Superecliptic pHluorin Research Articles

Measuring Calcium-Sensing Receptor Agonist-Driven Insertional Signaling (ADIS) and Trafficking by TIRF Microscopy.

The calcium-sensing receptor (CaSR), which regulates parathyroid hormone secretion by sensing serum calcium concentrations, has developed unique trafficking mechanisms to respond to constant exposure to its orthosteric ligand calcium. CaSR rapidly responds to fluctuations in serum calcium by driving forward trafficking of the receptor to cell surfaces in a mechanism known as agonist-driven insertional signaling (ADIS). This increase in CaSR at cell surfaces is counterbalanced by both constitutive and agonist-driven internalization of the receptor. Deciphering these mechanisms is important to understand how mutations in the CaSR and components of its signaling and trafficking pathways cause human disorders of calcium homeostasis.This chapter describes a protocol to measure CaSR ADIS and endocytosis in parallel using total internal reflection fluorescence (TIRF) microscopy. This utilizes a mammalian expression construct comprising the full-length human CaSR with an N-terminal bungarotoxin minimal-binding site that can be labeled with commercially available fluorescent ligands to measure endocytosis, and a super-ecliptic pHluorin (SEP) to measure total cell surface expression and exocytic events. This protocol could easily be adapted to simultaneously assess forward trafficking and endocytosis of other membrane proteins by TIRF microscopy.

Fluorescence lifetime imaging of AMPA receptor endocytosis in living neurons: effects of Aβ and PP1.

The relative amount of AMPA receptors expressed at the surface of neurons can be measured using superecliptic pHluorin (SEP) labeling at their N-terminus. However, the high signal variability resulting from protein overexpression in neurons and the low signal observed in intracellular vesicles make quantitative characterization of receptor trafficking difficult. Here, we establish a real-time live-cell assay of AMPAR trafficking based on fluorescence lifetime imaging (FLIM), which allows for simultaneous visualization of both surface and intracellular receptors. Using this assay, we found that elevating amyloid-beta (Aβ) levels leads to a strong increase in intracellular GluA1 and GluA2-containing receptors, indicating that Aβ triggers the endocytosis of these AMPARs. In APP/PS1 Alzheimer's disease model mouse neurons, FLIM revealed strikingly different AMPAR trafficking properties for GluA1- and GluA3-containing receptors, suggesting that chronic Aβ exposure triggered the loss of both surface and intracellular GluA3-containing receptors. Interestingly, overexpression of protein phosphatase 1 (PP1) also resulted in GluA1 endocytosis as well as depressed synaptic transmission, confirming the important role of phosphorylation in regulating AMPAR trafficking. This new approach allows for the quantitative measurement of extracellular pH, small changes in receptor trafficking, as well as simultaneous measurement of surface and internalized AMPARs in living neurons, and could therefore be applied to several different studies in the future.

The crystal structure of super ecliptic pHluorin reveals a novel pathway for pH to influence fluorescence

The crystal structure of super ecliptic pHluorin reveals a novel pathway for pH to influence fluorescence

Rational Engineering of an Improved Genetically Encoded pH Sensor Based on Superecliptic pHluorin.

Genetically encoded pH sensors based on fluorescent proteins are valuable tools for the imaging of cellular events that are associated with pH changes, such as exocytosis and endocytosis. Superecliptic pHluorin (SEP) is a pH-sensitive green fluorescent protein (GFP) variant widely used for such applications. Here, we report the rational design, development, structure, and applications of Lime, an improved SEP variant with higher fluorescence brightness and greater pH sensitivity. The X-ray crystal structure of Lime supports the mechanistic rationale that guided the introduction of beneficial mutations. Lime provides substantial improvements relative to SEP for imaging of endocytosis and exocytosis. Furthermore, Lime and its variants are advantageous for a broader range of applications including the detection of synaptic release and neuronal voltage changes.

Monitoring cell membrane recycling dynamics of proteins using whole-cell fluorescence recovery after photobleaching of pH-sensitive genetic tags.

Population behavior of signaling molecules on the cell surface is key to their adaptive function. Live imaging of proteins tagged with fluorescent molecules has been an essential tool in understanding this behavior. Typically, genetic or chemical tags are used to target molecules present throughout the cell, whereas antibody-based tags label the externally exposed molecular domains only. Both approaches could potentially overlook the intricate process of in-out membrane recycling in which target molecules appear or disappear on the cell surface. This limitation is overcome by using a pH-sensitive fluorescent tag, such as Super-Ecliptic pHluorin (SEP), because its emission depends on whether it resides inside or outside the cell. Here we focus on the main glial glutamate transporter GLT1 and describe a genetic design that equips GLT1 molecules with SEP without interfering with the transporter's main function. Expressing GLT1-SEP in astroglia in cultures or in hippocampal slices enables monitoring of the real-time dynamics of the cell-surface and cytosolic fractions of the transporter in living cells. Whole-cell fluorescence recovery after photobleaching and quantitative image-kinetic analysis of the resulting time-lapse images enables assessment of the rate of GLT1-SEP recycling on the cell surface, a fundamental trafficking parameter unattainable previously. The present protocol takes 15-20 d to set up cell preparations, and 2-3 d to carry out live cell experiments and data analyses. The protocol can be adapted to study different membrane molecules of interest, particularly those proteins whose lifetime on the cell surface is critical to their adaptive function.

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.

Toxoplasma gondii excretion of glycolytic products is associated with acidification of the parasitophorous vacuole during parasite egress.

The Toxoplasma gondii lytic cycle is a repetition of host cell invasion, replication, egress, and re-invasion into the next host cell. While the molecular players involved in egress have been studied in greater detail in recent years, the signals and pathways for triggering egress from the host cell have not been fully elucidated. A perforin-like protein, PLP1, has been shown to be necessary for permeabilizing the parasitophorous vacuole (PV) membrane or exit from the host cell. In vitro studies indicated that PLP1 is most active in acidic conditions, and indirect evidence using superecliptic pHluorin indicated that the PV pH drops prior to parasite egress. Using ratiometric pHluorin, a GFP variant that responds to changes in pH with changes in its bimodal excitation spectrum peaks, allowed us to directly measure the pH in the PV prior to and during egress by live-imaging microscopy. A statistically significant change was observed in PV pH during ionomycin or zaprinast induced egress in both wild-type RH and Δplp1 vacuoles compared to DMSO-treated vacuoles. Interestingly, if parasites are chemically paralyzed, a pH drop is still observed in RH but not in Δplp1 tachyzoites. This indicates that the pH drop is dependent on the presence of PLP1 or motility. Efforts to determine transporters, exchangers, or pumps that could contribute to the drop in PV pH identified two formate-nitrite transporters (FNTs). Auxin induced conditional knockdown and knockouts of FNT1 and FNT2 reduced the levels of lactate and pyruvate released by the parasites and lead to an abatement of vacuolar acidification. While additional transporters and molecules are undoubtedly involved, we provide evidence of a definitive reduction in vacuolar pH associated with induced and natural egress and characterize two transporters that contribute to the acidification.

Follicular lymphoma-associated mutations in the V-ATPase chaperone VMA21 activate autophagy creating a targetable dependency

The discovery of recurrent mutations in subunits and regulators of the vacuolar-type H+-translocating ATPase (V-ATPase) in follicular lymphoma (FL) highlights a role for macroautophagy/autophagy, amino-acid, and nutrient-sensing pathways in the pathogenesis of this disease. Here, we report on novel mutations in the ER-resident chaperone VMA21, which is involved in V-ATPase assembly in 12% of FL. Mutations in a novel VMA21 hotspot (p.93X) result in the removal of a C-terminal non-canonical ER retrieval signal thus causing VMA21 mislocalization to lysosomes. The resulting impairment in V-ATPase function prevents full lysosomal acidification and function, including impaired pH-dependent protein degradation as shown via lysosomal metabolomics and ultimately causes a degree of amino acid depletion in the cytoplasm. These deficiencies result in compensatory autophagy activation, as measured using multiple complementary assays in human and yeast cells. Of translational significance, the compensatory activation of autophagy creates a dependency for survival for VMA21-mutated primary human FL as shown using inhibitors to ULK1, the proximal autophagy-regulating kinase. Using high-throughput microscopy-based screening assays for autophagy-inhibiting compounds, we identify multiple clinical grade cyclin-dependent kinase inhibitors as promising drugs and thus provide new rationale for innovative clinical trials in FL harboring aberrant V-ATPase.

Auxiliary Subunits Regulate the Dendritic Turnover of AMPA Receptors in Mouse Hippocampal Neurons.

Different families of auxiliary subunits regulate the function and trafficking of native α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in the central nervous system. While a facilitatory role of auxiliary subunits in ER export and forward trafficking of newly synthesized AMPA receptors is firmly established, it is unclear whether auxiliary subunits also control endosomal receptor turnover in dendrites. Here, we manipulated the composition of AMPA receptor complexes in cultured hippocampal neurons by overexpression of two auxiliary subunits, transmembrane AMPAR regulatory protein (TARP) γ-8 or cysteine knot AMPAR-modulating protein (CKAMP) 44a, and monitored dendritic receptor cycling in live-cell imaging experiments. Receptor surface delivery was assayed using a modified AMPA receptor subunit carrying the pH-dependent fluorophore superecliptic pHluorin (SEP-GluA1), which regains its fluorescence during receptor exocytosis, when transiting from the acidic lumen of transport organelles to the neutral extracellular medium. Strikingly, we observed a dramatic reduction in the spontaneous fusion rate of AMPA receptor-containing organelles in neurons overexpressing either type of auxiliary subunit. An analysis of intracellular receptor distribution also revealed a decreased receptor pool in dendritic recycling endosomes, suggesting that incorporation of TARPγ-8 or CKAMP44a in receptor complexes generally diminishes cycling through the endosomal compartment. To directly analyze dendritic receptor turnover, we also generated a new reporter by N-terminal fusion of a self-labeling HaloTag to an AMPA receptor subunit (HaloTag-GluA1), which allows for selective, irreversible staining of surface receptors. Pulse chase-experiments with HaloTag-GluA1 indeed demonstrated that overexpression of TARPγ-8 or CKAMP44a reduces the constitutive internalization rate of surface receptors at extrasynaptic but not synaptic sites. Thus, our data point to a yet unrecognized regulatory function of TARPγ-8 and CKAMP44a, by which these structurally unrelated auxiliary subunits delay local recycling and increase surface lifetime of extrasynaptic AMPA receptors.

An optimized CRISPR/Cas9 approach for precise genome editing in neurons.

The efficient knock-in of large DNA fragments to label endogenous proteins remains especially challenging in non-dividing cells such as neurons. We developed Targeted Knock-In with Two (TKIT) guides as a novel CRISPR/Cas9 based approach for efficient, and precise, genomic knock-in. Through targeting non-coding regions TKIT is resistant to INDEL mutations. We demonstrate TKIT labeling of endogenous synaptic proteins with various tags, with efficiencies up to 42% in mouse primary cultured neurons. Utilizing in utero electroporation or viral injections in mice TKIT can label AMPAR subunits with Super Ecliptic pHluorin, enabling visualization of endogenous AMPARs in vivo using two-photon microscopy. We further use TKIT to assess the mobility of endogenous AMPARs using fluorescence recovery after photobleaching. Finally, we show that TKIT can be used to tag AMPARs in rat neurons, demonstrating precise genome editing in another model organism and highlighting the broad potential of TKIT as a method to visualize endogenous proteins.

PHmScarlet is a pH-sensitive red fluorescent protein to monitor exocytosis docking and fusion steps

pH-sensitive fluorescent proteins (FPs) are highly advantageous for the non-invasive monitoring of exocytosis events. Superecliptic pHluorin (SEP), a green pH-sensitive FP, has been widely used for imaging single-vesicle exocytosis. However, the docking step cannot be visualized using this FP, since the fluorescence signal inside vesicles is too low to be observed during docking process. Among the available red pH-sensitive FPs, none is comparable to SEP for practical applications due to unoptimized pH-sensitivity and fluorescence brightness or severe photochromic behavior. In this study, we engineer a bright and photostable red pH-sensitive FP, named pHmScarlet, which compared to other red FPs has higher pH sensitivity and enables the simultaneous detection of vesicle docking and fusion. pHmScarlet can also be combined with SEP for dual-color imaging of two individual secretory events. Furthermore, although the emission wavelength of pHmScarlet is red-shifted compared to that of SEP, its spatial resolution is high enough to show the ring structure of vesicle fusion pores using Hessian structured illumination microscopy (Hessian-SIM).

A new tool to sense pH changes at the neuromuscular junction synaptic cleft

Synaptic transmission triggers transient acidification of the synaptic cleft. Recently, it has been shown that pH affects the opening of postsynaptic channels and therefore the production of tools that allow to study these behaviors should result of paramount value. We fused α-bungarotoxin, a neurotoxin derived from the snake Bungarus multicinctus that binds irreversibly to the acetylcholine receptor extracellular domain, to the pH sensitive GFP Super Ecliptic pHluorin, and efficiently expressed it in Pichia pastoris. This sensor allows synaptic changes in pH to be measured without the need of incorporating transgenes into animal cells. Here, we show that incubation of the mouse levator auris muscle with a solution containing this recombinant protein is enough to fluorescently label the endplate post synaptic membrane. Furthermore, we could physiologically alter and measure post synaptic pH by evaluating changes in the fluorescent signal of pHluorin molecules bound to acetylcholine receptors. In fact, using this tool we were able to detect a drop in 0.01 to 0.05 pH units in the vicinity of the acetylcholine receptors following vesicle exocytosis triggered by nerve electrical stimulation. Further experiments will allow to learn the precise changes in pH during and after synaptic activation.

Inhibitors of synaptic vesicle exocytosis reduce surface expression of postsynaptic glutamate receptors

ABSTRACT Bafilomycin A1, a vacuolar H+-ATPase inhibitor, and botulinum toxin B and tetanus toxin, both vesicle fusion inhibitors, are widely known exocytosis blockers that have been used to inhibit the presynaptic release of neurotransmitters. However, protein trafficking mechanisms, such as the insertion of postsynaptic receptors and astrocytic glutamate-releasing channels into the plasma membrane, also require exocytosis. In our previous study, exocytosis inhibitors reduced the surface expression of astrocytic glutamate-releasing channels. Here, we further investigated whether exocytosis inhibitors influence the surface expression of postsynaptic receptors. Using pH-sensitive superecliptic pHluorin (SEP)-tagged postsynaptic glutamate receptors, including GluA1, GluA2, GluN1, and GluN2A, we found that bafilomycin A1, botulinum toxin B, and/or tetanus toxin reduce the SEP fluorescence of SEP-GluA1, SEP-GluA2, SEP-GluN1, and SEP-GluN2A. These findings indicate that presynaptic vesicle exocytosis inhibitors also affect the postsynaptic trafficking machinery for surface expression. Finally, this study provides profound insights assembling presynaptic, postsynaptic and astrocytic viewpoints into the interpretation of the data obtained using these synaptic vesicle exocytosis inhibitors.

Imaging endocytic vesicle formation at high spatial and temporal resolutions with the pulsed-pH protocol.

Endocytosis is a fundamental process occurring in all eukaryotic cells. Live cell imaging of endocytosis has helped to decipher many of its mechanisms and regulations. With the pulsed-pH (ppH) protocol, one can detect the formation of individual endocytic vesicles (EVs) with an unmatched temporal resolution of 2 s. The ppH protocol makes use of cargo protein (e.g., the transferrin receptor) coupled to a pH-sensitive fluorescent protein, such as superecliptic pHluorin (SEP), which is brightly fluorescent at pH 7.4 but not fluorescent at pH <6.0. If the SEP moiety is at the surface, its fluorescence will decrease when cells are exposed to a low pH (5.5) buffer. If the SEP moiety has been internalized, SEP will remain fluorescent even during application of the low pH buffer. Fast perfusion enables the complete exchange of low and high pH extracellular solutions every 2 s, defining the temporal resolution of the technique. Unlike other imaging-based endocytosis assays, the ppH protocol detects EVs without a priori hypotheses on the dynamics of vesicle formation. Here, we explain how the ppH protocol quantifies the endocytic activity of living cells and the recruitment of associated proteins in real time. We provide a step-by-step procedure for expression of the reporter proteins with transient transfection, live cell image acquisition with synchronized pH changes and automated analysis. The whole protocol can be performed in 2 d to provide quantitative information on the endocytic process being studied.

PHLuc, a Ratiometric Luminescent Reporter for in vivo Monitoring of Tumor Acidosis.

Even under normoxia, cancer cells exhibit increased glucose uptake and glycolysis, an occurrence known as the Warburg effect. This altered metabolism results in increased lactic acid production, leading to extracellular acidosis and contributing to metastasis and chemoresistance. Current pH imaging methods are invasive, costly, or require long acquisition times, and may not be suitable for high-throughput pre-clinical small animal studies. Here, we present a ratiometric pH-sensitive bioluminescence reporter called pHLuc for in vivo monitoring of tumor acidosis. pHLuc consists of a pH-sensitive GFP (superecliptic pHluorin or SEP), a pH-stable OFP (Antares), and Nanoluc luciferase. The resulting reporter produces a pH-responsive green 510nm emission (from SEP) and a pH-insensitive red-orange 580nm emission (from Antares). The ratiometric readout (R580/510) is indicative of changes in extracellular pH (pHe). In vivo proof-of-concept experiments with NSG mice model bearing human synovial sarcoma SW982 xenografts that stably express the pHLuc reporter suggest that the level of acidosis varies across the tumor. Altogether, we demonstrate the diagnostic value of pHLuc as a bioluminescent reporter for pH variations across the tumor microenvironment. The pHLuc reporter plasmids constructed in this work are available from Addgene.

TpHusion: An efficient tool for clonal pH determination in Drosophila.

Genetically encoded pH indicators (GEpHI) have emerged as important tools for investigating intracellular pH (pHi) dynamics in Drosophila. However, most of the indicators are based on the Gal4/UAS binary expression system. Here, we report the generation of a ubiquitously-expressed GEpHI. The fusion protein of super ecliptic pHluorin and FusionRed was cloned under the tubulin promoter (tpHusion) to drive it independently of the Gal4/UAS system. The function of tpHusion was validated in various tissues from different developmental stages of Drosophila. Differences in pHi were also indicated correctly in fixed tissues. Finally, we describe the use of tpHusion for comparative analysis of pHi in manipulated clones and the surrounding cells in epithelial tissues. Our findings establish tpHusion as a robust tool for studying pHi in Drosophila.

External Charges Influence Fluorescent Protein Proton Wires

Genetically encoded voltage indicators (GEVIs) based on the fluorescent protein Superecliptic pHluorin have been shown to alter fluorescent output in a voltage-dependent manner. The dimer affinity of the fluorescent proteins (FP) and specific charged residues in the dimer interface are involved in the mechanism of how the fluorescent output changes have been implicated in the optical signal. Here we show that the critical negative charge on the exterior of the β-can structure of the FP (A227D) can affect proton wires that ultimately affect the chromophore of the FP. When the negative charge is moved along the exterior of the β-barrel, the polarity of the voltage-dependent optical signal inverts. One hypothesis to explain this behavior is that the transient movement of the negative charge shifts the equilibrium of the protonation state of the chromophore. To test this hypothesis, alternating the excitation wavelength from 390 nm to 470 nm was performed revealing that the kinetics of the optical signal, voltage range of the optical signal, and the optimal linker length were wavelength dependent. These results suggest that the proton wires capable of responding to the external negative charge of a neighboring probe are different for the protonated chromophore and the anionic chromophore. To further investigate the effect on proton wires, several E222 mutations were tested which has been implicated as the proton release point for excited state proton transfer of GFP. The results imply that ArcLight-derived GEVIs can be used to alter and thereby study proton wires of fluorescent proteins. Combined with structural data, these GEVIs may enable the mapping of proton wires, which would enable the development of voltage sensors with large dynamic range.

Visualization of Exo- and Endocytosis of AMPA Receptors During Hippocampal Synaptic Plasticity Around Postsynaptic-Like Membrane Formed on Glass Surface.

Regulation of exo- and endocytosis of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptor (AMPAR) plays a critical role in the expression of synaptic plasticity such as long-term potentiation (LTP) and long-term depression (LTD) at excitatory central synapses. Enhanced AMPAR exocytosis or endocytosis has been suggested to contribute to LTP or LTD, respectively. However, several unsettled fundamental questions have remained about AMPAR exo- and endocytosis in the basal condition and during synaptic plasticity: (1) Does the size of each exo- or endocytosis event, and/or do the frequencies of these events change during LTP or LTD? If they change, what are the time courses of the respective changes? (2) Where does the exo- or endocytosis preferentially occur in each condition: inside or in the vicinity of postsynaptic membrane, or in the extrasynaptic membrane? (3) Do different types of AMPAR, such as GluA1 homo-tetramer, GluA1/2 hetero-tetramer and GluA2/3 hetero-tetramer, show distinct exo- and endocytosis changes? To address these questions, we developed new methods to observe individual events of AMPAR exo- or endocytosis with a high signal to noise (SN) ratio in a culture preparation using total internal reflection fluorescence microscopy (TIRFM). In these studies, hippocampal neurons were cultured on a neurexin (NRX)-coated glass coverslip, which induced formation of postsynaptic-like membrane (PSLM) directly on the glass surface. Then, a super-ecliptic pHluorin (SEP)-tagged AMPAR subunit such as GluA1 (GluA1-SEP) was expressed in neurons and its fluorescence changes during LTP induced by high frequency electrical field stimulation were observed with TIRFM, which showed different time courses of exocytosis changes of GluA1-, GluA2-, or GluA3-SEP in and around PSLM. In addition, a new method to detect individual endocytosis events of AMPAR was developed by combining TIFRM observation of GluA-SEP around PSLM with a rapid extracellular pH exchange method using a U-tube. Recent results on exo- and endocytosis changes of GluA-SEP during N-methyl-D-aspartate (NMDA)-induced LTD suggested that suppression of AMPAR exocytosis rather than enhancement of AMPAR endocytosis primarily contributes to LTD expression, although the NMDA application transiently enhances clathrin-dependent endocytosis of GluA1-containing AMPAR.

Visualization of Synchronous or Asynchronous Release of Single Synaptic Vesicle in Active-Zone-Like Membrane Formed on Neuroligin-Coated Glass Surface.

Fast repetitive synaptic transmission depends on efficient exocytosis and retrieval of synaptic vesicles around a presynaptic active zone. However, the functional organization of an active zone and regulatory mechanisms of exocytosis, endocytosis and reconstruction of release-competent synaptic vesicles have not been fully elucidated. By developing a novel visualization method, we attempted to identify the location of exocytosis of a single synaptic vesicle within an active zone and examined movement of synaptic vesicle protein synaptophysin (Syp) after exocytosis. Using cultured hippocampal neurons, we induced formation of active-zone-like membranes (AZLMs) directly adjacent and parallel to a glass surface coated with neuroligin, and imaged Syp fused to super-ecliptic pHluorin (Syp-SEP) after its translocation to the plasma membrane from a synaptic vesicle using total internal reflection fluorescence microscopy (TIRFM). An AZLM showed characteristic molecular and functional properties of a presynaptic active zone. It contained active zone proteins, cytomatrix at the active zone-associated structural protein (CAST), Bassoon, Piccolo, Munc13 and RIM, and showed an increase in intracellular Ca2+ concentration upon electrical stimulation. In addition, single-pulse stimulation sometimes induced a transient increase of Syp-SEP signal followed by lateral spread in an AZLM, which was considered to reflect an exocytosis event of a single synaptic vesicle. The diffusion coefficient of Syp-SEP on the presynaptic plasma membrane after the membrane fusion was estimated to be 0.17–0.19 μm2/s, suggesting that Syp-SEP diffused without significant obstruction. Synchronous exocytosis just after the electrical stimulation tended to occur at multiple restricted sites within an AZLM, whereas locations of asynchronous release occurring later after the stimulation tended to be more scattered.

Observing the Movement of Heterogeneous Voltage Sensing Domains via Intermolecular FRET

Fusion of a voltage sensitive domain (VSD) to the fluorescent protein (FP), Super Ecliptic pHluorin A227D, allows for the optical measurement of changes in membrane potential. However, the mechanism coupling fluorescence changes to changes in membrane potential is not fully understood. Primary studies of this mechanism suggest that dimerization via the cytoplasmic fluorescent protein domain is required. To further explore the dimerization effect of the genetically encoded voltage indicator (GEVI) via intermolecular Förster Resonance Energy Transfer (FRET), novel constructs fusing FRET donors or FRET acceptors to the VSD were constructed and co-transfected into HEK-293 cells. CFP-YFP FRET pairs showed a higher resting FRET than GFP-RFP FRET pairs presumably due to the dimerization of the CFP-YFP pairs. Robust voltage-dependent FRET signals were observed which suggested the movement of S4 transmembrane segment upon depolarization of the plasma membrane improved the FRET efficiency between the chromophore dipoles of VSDs tuned to different potentials. For instance, a FRET donor attached to a VSD tuned to extreme positive potentials can be paired with a FRET acceptor coupled to a VSD that responds to physiologically relevant potentials. This combination results in a GEVI that will only move one S4 transmembrane segment or both S4 transmembrane segments depending on the strength of the depolarization of the plasma membrane. Research reported in this publication was supported by the National Institute Of Neurological Disorders And Stroke of the National Institutes of Health under Award Number U01NS099691.