Mechanistic Studies of the Genetically Encoded Fluorescent Protein Voltage Probe ArcLight

ArcLight, a genetically encoded fluorescent protein voltage probe with a large ΔF/ΔV, is a fusion between the voltage sensing domain of the Ciona instestinalis voltage sensitive phosphatase and super ecliptic pHluorin carrying a single mutation (A227D in the fluorescent protein). Without this mutation the probe produces only a very small change in fluorescence in response to voltage deflections (∼1%). The large signal afforded by this mutation allows optical detection of action potentials and sub-threshold electrical events in single-trials in vitro and in vivo. However, it is unclear how this single mutation produces a probe with such a large modulation of its fluorescence output with changes in membrane potential. In this study, we identified which residues in super ecliptic pHluorin (vs eGFP) are critical for the ArcLight response, as a similarly constructed probe based on eGFP also exhibits large response amplitude if it carries these critical residues. We found that D147 is responsible for determining the pH sensitivity of the fluorescent protein used in these probes but by itself does not result in a voltage probe with a large signal. We also provide evidence that the voltage dependent signal of ArcLight is not simply sensing environmental pH changes. A two-photon polarization microscopy study showed that ArcLight''s response to changes in membrane potential includes a reorientation of the super ecliptic pHluorin. We also explored different changes including modification of linker length, deletion of non-essential amino acids in the super ecliptic pHluorin, adding a farnesylation site, using tandem fluorescent proteins and other pH sensitive fluorescent proteins.     

Effect of Voltage Sensitive Fluorescent Proteins on Neuronal Excitability

Fluorescent protein voltage sensors are recombinant proteins that are designed as genetically encoded cellular probes of membrane potential using mechanisms of voltage-dependent modulation of fluorescence. Several such proteins, including VSFP2.3 and VSFP3.1, were recently reported with reliable function in mammalian cells. They were designed as molecular fusions of the voltage sensor of Ciona intestinalis voltage sensor containing phosphatase with a fluorescence reporter domain. Expression of these proteins in cell membranes is accompanied by additional dynamic membrane capacitance, or “sensing capacitance”, with feedback effect on the native electro-responsiveness of targeted cells. We used recordings of sensing currents and fluorescence responses of VSFP2.3 and of VSFP3.1 to derive kinetic models of the voltage-dependent signaling of these proteins. Using computational neuron simulations, we quantitatively investigated the perturbing effects of sensing capacitance on the input/output relationship in two central neuron models, a cerebellar Purkinje and a layer 5 pyramidal neuron. Probe-induced sensing capacitance manifested as time shifts of action potentials and increased synaptic input thresholds for somatic action potential initiation with linear dependence on the membrane density of the probe. Whereas the fluorescence signal/noise grows with the square root of the surface density of the probe, the growth of sensing capacitance is linear. We analyzed the trade-off between minimization of sensing capacitance and signal/noise of the optical read-out depending on kinetic properties and cellular distribution of the probe. The simulation results suggest ways to reduce capacitive effects at a given level of signal/noise. Yet, the simulations indicate that significant improvement of existing probes will still be required to report action potentials in individual neurons in mammalian brain tissue in single trials.     

Enhanced Archaerhodopsin Fluorescent Protein Voltage Indicators

A longstanding goal in neuroscience has been to develop techniques for imaging the voltage dynamics of genetically defined subsets of neurons. Optical sensors of transmembrane voltage would enhance studies of neural activity in contexts ranging from individual neurons cultured in vitro to neuronal populations in awake-behaving animals. Recent progress has identified Archaerhodopsin (Arch) based sensors as a promising, genetically encoded class of fluorescent voltage indicators that can report single action potentials. Wild-type Arch exhibits sub-millisecond fluorescence responses to trans-membrane voltage, but its light-activated proton pump also responds to the imaging illumination. An Arch mutant (Arch-D95N) exhibits no photocurrent, but has a slower, ~40 ms response to voltage transients. Here we present Arch-derived voltage sensors with trafficking signals that enhance their localization to the neural membrane. We also describe Arch mutant sensors (Arch-EEN and -EEQ) that exhibit faster kinetics and greater fluorescence dynamic range than Arch-D95N, and no photocurrent at the illumination intensities normally used for imaging. We benchmarked these voltage sensors regarding their spike detection fidelity by using a signal detection theoretic framework that takes into account the experimentally measured photon shot noise and optical waveforms for single action potentials. This analysis revealed that by combining the sequence mutations and enhanced trafficking sequences, the new sensors improved the fidelity of spike detection by nearly three-fold in comparison to Arch-D95N.     

A Genetically Targetable Fluorescent Probe of Channel Gating with Rapid Kinetics

We have developed a genetically targetable, optical channel-gating reporter that converts rapid membrane potential changes into changes in fluorescence intensity. We have named this construct SPARC (sodium channel protein-based activity reporting construct). Green fluorescent protein was inserted into an intracellular loop of a reversibly nonconducting form of the rat μI skeletal muscle voltage-gated sodium channel. Rapid changes of the membrane potential modulate the fluorescence of the inserted green fluorescent protein. This change in fluorescence can faithfully report depolarizing pulses as short as 2 ms. The fluorescence signal does not inactivate during extended depolarizations. Several features of the probe’s response properties indicate that it likely reports gating charge movement of a single domain of rat μI skeletal muscle. This probe provides a new approach for studying rapid channel movements and may possibly act as a fluorescent activity reporter in excitable cells.     

A fluorescent, genetically-encoded voltage probe capable of resolving action potentials

There is a pressing need in neuroscience for genetically-encoded, fluorescent voltage probes that can be targeted to specific neurons and circuits to allow study of neural activity using fluorescent imaging. We created 90 constructs in which the voltage sensing portion (S1-S4) of Ciona intestinalis voltage sensitive phosphatase (CiVSP) was fused to circularly permuted eGFP. This led to ElectricPk, a probe that is an order of magnitude faster (taus ～1-2 ms) than any currently published fluorescent protein-based voltage probe. ElectricPk can follow the rise and fall of neuronal action potentials with a modest decrease in fluorescence intensity (～0.7% ΔF/F). The probe has a nearly linear fluorescence/membrane potential response to both hyperpolarizing and depolarizing steps. This is the first probe based on CiVSP that captures the rapid movements of the voltage sensor, suggesting that voltage probes designed with circularly permuted fluorescent proteins may have some advantages.     

Fluorescent Protein Voltage Probes Derived from ArcLight that Respond to Membrane Voltage Changes with Fast Kinetics

We previously reported the discovery of a fluorescent protein voltage probe, ArcLight, and its derivatives that exhibit large changes in fluorescence intensity in response to changes of plasma membrane voltage. ArcLight allows the reliable detection of single action potentials and sub-threshold activities in individual neurons and dendrites. The response kinetics of ArcLight (τ1-on ~10 ms, τ2-on ~ 50 ms) are comparable with most published genetically-encoded voltage probes. However, probes using voltage-sensing domains other than that from the Ciona intestinalis voltage sensitive phosphatase exhibit faster kinetics. Here we report new versions of ArcLight, in which the Ciona voltage-sensing domain was replaced with those from chicken, zebrafish, frog, mouse or human. We found that the chicken and zebrafish-based ArcLight exhibit faster kinetics, with a time constant (τ) less than 6ms for a 100 mV depolarization. Although the response amplitude of these two probes (8-9%) is not as large as the Ciona-based ArcLight (~35%), they are better at reporting action potentials from cultured neurons at higher frequency. In contrast, probes based on frog, mouse and human voltage sensing domains were either slower than the Ciona-based ArcLight or had very small signals.     

Growth Associated Protein 43 Is Expressed in Skeletal Muscle Fibers and Is Localized in Proximity of Mitochondria and Calcium Release Units

The neuronal Growth Associated Protein 43 (GAP43), also known as B-50 or neuromodulin, is involved in mechanisms controlling pathfinding and branching of neurons during development and regeneration. For many years this protein was classified as neuron-specific, but recent evidences suggest that a) GAP43 is expressed in the nervous system not only in neurons, but also in glial cells, and b) probably it is present also in other tissues. In particular, its expression was revealed in muscles from patients affected by various myopathies, indicating that GAP43 can no-longer considered only as a neuron-specific molecule. We have investigated the expression and subcellular localization of GAP43 in mouse satellite cells, myotubes, and adult muscle (extensor digitorum longus or EDL) using Western blotting, immuno-fluorescence combined to confocal microscopy and electron microscopy. Our in vitro results indicated that GAP43 is indeed expressed in both myoblasts and differentiating myotubes, and its cellular localization changes dramatically during maturation: in myoblasts the localization appeared to be mostly nuclear, whereas with differentiation the protein started to display a sarcomeric-like pattern. In adult fibers, GAP43 expression was evident with the protein labeling forming (in longitudinal views) a double cross striation reminiscent of the staining pattern of other organelles, such as calcium release units (CRUs) and mitochondria. Double immuno-staining and experiments done in EDL muscles fixed at different sarcomere lengths, allowed us to determine the localization, from the sarcomere Z-line, of GAP43 positive foci, falling between that of CRUs and of mitochondria. Staining of cross sections added a detail to the puzzle: GAP43 labeling formed a reticular pattern surrounding individual myofibrils, but excluding contractile elements. This work leads the way to further investigation about the possible physiological and structural role of GAP43 protein in adult fiber function and disease.     

Biophysical characterization of the fluorescent protein voltage probe VSFP2.3 based on the voltage-sensing domain of Ci-VSP

A voltage sensitive phosphatase was discovered in the ascidian Ciona intestinalis. The phosphatase, Ci-VSP, contains a voltage-sensing domain homologous to those known from voltage-gated ion channels, but unlike ion channels, the voltage-sensing domain of Ci-VSP can reside in the cell membrane as a monomer. We fused the voltage-sensing domain of Ci-VSP to a pair of fluorescent reporter proteins to generate a genetically encodable voltage-sensing fluorescent probe, VSFP2.3. VSFP2.3 is a fluorescent voltage probe that reports changes in membrane potential as a FRET (fluorescence resonance energy transfer) signal. Here we report sensing current measurements from VSFP2.3, and show that VSFP2.3 carries 1.2 e sensing charges, which are displaced within 1.5 ms. The sensing currents become faster at higher temperatures, and the voltage dependence of the decay time constants is temperature dependent. Neutralization of an arginine in S4, previously suggested to be a sensing charge, and measuring associated sensing currents indicate that this charge is likely to reside at the membrane-aqueous interface rather than within the membrane electric field. The data presented give us insights into the voltage-sensing mechanism of Ci-VSP, which will allow us to further improve the sensitivity and kinetics of the family of VSFP proteins.     

Using aequorin probes to measure Ca2+ in intracellular organelles

Aequorins are excellent tools for measuring intra-organellar Ca²⁺ and assessing its role in physiological and pathological functions. Here we review targeting strategies to express aequorins in various organelles. We address critical topics such as probe affinity tuning as well as normalization and calibration of the signal. We also focus on bioluminescent Ca²⁺ imaging in nucleus or mitochondria of living cells. Finally, recent advances with a new chimeric GFP-aequorin protein (GAP), which can be used either as luminescent or fluorescent Ca²⁺ probe, are presented. GAP is robustly expressed in transgenic flies and mice, where it has proven to be a suitable Ca²⁺ indicator for monitoring physiological Ca²⁺ signaling ex vivo and in vivo.     

Genetically Encoded Green Fluorescent Ca2+ Indicators with Improved Detectability for Neuronal Ca2+ Signals

Imaging the activities of individual neurons with genetically encoded Ca²⁺ indicators (GECIs) is a promising method for understanding neuronal network functions. Here, we report GECIs with improved neuronal Ca²⁺ signal detectability, termed G-CaMP6 and G-CaMP8. Compared to a series of existing G-CaMPs, G-CaMP6 showed fairly high sensitivity and rapid kinetics, both of which are suitable properties for detecting subtle and fast neuronal activities. G-CaMP8 showed a greater signal (F _max/F _min = 38) than G-CaMP6 and demonstrated kinetics similar to those of G-CaMP6. Both GECIs could detect individual spikes from pyramidal neurons of cultured hippocampal slices or acute cortical slices with 100% detection rates, demonstrating their superior performance to existing GECIs. Because G-CaMP6 showed a higher sensitivity and brighter baseline fluorescence than G-CaMP8 in a cellular environment, we applied G-CaMP6 for Ca²⁺ imaging of dendritic spines, the putative postsynaptic sites. By expressing a G-CaMP6-actin fusion protein for the spines in hippocampal CA3 pyramidal neurons and electrically stimulating the granule cells of the dentate gyrus, which innervate CA3 pyramidal neurons, we found that sub-threshold stimulation triggered small Ca²⁺ responses in a limited number of spines with a low response rate in active spines, whereas supra-threshold stimulation triggered large fluorescence responses in virtually all of the spines with a 100% activity rate.     

Ratiometric Biosensors that Measure Mitochondrial Redox State and ATP in Living Yeast Cells

Mitochondria have roles in many cellular processes, from energy metabolism and calcium homeostasis to control of cellular lifespan and programmed cell death. These processes affect and are affected by the redox status of and ATP production by mitochondria. Here, we describe the use of two ratiometric, genetically encoded biosensors that can detect mitochondrial redox state and ATP levels at subcellular resolution in living yeast cells. Mitochondrial redox state is measured using redox-sensitive Green Fluorescent Protein (roGFP) that is targeted to the mitochondrial matrix. Mito-roGFP contains cysteines at positions 147 and 204 of GFP, which undergo reversible and environment-dependent oxidation and reduction, which in turn alter the excitation spectrum of the protein. MitGO-ATeam is a Förster resonance energy transfer (FRET) probe in which the ε subunit of the F_oF₁-ATP synthase is sandwiched between FRET donor and acceptor fluorescent proteins. Binding of ATP to the ε subunit results in conformation changes in the protein that bring the FRET donor and acceptor in close proximity and allow for fluorescence resonance energy transfer from the donor to acceptor.     

Ratiometric fluorescence imaging of nuclear pH in living cells using Hoechst-tagged fluorescein

Small-molecule fluorescent sensors that allow specific measurement of nuclear pH in living cells will be valuable for biological research. Here we report that Hoechst-tagged fluorescein (hoeFL), which we previously developed as a green fluorescent DNA-staining probe, can be used for this purpose. Upon excitation at 405 nm, the hoeFL–DNA complex displayed two fluorescence bands around 460 nm and 520 nm corresponding to the Hoechst and fluorescein fluorescence, respectively. When pH was changed from 8.3 to 5.5, the fluorescence intensity ratio (F₅₂₀/F₄₆₀) significantly decreased, which allowed reliable pH measurement. Moreover, because hoeFL binds specifically to the genomic DNA in cells, it was applicable to visualize the intranuclear pH of nigericin-treated and intact living human cells by ratiometric fluorescence imaging.     

Modification of room-temperature picosecond chlorophyll fluorescence kinetics in Photosystem-II-enriched particles by photochemistry

Room-temperature single photon timing measurements on Photosystem-II (PS II-) enriched thylakoid fragments at low excitation energies indicate the presence of three kinetic decay components of chlorophyll fluorescence arising from PS-II-associated pigments. Closing the PS II reaction centres produced three variable components, with lifetime values of 0.02–0.25, 0.15–0.90 and 0.35–2.0 ns, between the initial (F₀) and maximal (F_m) fluorescence levels. The yield of each component paralleled the changes in their respective lifetimes, indicating the presence of well-connected PS II reaction centres favouring energy transfer between each other. These changes show that variable chlorophyll fluorescence (F_v) does not arise from one specific origin. The extent of the modifications and the observed relationship between component lifetime and yield, on closing PS II reaction centres, cannot be explained by either the delayed fluorescence (charge recombination) hypothesis of Klimov and co-workers (Klimov, V.V. et al. (1978) Dokl. Akad. Nauk. SSSR 242, 1204–1207) or the proposed changes and origins put forward by Holzwarth and co-workers (Holzwarth, A.R. (1986) Photochem. Photobiol. 43, 707–725; Holzwarth, A.R. et al. (1985) Biochim. Biophys. Acta 807, 155–167).     

Two-photon compatibility and single-voxel, single-trial detection of subthreshold neuronal activity by a two-component optical voltage sensor

Minimally invasive measurements of neuronal activity are essential for understanding how signal processing is performed by neuronal networks. While optical strategies for making such measurements hold great promise, optical sensors generally lack the speed and sensitivity necessary to record neuronal activity on a single-trial, single-neuron basis. Here we present additional biophysical characterization and practical improvements of a two-component optical voltage sensor (2cVoS), comprised of the neuronal tracer dye, DiO, and dipicrylamine (DiO/DPA). Using laser spot illumination we demonstrate that membrane potential-dependent fluorescence changes can be obtained in a wide variety of cell types within brain slices. We show a correlation between membrane labeling and the sensitivity of the magnitude of fluorescence signal, such that neurons with the brightest membrane labeling yield the largest ΔF/F values per action potential (AP; ∼40%). By substituting a blue-shifted donor for DiO we confirm that DiO/DPA works, at least in part, via a Förster resonance energy transfer (FRET) mechanism. We also describe a straightforward iontophoretic method for labeling multiple neurons with DiO and show that DiO/DPA is compatible with two-photon (2P) imaging. Finally, exploiting the high sensitivity of DiO/DPA, we demonstrate AP-induced fluorescence transients (fAPs) recorded from single spines of hippocampal pyramidal neurons and single-trial measurements of subthreshold synaptic inputs to granule cell dendrites. Our findings suggest that the 2cVoS, DiO/DPA, enables optical measurements of trial-to-trial voltage fluctuations with very high spatial and temporal resolution, properties well suited for monitoring electrical signals from multiple neurons within intact neuronal networks.     

The application of anti‐ESAT‐6 monoclonal antibody fluorescent probe in ex vivo near‐infrared fluorescence imaging in mice with pulmonary tuberculosis

Here, we aimed to assess the feasibility of anti‐ESAT‐6 monoclonal antibody (mAb) coupling with IR783 and rhodamine fluorescent probe in the detection of ESAT‐6 expression in tuberculosis tissue of mice using near‐infrared fluorescence imaging. IR783 and rhodamine were conjugated to the anti‐ESAT‐6 mAb or IgG. Mice in the experimental group were injected with fluorescence‐labeled mAb probe, and mice in the control group were injected with fluorescence‐labeled non‐specific IgG antibody. Twenty‐four hours later, the lung tissue of mice was examined using ex vivo near‐infrared fluorescence imaging. In addition, the contrast‐to‐noise ratio (CNR) was calculated by measuring the signal intensities of the pulmonary lesions, normal lung tissue and background noise. The frozen lung tissue section was examined under fluorescence microscopy and compared with hemoxylin and eosin (HE) staining. The ex vivo near‐infrared fluorescence imaging showed that the fluorescence signal in the lung tuberculosis lesions in the experimental group was significantly enhanced, whereas there was only a weak fluorescence signal or even no fluorescence signal in the control group. CNR values were 64.40 ± 7.02 (n = 6) and 8.75 ± 3.87 (n = 6), respectively (t = 17.01, p < 0.001). The fluorescence accumulation distribution detected under fluorescence microscopy was consistent with HE staining of the tuberculosis region. In conclusion, anti‐ESAT‐6 mAb fluorescent probe could target and be applied in specific ex vivo imaging of mice tuberculosis, and may be of further use in tuberculosis in living mice. Copyright © 2013 John Wiley & Sons, Ltd.     

Expression of Selenoproteins Is Maintained in Mice Carrying Mutations in SECp43, the tRNA Selenocysteine 1 Associated Protein (Trnau1ap)

Selenocysteine tRNA 1 associated protein (Trnau1ap) has been characterized as a tRNA^[Ser]Sec-binding protein of 43 kDa, hence initially named SECp43. Previous studies reported its presence in complexes containing tRNA^[Ser]Sec implying a role of SECp43 as a co-factor in selenoprotein expression. We generated two conditionally mutant mouse models targeting exons 3+4 and exons 7+8 eliminating parts of the first RNA recognition motif or of the tyrosine-rich domain, respectively. Constitutive inactivation of exons 3+4 of SECp43 apparently did not affect the mice or selenoprotein expression in several organs. Constitutive deletion of exons 7+8 was embryonic lethal. We therefore generated hepatocyte-specific Secp43 knockout mice and characterized selenoprotein expression in livers of mutant mice. We found no significant changes in the levels of ⁷⁵Se-labelled hepatic proteins, selenoprotein levels as determined by Western blot analysis, enzymatic activity or selenoprotein mRNA abundance. The methylation pattern of tRNA^[Ser]Sec remained unchanged. Truncated Secp43 ^Δ7,8mRNA increased in Secp43-mutant livers suggesting auto-regulation of Secp43 mRNA abundance. We found no signs of liver damage in Secp433-mutant mice, but neuron-specific deletion of exons 7+8 impaired motor performance, while not affecting cerebral selenoprotein expression or cerebellar development. These findings suggest that the targeted domains in the SECp43 protein are not essential for selenoprotein biosynthesis in hepatocytes and neurons. Whether the remaining second RNA recognition motif plays a role in selenoprotein biosynthesis and which other cellular process depends on SECp43 remains to be determined.     

Fluorescent sensors based on quinoline‐containing styrylcyanine: determination of ferric ions,hydrogen peroxide,and glucose,pH‐sensitive properties and bioimaging

A novel styrylcyanine‐based fluorescent probe 1 was designed and synthesized via facile methods. Ferric ions quenched the fluorescence of probe 1, whereas the addition of ferrous ions led to only small changes in the fluorescence signal. When hydrogen peroxide was introduced into the solution containing probe 1 and Fe²⁺, Fe²⁺ was oxidized to Fe³⁺, resulting in the quenching of the fluorescence. The probe 1/Fe²⁺ solution fluorescence could also be quenched by H₂O₂ released from glucose oxidation by glucose oxidase (GOD), which means that probe 1/Fe²⁺ platform could be used to detect glucose. Probe 1 is fluorescent in basic and neutral media but almost non‐fluorescent in strong acidic environments. Such behaviour enables it to work as a fluorescent pH sensor in both the solution and solid states and as a chemosensor for detecting volatile organic compounds with high acidity and basicity. Subsequently, the fluorescence microscopic images of probe 1 in live cells and in zebrafish were achieved successfully, suggesting that the probe has good cell membrane permeability and a potential application for imaging in living cells and living organisms. Copyright © 2014 John Wiley & Sons, Ltd.     

Fluorescence transients in chloroplasts of Chara corallina cells during transmission of photoinduced signal with the streaming cytoplasm

Intracellular transport assisted by rotatory cytoplasmic movement in characean green algae exerts regulatory influence on plasmalemmal ion channels and photosynthetic activity of chloroplasts. In internodal cells of Chara corallina Klein ex Willd., the photoinduced signal transmitted with the flow of streaming cytoplasm for a distance of 1–3 mm from the site of its emergence was found to release or enhance non-photochemical quenching of chlorophyll fluorescence, depending on the intensity of background illumination in the analyzed area. Under dim background irradiance (10–30 μmol quanta/(m²s)), the distant signal transferred from brightly illuminated 0.4-mm-wide area elicited a transient increase in maximal (F′_m) and actual (F) fluorescence. However, at higher background irradiances, the arrival of the same signal resulted in strong quenching of F′_m and in transitory changes of F. The transformation of “low light response” to F′_m changes of opposite polarity occurred at some threshold irradiance. Hence, even slight variations in irradiance at the chloroplast layer, attributed to structural features of characean internodes, might promote formation of uneven photosynthetic profile under the influence of signals transmitted along the cell with the cytoplasmic flow. Analysis of chloroplast fluorescence in situ as a function of pH in experiments with intracellular perfusion indicated that the initial response to a distant light stimulus is caused by the transient increase in cytoplasmic pH in the area of fluorescence measurements.     

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 β-can structure can affect the fluorescence of the chromophore and transiently affect those pathways via conformational changes mediated by manipulating membrane potential.     

Synaptic localization of growth‐associated protein 43 in cultured hippocampal neurons during synaptogenesis

Growth‐associated protein 43 (GAP‐43), a novel axonal phosphoprotein, is originally identified as a growth‐cone‐specific protein of developing neurons in vitro. The expression of GAP‐43 is also shown to be up‐regulated concomitant with increased synaptic plasticity in the brains in vivo, but how GAP‐43 is concerned with synaptic plasticity is not well understood. In the present study, therefore, we aimed to elucidate subcellular localization of GAP‐43 as culture development of rat hippocampal neurons. Western blotting showed that the expression of GAP‐43 in the cerebral and hippocampal tissues was prominently high at postnatal days 14 and 21 or the active period of synaptogenesis. Double‐labelling immunohistochemistry with an axonal marker Tau revealed that the immunoreactivity of GAP‐43 was seen throughout axons of cultured hippocampal neurons but stronger at axonal puncta of developing neurons than axonal processes. Double‐labelling immunohistochemistry with presynaptic terminal markers of synapsin and synaptotagmin revealed that the immunoreactivity of GAP‐43 was observed mostly at weak synapsin‐ and synaptotagmin‐positive puncta rather than strong ones. The quantitative analysis of immunofluorescent intensity showed a clear inverse correlation between GAP‐43 and either synapsin or synaptotagmin expression. These data indicate that GAP‐43 is highly expressed at immature growing axonal terminals and its expression is decreased along with the maturation of synaptogenesis. Copyright © 2012 John Wiley & Sons, Ltd.