBP that track with alterations of flux ( 30 ) make this sensor an especially useful tool for monitoring metabolism in live cells. HYlight thus complements existing fluorescent biosensors covering the major glycolytic pathway inputs [glucose ( 31 )], outputs [pyruvate ( 32 ) and lactate ( 33 , 34 )], and cofactors [NAD + :NADH ( 35 ) and ATP:ADP ( 8 )].…”

Synthesis and application of a photocaged L-lactate

“…We and others have recently reported a variety of genetically encoded biosensors for the measurement and imaging of lactate in tissues. [11][12][13][14][15][16][17][18][19][20][21][22][23][24] Applications of these measurement tools would be complemented by a tool for perturbing lactate concentrations. A molecule that enables the photorelease of lactate (that is, a 'caged' lactate) could be a particularly powerful tool for the spatiotemporal manipulation of lactate concentration in tissues.…”

“…13 C-NMR (101 MHz, CDCl3) δ 172.8, 158.8, 137.7, 135.5, 125.4, 123.1, 106.3, 77.3, 77.0, 76.7, 71.4, 55.9, 50.0, 27.0, 25.7, 21.2, 18.1, -4.9, -5.2 HRMS (ESI) m/z calcd for C18H28N2O5Si+Na + : 403.1659 [M+Na] + ; found: 403.1659.…”

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

“…Relative to FRET-based indicators, single FP-based indicators can typically be engineered to have much larger intensiometric fluorescent responses and are suitable for multiplexed imaging applications. In efforts aimed at realizing these advantages, a number of single FP-based L-lactate biosensors have been engineered in recent years [22][23][24][25][26][27] . These single FP-based biosensors share a general design in which the L-lactate-binding protein is genetically linked to an FP such that the binding protein is located close to the chromophore 28 .…”

“…. Other recently reported GFP-based L-lactate biosensors have ∆F/F values of <1, 3.0, 4.2, and 6.0 (refs [22][23][24]26)…”