Glucose is arguably the most important molecule in metabolism, and its dysregulation underlies diabetes. We describe a family of single-wavelength genetically encoded glucose sensors with a high signal-to-noise ratio, fast kinetics, and affinities varying over four orders of magnitude (1 μM to 10 mM). The sensors allow mechanistic characterization of glucose transporters expressed in cultured cells with high spatial and temporal resolution. Imaging of neuron/glia co-cultures revealed ∼3-fold faster glucose changes in astrocytes. In larval Drosophila central nervous system explants, intracellular neuronal glucose fluxes suggested a rostro-caudal transport pathway in the ventral nerve cord neuropil. In zebrafish, expected glucose-related physiological sequelae of insulin and epinephrine treatments were directly visualized. Additionally, spontaneous muscle twitches induced glucose uptake in muscle, and sensory and pharmacological perturbations produced large changes in the brain. These sensors will enable rapid, high-resolution imaging of glucose influx, efflux, and metabolism in behaving animals.

Fold-switching proteins challenge the one-sequence-one-structure paradigm by adopting multiple stable folds. Nevertheless, it is uncertain whether fold switchers are naturally pervasive or rare exceptions to the well-established rule. To address this question, we developed a predictive method and applied it to the NusG superfamily of >15,000 transcription factors. We predicted that a substantial population (25%) of the proteins in this family switch folds. Circular dichroism and nuclear magnetic resonance spectroscopies of 10 sequence-diverse variants confirmed our predictions. Thus, we leveraged family-wide predictions to determine both conserved contacts and taxonomic distributions of fold-switching proteins. Our results indicate that fold switching is pervasive in the NusG superfamily and that the single-fold paradigm significantly biases structure-prediction strategies.

Hippocampal place cells contribute to mammalian spatial navigation and memory formation. Numerous models have been proposed to explain the location-specific firing of this cognitive representation, but the pattern of excitatory synaptic input leading to place firing is unknown, leaving no synaptic-scale explanation of place coding. Here we used resonant scanning two-photon microscopy to establish the pattern of synaptic glutamate input received by CA1 place cells in behaving mice. During traversals of the somatic place field, we found increased excitatory dendritic input, mainly arising from inputs with spatial tuning overlapping the somatic field, and functional clustering of this input along the dendrites over ~10 µm. These results implicate increases in total excitatory input and co-activation of anatomically clustered synaptic input in place firing. Since they largely inherit their fields from upstream synaptic partners with similar fields, many CA1 place cells appear to be part of multi-brain-region cell assemblies forming representations of specific locations.

Genetically encoded Ca indicators (GECIs) enable long-term monitoring of cellular and subcellular dynamics of this second messenger in response to environmental and developmental cues without relying on exogenous dyes. Continued development and optimization in GECIs, combined with advances in gene manipulation, offer new opportunities for investigating the mechanism of Ca signaling in fungi, ranging from documenting Ca signatures under diverse conditions and genetic backgrounds to evaluating how changes in Ca signature impact calcium-binding proteins and subsequent cellular changes. Here, we attempted to express multi-color (green, yellow, blue, cyan, and red) circularly permuted fluorescent protein (FP)-based Ca indicators driven by multiple fungal promoters in Fusarium oxysporum, F. graminearum, and Neurospora crassa. Several variants were successfully expressed, with GCaMP5G driven by the Magnaporthe oryzae ribosomal protein 27 (P) and F. verticillioides elongation factor-1α (P) gene promoters being optimal for F. graminearum and F. oxysporum, respectively. Transformants expressing GCaMP5G were compared with those expressing YC3.60, a ratiometric Cameleon Ca indicator. Wild-type and three Ca signaling mutants of F. graminearum expressing GCaMP5G exhibited improved signal-to-noise and increased temporal and spatial resolution and are also more amenable to studies involving multiple FPs compared to strains expressing YC3.60.

Focal epilepsy is associated with intermittent brief population discharges (interictal spikes), which resemble sentinel spikes that often occur at the onset of seizures. Why interictal spikes self-terminate whilst seizures persist and propagate is incompletely understood. Here we use fluorescent glutamate and GABA sensors in an awake rodent model of neocortical seizures to resolve the spatiotemporal evolution of both neurotransmitters in the extracellular space. Interictal spikes are accompanied by brief glutamate transients which are maximal at the initiation site and rapidly propagate centrifugally. GABA transients last longer than glutamate transients and are maximal ~1.5 mm from the focus where they propagate centripetally. At the transition to seizures, GABA transients are attenuated, whilst glutamate transients increase in spatial extent. The data imply that an annulus of feed-forward GABA release intermittently collapses, allowing seizures to escape from local inhibitory restraint.

Light-inducible dimerization protein modules enable precise temporal and spatial control of biological processes in non-invasive fashion. Among them, Magnets are small modules engineered from the photoreceptor Vivid by orthogonalizing the homodimerization interface into complementary heterodimers. Both Magnets components, which are well-tolerated as protein fusion partners, are photoreceptors requiring simultaneous photoactivation to interact, enabling high spatiotemporal confinement of dimerization with a single-excitation wavelength. However, Magnets require concatemerization for efficient responses and cell preincubation at 28C to be functional. Here we overcome these limitations by engineering an optimized Magnets pair requiring neither concatemerization nor low temperature preincubation. We validated these 'enhanced' Magnets (eMags) by using them to rapidly and reversibly recruit proteins to subcellular organelles, to induce organelle contacts, and to reconstitute OSBP-VAP ER-Golgi tethering implicated in phosphatidylinositol-4-phosphate transport and metabolism. eMags represent a very effective tool to optogenetically manipulate physiological processes over whole cells or in small subcellular volumes.

How neuromodulatory transmitters diffuse into the extracellular space remains an unsolved fundamental biological question, despite wide acceptance of the volume transmission model. Here, we report development of a method combining genetically encoded fluorescent sensors with high-resolution imaging and analysis algorithms which permits the first direct visualization of neuromodulatory transmitter diffusion at various neuronal and non-neuronal cells. Our analysis reveals that acetylcholine and monoamines diffuse at individual release sites with a spread length constant of ∼0.75 μm. These transmitters employ varied numbers of release sites, and when spatially close-packed release sites coactivate they can spillover into larger subcellular areas. Our data indicate spatially restricted (i.e., nonvolume) neuromodulatory transmission to be a prominent intercellular communication mode, reshaping current thinking of control and precision of neuromodulation crucial for understanding behaviors and diseases.

Femtosecond lasers at fixed wavelengths above 1,000 nm are powerful, stable and inexpensive, making them promising sources for two-photon microscopy. Biosensors optimized for these wavelengths are needed for both next-generation microscopes and affordable turn-key systems. Here we report jYCaMP1, a yellow variant of the calcium indicator jGCaMP7 that outperforms its parent in mice and flies at excitation wavelengths above 1,000 nm and enables improved two-color calcium imaging with red fluorescent protein-based indicators.

In many reptiles, including the red-eared slider turtle (), sex is determined by ambient temperature during embryogenesis. We previously showed that the epigenetic regulator is elevated at the male-producing temperature and essential to activate the male pathway. In this work, we established a causal link between temperature and transcriptional regulation of We show that signal transducer and activator of transcription 3 (STAT3) is phosphorylated at the warmer, female-producing temperature, binds the locus, and represses transcription, blocking the male pathway. Influx of Ca, a mediator of STAT3 phosphorylation, is elevated at the female temperature and acts as a temperature-sensitive regulator of STAT3 activation.

Here we design and optimize a genetically encoded fluorescent indicator, iAChSnFR, for the ubiquitous neurotransmitter acetylcholine, based on a bacterial periplasmic binding protein. iAChSnFR shows large fluorescence changes, rapid rise and decay kinetics, and insensitivity to most cholinergic drugs. iAChSnFR revealed large transients in a variety of slice and in vivo preparations in mouse, fish, fly and worm. iAChSnFR will be useful for the study of acetylcholine in all animals.