Hydrophobic residues in S1 modulate enzymatic function and voltage sensing in voltage-sensing phosphatase.

The voltage-sensing domain (VSD) is a four-helix modular protein domain that converts electrical signals into conformational changes, leading to open pores and active enzymes. In most voltage-sensing proteins, the VSDs do not interact with one another, and the S1-S3 helices are considered mainly scaffolding, except in the voltage-sensing phosphatase (VSP) and the proton channel (Hv). To investigate its contribution to VSP function, we mutated four hydrophobic amino acids in S1 to alanine (F127, I131, I134, and L137), individually or in combination. Most of these mutations shifted the voltage dependence of activity to higher voltages; however, not all substrate reactions were the same. The kinetics of enzymatic activity were also altered, with some mutations significantly slowing down dephosphorylation. The voltage dependence of VSD motions was consistently shifted to lower voltages and indicated a second voltage-dependent motion. Additionally, none of the mutations broke the VSP dimer, indicating that the S1 impact could stem from intra- and/or intersubunit interactions. Lastly, when the same mutations were introduced into a genetically encoded voltage indicator, they dramatically altered the optical readings, making some of the kinetics faster and shifting the voltage dependence. These results indicate that the S1 helix in VSP plays a critical role in tuning the enzyme's conformational response to membrane potential transients and influencing the function of the VSD.

S1 hydrophobic residues modulate voltage sensing phosphatase enzymatic function and voltage sensing.

The voltage sensing domain (VSD) is a four-helix modular protein domain that converts electrical signals into conformational changes, leading to open pores and active enzymes. In most voltage sensing proteins, the VSDs do not interact with one another and the S1-S3 helices are considered mainly as scaffolding. The two exceptions are the voltage sensing phosphatase (VSP) and the proton channel (Hv). VSP is a voltage-regulated enzyme and Hvs are channels that only have VSDs. To investigate the S1 contribution to VSP function, we individually mutated four hydrophobic amino acids in S1 to alanine (F127, I131, I134 and L137). We also combined these mutations to generate quadruple mutation designated S1-Q. Most of these mutations shifted the voltage dependence of activity to higher voltages though interestingly, not all substrate reactions were the same. The kinetics of enzymatic activity were also altered with some mutations significantly slowing down dephosphorylation. The voltage dependence of VSD motions were consistently shifted to lower voltages and indicated a second voltage dependent motion. Co-immunoprecipitation demonstrated that none of the mutations broke the VSP dimer indicating that the S1 impact could stem from intrasubunit and/or intersubunit interactions. Lastly, when the same alanine mutations were introduced into a genetically encoded voltage indicator, they dramatically altered the optical readings, making some of the kinetics faster and shifting the voltage dependence. These results indicate that the S1 helix in VSP plays a critical role in tuning the enzymes conformational response to membrane potential transients and influencing the function of the VSD.

We don't know what you did last summer. On the importance of transparent reporting of reaction time data pre-processing.

In behavioral, cognitive, and social sciences, reaction time measures are an important source of information. However, analyses on reaction time data are affected by researchers' analytical choices and the order in which these choices are applied. The results of a systematic literature review, presented in this paper, revealed that the justification for and order in which analytical choices are conducted are rarely reported, leading to difficulty in reproducing results and interpreting mixed findings. To address this methodological shortcoming, we created a checklist on reporting reaction time pre-processing to make these decisions more explicit, improve transparency, and thus, promote best practices within the field. The importance of the pre-processing checklist was additionally supported by an expert consensus survey and a multiverse analysis. Consequently, we appeal for maximal transparency on all methods applied and offer a checklist to improve replicability and reproducibility of studies that use reaction time measures.

Teaching open and reproducible scholarship: a critical review of the evidence base for current pedagogical methods and their outcomes.

In recent years, the scientific community has called for improvements in the credibility, robustness and reproducibility of research, characterized by increased interest and promotion of open and transparent research practices. While progress has been positive, there is a lack of consideration about how this approach can be embedded into undergraduate and postgraduate research training. Specifically, a critical overview of the literature which investigates how integrating open and reproducible science may influence student outcomes is needed. In this paper, we provide the first critical review of literature surrounding the integration of open and reproducible scholarship into teaching and learning and its associated outcomes in students. Our review highlighted how embedding open and reproducible scholarship appears to be associated with (i) students' scientific literacies (i.e. students' understanding of open research, consumption of science and the development of transferable skills); (ii) student engagement (i.e. motivation and engagement with learning, collaboration and engagement in open research) and (iii) students' attitudes towards science (i.e. trust in science and confidence in research findings). However, our review also identified a need for more robust and rigorous methods within pedagogical research, including more interventional and experimental evaluations of teaching practice. We discuss implications for teaching and learning scholarship.

What's in a Badge? A Computational Reproducibility Investigation of the Open Data Badge Policy in One Issue of Psychological Science.

In April 2019, Psychological Science published its first issue in which all Research Articles received the Open Data badge. We used that issue to investigate the effectiveness of this badge, focusing on the adherence to its aim at Psychological Science: sharing both data and code to ensure reproducibility of results. Twelve researchers of varying experience levels attempted to reproduce the results of the empirical articles in the target issue (at least three researchers per article). We found that all 14 articles provided at least some data and six provided analysis code, but only one article was rated to be exactly reproducible, and three were rated as essentially reproducible with minor deviations. We suggest that researchers should be encouraged to adhere to the higher standard in force at Psychological Science. Moreover, a check of reproducibility during peer review may be preferable to the disclosure method of awarding badges.

A trafficking motif alters GEVI activity implicating persistent protein interactions at the membrane.

Efficient plasma-membrane expression is critical for genetically encoded voltage indicators (GEVIs). To improve the plasma-membrane expression, we introduced multiple combinations of plasma-membrane trafficking motifs at different positions to members of the Bongwoori family of GEVIs. An improvement from 20% to 27% in the ΔF/F/100 mV depolarization of the plasma membrane was observed when a Golgi transport motif was inserted near the N-terminus in conjunction with an endoplasmic reticulum release motif near the C-terminus of the protein. Unfortunately, this variant was also slower. The weighted tau on of the variant (25 ms) was more than double the original construct (11 ms). The weighted tau off was >20 ms compared with 10 ms for the original GEVI. The voltage range of the GEVI was also shifted to more negative potentials. Insertion of spacer amino acids between the fluorescent-protein domain and the endoplasmic reticulum release motif at the C-terminus rescued the speed of both the tau on and tau off while restoring the voltage range and maintaining the improved voltage-dependent optical signal. These results suggest that while trafficking motifs do improve plasma-membrane expression, they may also mediate persistent associations that affect the functioning of the protein.

Conserved Amino Acids Residing Outside the Voltage Field Can Shift the Voltage Sensitivity and Increase the Signal Speed and Size of Ciona Based GEVIs.

To identify potential regions of the voltage-sensing domain that could shift the voltage sensitivity of Ciona intestinalis based Genetically Encoded Voltage Indicators (GEVIs), we aligned the amino acid sequences of voltage-gated sodium channels from different organisms. Conserved polar residues were identified at multiple transmembrane/loop junctions in the voltage sensing domain. Similar conservation of polar amino acids was found in the voltage-sensing domain of the voltage-sensing phosphatase gene family. These conserved residues were mutated to nonpolar or oppositely charged amino acids in a GEVI that utilizes the voltage sensing domain of the voltage sensing phosphatase from Ciona fused to the fluorescent protein, super ecliptic pHluorin (A227D). Different mutations shifted the voltage sensitivity to more positive or more negative membrane potentials. Double mutants were then created by selecting constructs that shifted the optical signal to a more physiologically relevant voltage range. Introduction of these mutations into previously developed GEVIs resulted in Plos6-v2 which improved the dynamic range to 40% ΔF/F/100 mV, a 25% increase over the parent, ArcLight. The onset time constant of Plos6-v2 is also 50% faster than ArcLight. Thus, Plos6-v2 appears to be the GEVI of choice.

A Multimodal Multi-Shank Fluorescence Neural Probe for Cell-Type-Specific Electrophysiology in Multiple Regions across a Neural Circuit.

Cell-type-specific, activity-dependent electrophysiology can allow in-depth analysis of functional connectivity inside complex neural circuits composed of various cell types. To date, optics-based fluorescence recording devices enable monitoring cell-type-specific activities. However, the monitoring is typically limited to a single brain region, and the temporal resolution is significantly low. Herein, a multimodal multi-shank fluorescence neural probe that allows cell-type-specific electrophysiology from multiple deep-brain regions at a high spatiotemporal resolution is presented. A photodiode and an electrode-array pair are monolithically integrated on each tip of a minimal-form-factor silicon device. Both fluorescence and electrical signals are successfully measured simultaneously in GCaMP6f expressing mice, and the cell type from sorted neural spikes is identified. The probe's capability of combined electro-optical recordings for cell-type-specific electrophysiology at multiple brain regions within a neural circuit is demonstrated. The new experimental paradigm to enable the precise investigation of functional connectivity inside and across complex neural circuits composed of various cell types is expected.

Visualizing Oscillations in Brain Slices With Genetically Encoded Voltage Indicators.

Genetically encoded voltage indicators (GEVIs) expressed pan-neuronally were able to optically resolve bicuculline induced spontaneous oscillations in brain slices of the mouse motor cortex. Three GEVIs were used that differ in their timing of response to voltage transients as well as in their voltage ranges. The duration, number of cycles, and frequency of the recorded oscillations reflected the characteristics of each GEVI used. Multiple oscillations imaged in the same slice never originated at the same location, indicating the lack of a "hot spot" for induction of the voltage changes. Comparison of pan-neuronal, Ca2+/calmodulin-dependent protein kinase II α restricted, and parvalbumin restricted GEVI expression revealed distinct profiles for the excitatory and inhibitory cells in the spontaneous oscillations of the motor cortex. Resolving voltage fluctuations across space, time, and cell types with GEVIs represent a powerful approach to dissecting neuronal circuit activity.

GEVI cell-type specific labelling and a manifold learning approach provide evidence for lateral inhibition at the population level in the mouse hippocampal CA1 area.

The CA1 area in the mammalian hippocampus is essential for spatial learning. Pyramidal cells are the hippocampus output neurons and their activities are regulated by inhibition exerted by a diversified population of interneurons. Lateral inhibition has been suggested as the mechanism enabling the reconfiguration of pyramidal cell assembly activity observed during spatial learning tasks in rodents. However, lateral inhibition in the CA1 lacks the overwhelming evidence reported in other hippocampal areas such as the CA3 and the dentate gyrus. The use of genetically encoded voltage indicators and fast optical recordings permits the construction of cell-type specific response maps of neuronal activity. Here, we labelled mouse CA1 pyramidal neurons with the genetically encoded voltage indicator ArcLight and optically recorded their response to Schaffer Collaterals stimulation in vitro. By undertaking a manifold learning approach, we report a hyperpolarization-dominated area focused in the perisomatic region of pyramidal cells receiving late excitatory synaptic input. Functional network organization metrics revealed that information transfer was higher in this area. The localized hyperpolarization disappeared when GABAA receptors were pharmacologically blocked. This is the first report where the spatiotemporal pattern of lateral inhibition is visualized in the CA1 by expressing a genetically encoded voltage indicator selectively in principal neurons. Our analysis suggests a fundamental role of lateral inhibition in CA1 information processing.

Improving the flexibility of genetically encoded voltage indicators via intermolecular FRET.

A new family of genetically encoded voltage indicators (GEVIs) has been developed based on intermolecular Förster resonance energy transfer (FRET). To test the hypothesis that the GEVI ArcLight functions via interactions between the fluorescent protein (FP) domains of neighboring probes, the FP of ArcLight was replaced with either a FRET donor or acceptor FP. We discovered relatively large FRET signals only when cells were cotransfected with both the FRET donor and acceptor GEVIs. Using a cyan fluorescent protein donor and an RFP acceptor, we were able to observe a voltage-dependent signal with an emission peak separated by over 200 nm from the excitation wavelength. The intermolecular FRET strategy also works for rhodopsin-based probes, potentially improving their flexibility as well. Separating the FRET pair into two distinct proteins has important advantages over intramolecular FRET constructs. The signals are larger because the voltage-induced conformational change moves two FPs independently. The expression of the FRET donor and acceptor can also be restricted independently, enabling greater cell type specificity as well as refined subcellular voltage reporting.

Mechanism of ArcLight derived GEVIs involves electrostatic interactions that can affect proton wires.

The genetically encoded voltage indicators ArcLight and its derivatives mediate voltage-dependent optical signals by intermolecular, electrostatic interactions between neighboring fluorescent proteins (FPs). A random mutagenesis event placed a negative charge on the exterior of the FP, resulting in a greater than 10-fold improvement of the voltage-dependent optical signal. Repositioning this negative charge on the exterior of the FP reversed the polarity of voltage-dependent optical signals, suggesting the presence of "hot spots" capable of interacting with the negative charge on a neighboring FP, thereby changing the fluorescent output. To explore the potential effect on the chromophore state, voltage-clamp fluorometry was performed with alternating excitation at 390 nm followed by excitation at 470 nm, resulting in several mutants exhibiting voltage-dependent, ratiometric optical signals of opposing polarities. However, the kinetics, voltage ranges, and optimal FP fusion sites were different depending on the wavelength of excitation. These results suggest that the FP has external, electrostatic pathways capable of quenching fluorescence that are wavelength specific. One mutation to the FP (E222H) showed a voltage-dependent increase in fluorescence when excited at 390 nm, indicating the ability to affect the proton wire from the protonated chromophore to the H222 position. ArcLight-derived sensors may therefore offer a novel way to map how conditions external to the ß-can structure can affect the fluorescence of the chromophore and transiently affect those pathways via conformational changes mediated by manipulating membrane potential.

Biophysical Parameters of GEVIs: Considerations for Imaging Voltage.

Genetically encoded voltage indicators (GEVIs) continue to evolve, resulting in many different probes with varying strengths and weaknesses. Developers of new GEVIs tend to highlight their positive features. A recent article from an independent laboratory has compared the signal/noise ratios of a number of GEVIs. Such a comparison can be helpful to investigators eager to try to image the voltage of excitable cells. In this perspective, we will present examples of how the biophysical features of GEVIs affect the imaging of excitable cells in an effort to assist researchers when considering probes for their specific needs.

Engineering Photoactivatability in Genetically Encoded Voltage and pH Indicators.

Genetically-encoded indicators of neuronal activity enable the labeling of a genetically defined population of neurons to optically monitor their activities. However, researchers often find difficulties in identifying relevant signals from excessive background fluorescence. A photoactivatable version of a genetically encoded calcium indicator, sPA-GCaMP6f is a good example of circumventing such an obstacle by limiting the fluorescence to a region of interest defined by the user. Here, we apply this strategy to genetically encoded voltage (GEVI) and pH (GEPI) indicators. Three photoactivatable GEVI candidates were considered. The first one used a circularly-permuted fluorescent protein, the second design involved a Förster resonance energy transfer (FRET) pair, and the third approach employed a pH-sensitive variant of GFP, ecliptic pHluorin. The candidate with a variant of ecliptic pHluorin exhibited photoactivation and a voltage-dependent fluorescence change. This effort also yielded a pH-sensitive photoactivatable GFP that varies its brightness in response to intracellular pH changes.

Optical consequences of a genetically-encoded voltage indicator with a pH sensitive fluorescent protein.

Genetically-Encoded Voltage Indicators (GEVIs) are capable of converting changes in membrane potential into an optical signal. Here, we focus on recent insights into the mechanism of ArcLight-type probes and the consequences of utilizing a pH-dependent Fluorescent Protein (FP). A negative charge on the exterior of the ß-can of the FP combined with a pH-sensitive FP enables voltage-dependent conformational changes to affect the fluorescence of the probe. This hypothesis implies that interaction/dimerization of the FP creates a microenvironment for the probe that is altered via conformational changes. This mechanism explains why a pH sensitive FP with a negative charge on the outside of the ß-can is needed, but also suggests that pH could affect the optical signal as well. To better understand the effects of pH on the voltage-dependent signal of ArcLight, the intracellular pH (pHi) was tested at pH 6.8, 7.2, or 7.8. The resting fluorescence of ArcLight gets brighter as the pHi increases, yet only pH 7.8 significantly affected the ΔF/F. ArcLight could also simultaneously report voltage and pH changes during the acidification of a neuron firing multiple action potentials revealing different buffering capacities of the soma versus the processes of the cell.

A dimeric fluorescent protein yields a bright, red-shifted GEVI capable of population signals in brain slice.

A bright, red-shifted Genetically Encoded Voltage Indicator (GEVI) was developed using a modified version of the fluorescent protein, tdTomato. Dimerization of the fluorescent domain for ArcLight-type GEVIs has been shown to affect the signal size of the voltage-dependent optical signal. For red-shifted GEVI development, tdTomato was split fusing a single dTomato chromophore to the voltage sensing domain. Optimization of the amino acid length and charge composition of the linker region between the voltage sensing domain and the fluorescent protein resulted in a probe that is an order of magnitude brighter than FlicR1 at a resting potential of -70 mV and exhibits a ten-fold larger change in fluorescence (ΔF) upon 100 mV depolarization of the plasma membrane in HEK 293 cells. Unlike ArcLight, the introduction of charged residues to the exterior of dTomato did not substantially improve the dynamic range of the optical signal. As a result, this new GEVI, Ilmol, yields a 3-fold improvement in the signal-to-noise ratio compared to FlicR1 despite a smaller fractional change in fluorescence of 4% per 100 mV depolarization of the plasma membrane. Ilmol expresses well in neurons resolving action potentials in neuronal cultures and reporting population signals in mouse hippocampal acute brain slice recordings. Ilmol is the brightest red-shifted GEVI to date enabling imaging with 160-fold less light than Archon1 for primary neuron recordings (50 mW/cm2 versus 8 W/cm2) and 600-fold less light than QuasAr2 for mouse brain slice recordings (500 mW/cm2 versus 300 W/cm2). This new GEVI uses a distinct mechanism from other approaches, opening an alternate engineering path to improve sensitivity and speed.

Monitoring voltage fluctuations of intracellular membranes.

In eukaryotic cells, the endoplasmic reticulum (ER) is the largest continuous membrane-enclosed network which surrounds a single lumen. Using a new genetically encoded voltage indicator (GEVI), we applied the patch clamp technique to cultured HEK293 cells and neurons and found that there is a very fast electrical interaction between the plasma membrane and internal membrane(s). This discovery suggests a novel mechanism for interaction between the external membrane and internal membranes as well as mechanisms for interactions between the various internal membranes. The ER may transfer electrical signals between the plasma membrane and other internal organelles. The internal membrane optical signal is reversed in polarity but has a time course similar to that of the plasma membrane signal. The optical signal of the GEVI in the plasma membrane is consistent from trial to trial. However, the internal signal decreases in size with repeated trials suggesting that the electrical coupling is degrading and/or the resistance of the internal membrane is decaying.

Mapping of excitatory and inhibitory postsynaptic potentials of neuronal populations in hippocampal slices using the GEVI, ArcLight.

To understand the circuitry of the brain, it is essential to clarify the functional connectivity among distinct neuronal populations. For this purpose, neuronal activity imaging using genetically-encoded calcium sensors such as GCaMP has been a powerful approach due to its cell-type specificity. However, calcium (Ca2+) is an indirect measure of neuronal activity. A more direct approach would be to use genetically encoded voltage indicators (GEVIs) to observe subthreshold, synaptic activities. The GEVI, ArcLight, which exhibits large fluorescence transients in response to voltage, was expressed in excitatory neurons of the mouse CA1 hippocampus. Fluorescent signals in response to the electrical stimulation of the Schaffer collateral axons were observed in brain slice preparations. ArcLight was able to map both excitatory and inhibitory inputs projected to excitatory neurons. In contrast, the Ca2+ signal detected by GCaMP6f, was only associated with excitatory inputs. ArcLight and similar voltage sensing probes are also becoming powerful paradigms for functional connectivity mapping of brain circuitry.

Modulating the Voltage-sensitivity of a Genetically Encoded Voltage Indicator.

Saturation mutagenesis was performed on a single position in the voltage-sensing domain (VSD) of a genetically encoded voltage indicator (GEVI). The VSD consists of four transmembrane helixes designated S1-S4. The V220 position located near the plasma membrane/extracellular interface had previously been shown to affect the voltage range of the optical signal. Introduction of polar amino acids at this position reduced the voltage-dependent optical signal of the GEVI. Negatively charged amino acids slightly reduced the optical signal by 33 percent while positively charge amino acids at this position reduced the optical signal by 80%. Surprisingly, the range of V220D was similar to that of V220K with shifted optical responses towards negative potentials. In contrast, the V220E mutant mirrored the responses of the V220R mutation suggesting that the length of the side chain plays in role in determining the voltage range of the GEVI. Charged mutations at the 219 position all behaved similarly slightly shifting the optical response to more negative potentials. Charged mutations to the 221 position behaved erratically suggesting interactions with the plasma membrane and/or other amino acids in the VSD. Introduction of bulky amino acids at the V220 position increased the range of the optical response to include hyperpolarizing signals. Combining The V220W mutant with the R217Q mutation resulted in a probe that reduced the depolarizing signal and enhanced the hyperpolarizing signal which may lead to GEVIs that only report neuronal inhibition.