Single-Fluorophore Indicator to Explore Cellular and Sub-cellular Lactate Dynamics.

Lactate is an energy substrate and an intercellular signal, which can be monitored in intact cells with the genetically encoded FRET indicator Laconic. However, the structural complexity, need for sophisticated equipment, and relatively small fluorescent change limit the use of FRET indicators for subcellular targeting and development of high-throughput screening methodologies. Using the bacterial periplasmic binding protein TTHA0766 from Thermus thermophilus, we have now developed a single-fluorophore indicator for lactate, CanlonicSF. This indicator exhibits a maximal fluorescence change of 200% and a KD of ∼300 µM. The fluorescence is not affected by other monocarboxylates. The lactate indicator was not significantly affected by Ca2+ at the physiological concentrations prevailing in the cytosol, endoplasmic reticulum, and extracellular space, but was affected by Ca2+ in the low micromolar range. Targeting the indicator to the endoplasmic reticulum revealed for the first time sub-cellular lactate dynamics. Its improved lactate-induced fluorescence response permitted the development of a multiwell plate assay to screen for inhibitors of the monocarboxylate transporters MCTs, a pharmaceutical target for cancer and inflammation. The functionality of the indicator in living tissue was demonstrated in the brain of Drosophila melanogaster larvae. CanlonicSF is well suited to explore lactate dynamics with sub-cellular resolution in intact systems.

The long noncoding RNA mimi scaffolds neuronal granules to maintain nervous system maturity.

RNA binding proteins and messenger RNAs (mRNAs) assemble into ribonucleoprotein granules that regulate mRNA trafficking, local translation, and turnover. The dysregulation of RNA-protein condensation disturbs synaptic plasticity and neuron survival and has been widely associated with human neurological disease. Neuronal granules are thought to condense around particular proteins that dictate the identity and composition of each granule type. Here, we show in Drosophila that a previously uncharacterized long noncoding RNA, mimi, is required to scaffold large neuronal granules in the adult nervous system. Neuronal ELAV-like proteins directly bind mimi and mediate granule assembly, while Staufen maintains condensate integrity. mimi granules contain mRNAs and proteins involved in synaptic processes; granule loss in mimi mutant flies impairs nervous system maturity and neuropeptide-mediated signaling and causes phenotypes of neurodegeneration. Our work reports an architectural RNA for a neuronal granule and provides a handle to interrogate functions of a condensate independently of those of its constituent proteins.