CRISPR-free, programmable RNA pseudouridylation to suppress premature termination codons.

Nonsense mutations, accounting for >20% of disease-associated mutations, lead to premature translation termination. Replacing uridine with pseudouridine in stop codons suppresses translation termination, which could be harnessed to mediate readthrough of premature termination codons (PTCs). Here, we present RESTART, a programmable RNA base editor, to revert PTC-induced translation termination in mammalian cells. RESTART utilizes an engineered guide snoRNA (gsnoRNA) and the endogenous H/ACA box snoRNP machinery to achieve precise pseudouridylation. We also identified and optimized gsnoRNA scaffolds to increase the editing efficiency. Unexpectedly, we found that a minor isoform of pseudouridine synthase DKC1, lacking a C-terminal nuclear localization signal, greatly improved the PTC-readthrough efficiency. Although RESTART induced restricted off-target pseudouridylation, they did not change the coding information nor the expression level of off-targets. Finally, RESTART enables robust pseudouridylation in primary cells and achieves functional PTC readthrough in disease-relevant contexts. Collectively, RESTART is a promising RNA-editing tool for research and therapeutics.

Orange/far-red hybrid voltage indicators with reduced phototoxicity enable reliable long-term imaging in neurons and cardiomyocytes.

Hybrid voltage indicators (HVIs) are chemogenetic sensors that combines the superior photophysical properties of organic dyes and the genetic targetability of protein sensors to report transient membrane voltage changes. They exhibit boosted sensitivity in excitable cells such as neurons and cardiomyocytes. However, the voltage signals recorded during long-term imaging are severely diminished or distorted due to phototoxicity and photobleaching issues. To capture stable electrophysiological activities over a long time, we employ cyanine dyes conjugated with a cyclooctatetraene (COT) molecule as the fluorescence reporter of HVI. The resulting orange-emitting HVI-COT-Cy3 enables high-fidelity voltage imaging for up to 30 min in cultured primary neurons with a sensitivity of ~ -30% ΔF/F0 per action potential (AP). It also maximally preserves the signal of individual APs in cardiomyocytes. The far-red-emitting HVI-COT-Cy5 allows two-color voltage/calcium imaging with GCaMP6s in neurons and cardiomyocytes for 15 min. We leverage the HVI-COT series with reduced phototoxicity and photobleaching to evaluate the impact of drug candidates on the electrophysiology of excitable cells.

Hybrid Indicators for Fast and Sensitive Voltage Imaging.

Membrane voltage is an important biophysical signal that underlies intercellular electrical communications. A fluorescent voltage indicator is presented that enables the investigation of electrical signaling at high spatial resolution. The method is built upon the site-specific modification of microbial rhodopsin proteins with organic fluorophores, resulting in a hybrid indicator scaffold that is one of the most sensitive and fastest orange-colored voltage indicators developed to date. We applied this technique to optically map electrical connectivity in cultured cells, which revealed gap junction-mediated long-range coupling that spanned over hundreds of micrometers.

Profiling stress-triggered RNA condensation with photocatalytic proximity labeling.

Stress granules (SGs) are highly dynamic cytoplasmic membrane-less organelles that assemble when cells are challenged by stress. RNA molecules are sorted into SGs where they play important roles in maintaining the structural stability of SGs and regulating gene expression. Herein, we apply a proximity-dependent RNA labeling method, CAP-seq, to comprehensively investigate the content of SG-proximal transcriptome in live mammalian cells. CAP-seq captures 457 and 822 RNAs in arsenite- and sorbitol-induced SGs in HEK293T cells, respectively, revealing that SG enrichment is positively correlated with RNA length and AU content, but negatively correlated with translation efficiency. The high spatial specificity of CAP-seq dataset is validated by single-molecule FISH imaging. We further apply CAP-seq to map dynamic changes in SG-proximal transcriptome along the time course of granule assembly and disassembly processes. Our data portray a model of AU-rich and translationally repressed SG nanostructure that are memorized long after the removal of stress.

Immunoproximity biotinylation reveals the axon initial segment proteome.

The axon initial segment (AIS) is a specialized neuronal compartment required for action potential generation and neuronal polarity. However, understanding the mechanisms regulating AIS structure and function has been hindered by an incomplete knowledge of its molecular composition. Here, using immuno-proximity biotinylation we further define the AIS proteome and its dynamic changes during neuronal maturation. Among the many AIS proteins identified, we show that SCRIB is highly enriched in the AIS both in vitro and in vivo, and exhibits a periodic architecture like the axonal spectrin-based cytoskeleton. We find that ankyrinG interacts with and recruits SCRIB to the AIS. However, loss of SCRIB has no effect on ankyrinG. This powerful and flexible approach further defines the AIS proteome and provides a rich resource to elucidate the mechanisms regulating AIS structure and function.

Dendritic Morphology Affects the Velocity and Amplitude of Back-propagating Action Potentials.

The back-propagating action potential (bpAP) is crucial for neuronal signal integration and synaptic plasticity in dendritic trees. Its properties (velocity and amplitude) can be affected by dendritic morphology. Due to limited spatial resolution, it has been difficult to explore the specific propagation process of bpAPs along dendrites and examine the influence of dendritic morphology, such as the dendrite diameter and branching pattern, using patch-clamp recording. By taking advantage of Optopatch, an all-optical electrophysiological method, we made detailed recordings of the real-time propagation of bpAPs in dendritic trees. We found that the velocity of bpAPs was not uniform in a single dendrite, and the bpAP velocity differed among distinct dendrites of the same neuron. The velocity of a bpAP was positively correlated with the diameter of the dendrite on which it propagated. In addition, when bpAPs passed through a dendritic branch point, their velocity decreased significantly. Similar to velocity, the amplitude of bpAPs was also positively correlated with dendritic diameter, and the attenuation patterns of bpAPs differed among different dendrites. Simulation results from neuron models with different dendritic morphology corresponded well with the experimental results. These findings indicate that the dendritic diameter and branching pattern significantly influence the properties of bpAPs. The diversity among the bpAPs recorded in different neurons was mainly due to differences in dendritic morphology. These results may inspire the construction of neuronal models to predict the propagation of bpAPs in dendrites with enormous variation in morphology, to further illuminate the role of bpAPs in neuronal communication.

Differences in action potential propagation speed and axon initial segment plasticity between neurons from Sprague-Dawley rats and C57BL/6 mice.

Action potentials (APs) in neurons are generated at the axon initial segment (AIS). AP dynamics, including initiation and propagation, are intimately associated with neuronal excitability and neurotransmitter release kinetics. Most learning and memory studies at the single-neuron level have relied on the use of animal models, most notably rodents. Here, we studied AP initiation and propagation in cultured hippocampal neurons from Sprague-Dawley (SD) rats and C57BL/6 (C57) mice with genetically encoded voltage indicator (GEVI)-based voltage imaging. Our data showed that APs traveled bidirectionally in neurons from both species; forward-propagating APs (fpAPs) had a different speed than backpropagating APs (bpAPs). Additionally, we observed distinct AP propagation characteristics in AISs emerging from the somatic envelope compared to those originating from dendrites. Compared with rat neurons, mouse neurons exhibited higher bpAP speed and lower fpAP speed, more distally located ankyrin G (AnkG) in AISs, and longer Nav1.2 lengths in AISs. Moreover, during AIS plasticity, AnkG and Nav1.2 showed distal shifts in location and shorter lengths of labeled AISs in rat neurons; in mouse neurons, however, they showed a longer AnkG-labeled length and more distal Nav1.2 location. Our findings suggest that hippocampal neurons in SD rats and C57 mice may have different AP propagation speeds, different AnkG and Nav1.2 patterns in the AIS, and different AIS plasticity properties, indicating that comparisons between these species must be carefully considered.

O-GlcNAcylation modulates liquid-liquid phase separation of SynGAP/PSD-95.

Liquid-liquid phase separation (LLPS) of SynGAP and PSD-95, two abundant proteins that interact in the postsynaptic density (PSD) of neurons, has been implicated in modulating SynGAP PSD enrichment in excitatory synapses. However, the underlying regulatory mechanisms remain enigmatic. Here we report that O-GlcNAcylation of SynGAP acts as a suppressor of LLPS of the SynGAP/PSD-95 complex. We identified multiple O-GlcNAc modification sites for the endogenous SynGAP isolated from rat brain and the recombinantly expressed protein. Protein semisynthesis was used to generate site-specifically O-GlcNAcylated forms of SynGAP, and in vitro and cell-based LLPS assays demonstrated that T1306 O-GlcNAc of SynGAP blocks the interaction with PSD-95, thus inhibiting LLPS. Furthermore, O-GlcNAcylation suppresses SynGAP/PSD-95 LLPS in a dominant-negative manner, enabling sub-stoichiometric O-GlcNAcylation to exert effective regulation. We also showed that O-GlcNAc-dependent LLPS is reversibly regulated by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). These findings demonstrate that OGT- and OGA-catalysed O-GlcNAc cycling may serve as an LLPS-regulating post-translational modification.

A far-red hybrid voltage indicator enabled by bioorthogonal engineering of rhodopsin on live neurons.

Membrane potential is a key aspect of cellular signalling and is dynamically regulated by an array of ion-selective pumps and channels. Fluorescent voltage indicators enable non-invasive optical recording of the cellular membrane potential with high spatial resolution. Here, we report a palette of bright and sensitive hybrid voltage indicators (HVIs) with fluorescence intensities sensitive to changes in membrane potential via electrochromic Förster resonance energy transfer. Enzyme-mediated site-specific incorporation of a probe, followed by an inverse-electron-demand Diels-Alder cycloaddition, was used to create enhanced voltage-sensing rhodopsins with hybrid dye-protein architectures. The most sensitive indicator, HVI-Cy3, displays high voltage sensitivity (-39% ΔF/F0 per 100 mV) and millisecond response kinetics, enabling optical recording of action potentials at a sampling rate of 400 Hz over 10 min across a large neuronal population. The far-red indicator HVI-Cy5 could be paired with optogenetic actuators and green/red-emitting fluorescent indicators, allowing an all-optical investigation of neuronal electrophysiology.

Imaging Neuronal Activity with Fast and Sensitive Red-Shifted Electrochromic FRET Indicators.

Genetically encoded voltage indicators (GEVIs) allow optical recording of neuronal activities with high spatial resolution. While most existing GEVIs emit in the green range, red-shifted GEVIs are highly sought after because they would enable simultaneous stimulation and recording of neuronal activities when paired with optogenetic actuators, or two-color imaging of signaling and neuronal activities when used along with GFP-based indicators. In this study, we present several improved red-shifted GEVIs based on the electrochromic Förster resonance energy transfer (eFRET) between orange/red fluorescent proteins/dyes and rhodopsin mutants. Through structure-guided mutagenesis and cell-based sensitivity screening, we identified a mutant rhodopsin with a single mutation that exhibited more than 2-fold improvement in voltage sensitivity. Notably, this mutation has been independently discovered by Pieribone et al. ( Pieribone, V. A. et al. Nat Methods 2018 , 15 ( 12 ), 1108 - 1116 ). In cultured rat hippocampal neurons, our sensors faithfully reported action potential waveforms and subthreshold activities. We also demonstrated that this mutation could enhance the sensitivity of hybrid indicators, thus providing insights for future development.

MRGPRX4 is a bile acid receptor for human cholestatic itch.

Patients with liver diseases often suffer from chronic itch, yet the pruritogen(s) and receptor(s) remain largely elusive. Here, we identify bile acids as natural ligands for MRGPRX4. MRGPRX4 is expressed in human dorsal root ganglion (hDRG) neurons and co-expresses with itch receptor HRH1. Bile acids elicited Ca2+ responses in cultured hDRG neurons, and bile acids or a MRGPRX4 specific agonist induced itch in human subjects. However, a specific agonist for another bile acid receptor TGR5 failed to induce itch in human subjects and we find that human TGR5 is not expressed in hDRG neurons. Finally, we show positive correlation between cholestatic itch and plasma bile acids level in itchy patients and the elevated bile acids is sufficient to activate MRGPRX4. Taken together, our data strongly suggest that MRGPRX4 is a novel bile acid receptor that likely underlies cholestatic itch in human, providing a promising new drug target for anti-itch therapies.