A general approach to engineer positive-going eFRET voltage indicators

Abstract

Imaging membrane voltage from genetically defined cells offers the unique ability to report spatial and temporal dynamics of electrical signaling at cellular and circuit levels. Here, we present a general approach to engineer electrochromic fluorescence resonance energy transfer (eFRET) genetically encoded voltage indicators (GEVIs) with positive-going fluorescence response to membrane depolarization through rational manipulation of the native proton transport pathway in microbial rhodopsins. We transform the state-of-the-art eFRET GEVI Voltron into Positron, with kinetics and sensitivity equivalent to Voltron but flipped fluorescence signal polarity. We further apply this general approach to GEVIs containing different voltage sensitive rhodopsin domains and various fluorescent dye and fluorescent protein reporters.

Fig. 1. Engineering a positive-going rhodopsin eFRET GEVI.

a Schematic (top) showing the hypothetical path of proton transport (arrows) through the Ace2 rhodopsin proton pump. The proton acceptor (PA), proton donor (PD), and proton release (PR) positions are represented as yellow spheres and labeled, retinal is shown as blue sticks. Photocurrent measurements (bottom) for the Ace2 rhodopsin proton pump. Steady-state photocurrent = 46 ± 11 pA (mean ± SD, N = 4 cells). b Schematic representing the proposed mechanism of negative-going rhodopsin eFRET GEVIs. Spectra in the lower panels have been scaled to illustrate the direction of absorbance and fluorescence change with membrane voltage change. We were not able to quantitatively measure these spectra in cell membranes at different voltages. At resting membrane potential, rhodopsin absorbance is low and therefore fluorophore emission is high. When the membrane depolarizes, rhodopsin absorbance increases leading to a decrease in fluorophore emission. c–f Schematic of amino acid substitutions (top), simultaneous fluorescence imaging (second row), and whole-cell patch-clamp membrane voltage measurements (third row), as well as photocurrent measurements (bottom) from rat hippocampal neurons in culture expressing Voltron, Voltron D92N, Voltron D92N N81D, and Positron labeled with JF₅₂₅. Blue bar denotes time of light illumination ((508 nm–522 nm) at an irradiance of 70 mW mm⁻²) for photocurrent measurements. Steady-state photocurrents for all variants are negligible (−0.7 ± 3 pA, −0.1 ± 1 pA, 0.0 ± 1.5 pA, 0.1 ± 1 pA (mean ± SD, N = 5–7 cells), respectively). Simultaneous voltage and fluorescence traces are representative of N ≥ 3 cells.

Fig. 2. Characterization of positron neuron culture and larval zebrafish.

a Graph of fluorescence vs. membrane voltage for Voltron (N = 6 cells), Voltron D92N N81D (N = 5 cells), and Positron (N = 5 cells) expressed in voltage-clamped rat hippocampal neurons in culture. ΔF/F₀ measurements are mean ± SD. b Box and whisker plot showing the fluorescence of Voltron and Positron at resting membrane potential in neuron culture. Box represents interquartile range, center represents median, and whiskers represent 1st–99th percentile. Outliers shown as dots. N = 633 cells and 686 cells. ***P < 0.001 for two-tailed unpaired t-test. c Comparison of photobleaching of Positron and Voltron. Mean normalized fluorescence from a field of view of labeled, GEVI-expressing neurons is shown ±SEM. N = 19 fields of view each from N = 4 independent neuron cultures each. d Schematic representing the proposed mechanism of positive-going rhodopsin eFRET GEVIs. Spectra in the lower panels have been scaled to illustrate the direction of absorbance and fluorescence change with membrane voltage change. We were not able to quantitatively measure these spectra in cell membranes at different voltages. At resting membrane potential, rhodopsin absorbance is high and therefore fluorophore emission is quenched. When the membrane depolarizes, rhodopsin absorbance decreases leading to an increase in fluorophore emission. e Imaging of voltage signals from five neurons in the forebrain of live larval zebrafish. (top) Imaged field of view showing fluorescence from five individual neurons expressing Positron and labeled with JF₅₂₅. Inset shows an overview of the larval zebrafish brain and the imaged area (blue rectangle). (bottom) Fluorescence of the five visible neurons over time. Scale bar: 20 μm Data are representative of N = 3 fish.

Fig. 3. Generality of the approach to engineer positive-going eFRET GEVIs.

Each panel shows a schematic of the voltage indicator construct (left), a fluorescence image of a neuron in culture expressing this voltage indicator (center), and simultaneous recording of fluorescence (right top) and membrane potential (right bottom) in response to current injection. Fluorescence image scale bar = 20 μm. Simultaneous voltage and fluorescence traces are representative of N ≥ 3 cells. a Positron labeled with two different colors of fluorescent dye, JF₅₂₅ (top) and JF₅₈₅ (bottom). b Ace2 rhodopsin bearing the signal-inverting mutations described and fused to the different color FP domains mNeonGreen (top) and mRuby3 (bottom). c Different rhodopsin domains (Mac, top and Ace1, bottom) bearing mutations analogous to the described signal-inverting mutations described, fused to HaloTag, and labeled with JF₅₂₅.