A red fluorescent protein with improved monomericity enables ratiometric voltage imaging with ASAP3

Abstract

A ratiometric genetically encoded voltage indicator (GEVI) would be desirable for tracking transmembrane voltage changes in the presence of sample motion. We performed combinatorial multi-site mutagenesis on a cyan-excitable red fluorescent protein to create the bright and monomeric mCyRFP3, which proved to be uniquely non-perturbing when fused to the GEVI ASAP3. The green/red ratio from ASAP3-mCyRFP3 (ASAP3-R3) reported voltage while correcting for motion artifacts, allowing the visualization of membrane voltage changes in contracting cardiomyocytes and throughout the cell cycle of motile cells.

Figure 1

Engineering and characterization of mCyRFP3. Engineering of mCyRFP3. (a) Crystal structure of dimeric predecessor CyOFP1 displaying interacting residues Y148 and A161. Image was generated in Schrödinger MacPyMol 1.8. (b) Size-exclusion chromatography of EGFP, tdTomato, CyRFP1, and mCyRFP3. EGFP was used as a monomeric standard, while tdTomato was used as a dimeric standard. (c) Excitation and emission spectra of mCyRFP3 compared to its parent mCyRFP1. (d) pH dependence of mCyRFP3 fluorescence, demonstrating a pKa of 4.1. Error bars are s.em.m of triplicate measurements. (e) Photobleaching kinetics of purified cyan- excitable red fluorescent proteins under illumination by a 120-W metal-halide arc lamp through a 490/20-nm excitation filter. The time-axis was adjusted for each fluorophore to simulate excitation conditions producing 1000 photons per s per molecule. Lighter shading on CyRFP1, mCyRFP1, and mCyRFP3 lines represents standard deviation of five measurements. (f) Maturation kinetics of mCyRFP3 demonstrating a half-life of t = 12.5 min. Graphs in (a) through (f) were generated in Microsoft Excel for Mac 16.

Figure 2

Performance of mCyRFP3 in mammalian cells. (a) HeLa cells expressing mCyRFP3 fused various subcellular proteins. For each fusion, the original of the fusion partner and its normal subcellular location are indicated in parentheses. (i) mCyRFP3-2aa-tubulin (human, microtubules), (ii) mCyRFP3-7aa-actin (human, actin cytoskeleton), (iii) Calnexin-14aa-mCyRFP3 (human, endoplasmic reticulum), (iv) mannosidaseII-10aa-mCyRFP3 (mouse, Golgi complex), (v) mCyRFP3-10aa-lamin B1 (human, nuclear envelope) (vi) PDHA-10aa-mCyRFP3 (human, mitochondrial pyruvate dehydrogenase), (vii) connexin43-7aa-mCyRFP3 (rat, cell–cell adhesion junctions), (viii) paxillin-22aa-mCyRFP3 (chicken, focal adhesions), (ix). mCyRFP3-2aa-CAAX. Scale bar, 10 µm. (b) mCyRFP3-10aa-H2B (human, nucleosomes) in (i) interphase, (ii) prophase, (iii) metaphase, (iv) anaphase. Spinning-disk confocal image stacks were acquired using Improvision Volocity 6.0 and flattened and scaled in NIH Fiji 2.1. (c) Performance of mCyRFP3 compared to mCyRFP1, mEGFP and mCardinal on CytERM monomericity assay. Error bars represent standard deviation of measurements from > 150 cells in each of three separate experiments. (d) mCyRFP3 characterization in mammalian cells. Brightness comparison of mCyRFP3 in HEK293A and HeLa cells expressing a bicistronic construct EGFP-P2A-RFP where RFP = CyRFP1, mCyRFP1, and mCyRFP3. The red fluorescence generated from each of the RFPs were normalized to the fluorescence of EGFP to normalize for expression and were charted relative to the value of CyRFP1. Excitation was performed with a 480/10-nm filter and emission was collected from 580 to 800 nm. Integrated red emission relative to the integrated GFP emission is shown as mean ± s.e.m. of 6–8 biological replicates. Graphing and two-tailed t tests with Bonferroni correction were performed in Microsoft Excel for Mac 16.

Figure 3

Characterization of ASAP3-mCyRFP3 (ASAP3-R3). (a) Representative images of ASAP3 fusions to various RFPs expressed in primary rat hippocampal neurons acquired on a spinning-disk confocal microscope using Improvision Volocity 6.0 and processed in NIH Fiji 2.1. (b) Laser-scanning confocal images of ASAP3-R3 in a rat hippocampal neuron. Images were acquired with Zeiss Zen Blue software and processed in Fiji 2.1. (c) Above, normalized excitation spectra of mCyRFP3 and mEGFP, showing a single excitation wavelength can be used. Below, normalized emission spectra of mCyRFP3 and mEGFP, showing their emissions are separable (d) Steady-state voltage responsivity of ASAP3-R3. Error bars represent standard error of the mean (n = 8 HEK293 cells). Graphs were made in Microsoft Excel for Mac 16.

Figure 4

ASAP3-R3 performance in cardiomyocytes. (a) Representative induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs) expressing ASAP3 or ASAP3-R3. Epifluorescence images were acquired using Hamamatsu HCImage software and color channels assigned in NIH Fiji 2.1. (b) Representative single-trial ∆R/R or ∆F/F responses to spontaneous cardiac APs of cells expressing ASAP3, expressing ASAP3-R3, or expressing ASAP3-R3 and treated with isoproterenol (ISO). Graphs were generated in Microsoft Excel for Mac 16. (c) Characterization of traces obtained from ASAP3 (n = 6), ASAP3-R3 (n = 10), or ASAP3-R3 + ISO (n = 7). Graphing and statistical analyses were performed in GraphPad Prism 7.

Figure 5

ASAP3-R3 accurately reports voltage in motile cells and detects voltage fluctuations during the cell cycle. (a) Top, one segment of membrane in a beating CM was analyzed. Middle, raw traces of green and red signals in the selected region. Red trace indicates significant cell movement in and out of selected region detected by the voltage-independent mCyRFP. Bottom, relative change in green fluorescence alone (− ∆F/F) and the relative change in the green/red ratio (− ∆R/R). While green intensity changes are larger than those observed previously in non-contracting CMs due to these motion artifacts, ratio changes are similar in magnitude to those observed previously. (b) Similar analysis on a different membrane segment of the same cell. Here, the green fluorescence changes are opposite in direction from that expected due to movement, which is detected in the red channel. While green intensity traces do not resemble AP waveforms, ratio traces are indistinguishable from AP waveforms in non-motile CMs and similar in shape to those in (a). Epifluorescence images were acquired using Hamamatsu HCImage software and color channels assigned and overlaid in NIH Fiji 2.1. Graphs were generated in Microsoft Excel for Mac 16. (c) Top, images of the ASAP3-R3 green/red ratio at different time points relative to cytokinesis in a HeLa cell. Middle, individual green/red ratio time courses of three HeLa cells during cell division. The cell in the images is represented by the blue trace. Cytokinesis occurs immediately before the 0-h time point. Below, mean green/red ratios of the three cells. Error bars represent standard deviation. Epifluorescence images were acquired using Hamamatsu HCImage software and pseudocoloring with a ratiometric lookup table was performed in NIH Fiji 2.1. Graphs were generated in Microsoft Excel for Mac 16.