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. IntroductionAcetylcholine (ACh) is a critical neurotransmitter in all animals. Among invertebrates, it is the most prevalent excitatory transmitter in the brain, sensory ganglia, and frequently the neuromuscular junction (NMJ). Among vertebrates, only a minority of neurons release ACh, but these signals play varying key roles. For instance, ACh signals at the NMJ, in the autonomic nervous system, and in subsets of the central nervous system, particularly projections arising from the brainstem and basal forebrain. Other cholinergic neuron populations in the brain include striatal interneurons, the stria vascularis-medial habenula-interpeduncular nucleus pathway, and sparse, incompletely characterized cell types such as intrinsic cholinergic interneurons in cortex 1 and hippocampus 2 . ACh helps to regulate attention 3 and wakefulness 4 , and participates in memory formation and consolidation 5 . ACh is also an important transmitter in glia, and between the nervous and immune systems 6 .Acetylcholine is synthesized pre-synaptically from choline and acetyl-CoA by choline acetyltransferase (ChAT), then packaged into synaptic vesicles by the vesicular acetylcholine transporter (VAChT). A key, partially understood aspect of cholinergic signaling is co-release with other neurotransmitters, including GABA, ATP, and glutamate 7,8 . To understand the role of co-release, one must measure ACh release alongside emerging measurements of other neurotransmitters.Acetylcholine receptors are among the most diverse neurotransmitter receptor families. Humans possess five muscarinic G protein-coupled receptors (GPCRs) for ACh (mAChRs) with diverse expression in the brain and smooth, cardiac, and skeletal muscle. Vertebrate nicotinic ACh receptors (nAChRs) are pentameric ligand-gated cation channels. Humans have a total of 17 nAChR subunit genes, in five classes: 10 a, 4 b, and one each of g, d, and e. nAChRs occur with many subunit combinations 9 , and others may be undiscovered. Invertebrates also have AChgated chloride channels. On neurons, receptors can be localized pre-, post-, and extrasynaptically, often with different isoforms in each place 10

Information processing by brain circuits depends on Ca 2+-dependent, stochastic release of the excitatory neurotransmitter glutamate. Recently developed optical sensors have enabled detection of evoked and spontaneous release at common glutamatergic synapses. However, monitoring synaptic release probability, its use-dependent changes, and its underpinning presynaptic machinery in situ requires concurrent, intensity-independent readout of presynaptic Ca 2+ and glutamate release. Here, we find that the red-shifted Ca 2+indicator Cal-590 shows Ca 2+-sensitive fluorescence lifetime, and employ it in combination with the novel green glutamate sensor SF-iGluSnFR variant to document quantal release of glutamate together with presynaptic Ca 2+ concentration, in multiple synapses in an identified neural circuit. At the level of individual presynaptic boutons, we use multiexposure and stochastic reconstruction procedures to reveal nanoscopic co-localisation of presynaptic Ca 2+ entry and glutamate release, a fundamental unknown in modern neurobiology. This approach opens a new horizon in the quest to understand release machinery of central synapses. Stochastic, Ca2+ -dependent release of the excitatory neurotransmitter glutamate by individual synapses is what underpins information handling and storage by neural networks. However, in many central circuits glutamate release occurs with a low probability and a high degree of heterogeneity among synapses 1, 2 . Therefore, methods to probe presynaptic function in an intact brain aim to reliably detect presynaptic action potentials, record the presynaptic Ca 2+ dynamics, and register release of individual glutamate quanta with high temporal resolution and broad dynamic range. The optical quantal analysis method went some way toward this goal, by providing quantification of release probability at individual synapses in brain slices 3,4 . In parallel, advances in the imaging techniques suited to monitor membrane retrieval at presynaptic terminals have enabled detection of synaptic vesicle exocytosis in cultured neurons 5 . Recently developed optical glutamate sensors 6,7 have drastically expanded the sensitivity and the dynamic range of glutamate discharge detection in organised brain tissue 8 . However, such methods on their own cannot relate neurotransmitter release to presynaptic Ca 2+ dynamics, which . CC-BY-NC-ND 4.0 International license It is made available under a was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint (which . http://dx.doi.org/10.1101/336891 doi: bioRxiv preprint first posted online Jun. 2, 2018; 2 is the key to understanding presynaptic release machinery, as demonstrated in elegant studies of giant synapses permitting direct electrophysiological probing 9-11 . In parallel, we have developed a glutamate sensor variant SF-iGluSnFR.A184S whose kinetic properties permit reliable registration of individual quanta of released neurotransmitter at multiple gl...

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.