Genetically encoded voltage indicators (GEVIs) are promising tools for directly imaging voltage dynamics and/or spiking activity of genetically defined cell types1–3. However, the strong excitation light, which is usually required for voltage imaging causes severe autofluorescence especially in the tissue sample. Furthermore, it often poses problems such as photobleaching and phototoxicity, the latter being particularly limiting in fragile cells4,5.
The best way to avoid these problems is to perform voltage imaging that doesn’t require excitation light, such as by using bioluminescent proteins. We previously developed the Nanolantern series6, bioluminescent proteins consisting of a Renilla luciferase (RLuc) variant and the yellow fluorescent protein (Venus)7, in which the bioluminescence intensity is enhanced by Förster resonance energy transfer (FRET). Then, we applied the Nano-lantern to develop several indicators for biological elements including Ca2+ and ATP, thereby succeeded Ca2+imaging with the optical stimulation of Channelrhodopsin-2, and ATP imaging during photosynthesis in a plant leaf. In line with this trend, we expanded the application of bioluminescent indicators to voltage imaging.
Material and Methods
Development of a bioluminescent voltage indicator
The design of the bioluminescent GEVI (bGEVI) was inspired with the FRET-based GEVIs such as VSFP BF1.2 and Mermaid28,9. To develop a bright bGEVI, we used NanoLuc which produces approximately 150-times the luminescence of RLuc10 and Venus as a FRET donor and acceptor, respectively. Similar to VSFP BF1.2 and Mermaid2, the working principle of our bGEVI is that a change in FRET is induced by the voltage-dependent movement of the voltage sensitive domain (VSD(R217Q)) of Ci-VSP11 on the plasma membrane (Fig. a). Finally, we designated this bGEVI as LOTUS-V (Luminescent Optical Tool for Universal Sensing of Voltage).
Results and Discussion
Comparison with other fluorescent GEVIs
When we added the bioluminescent substrate, furimazine to the imaging medium, intense bioluminescence from rat pituitary epithelial-like tumor (GH3) cells was observed. (Fig. b). Comparing with VSFP BF1.2 or Mermaid2, the signal change in LOTUS-V upon KCl-induced depolarization was more than four times that seen with them (Fig. c).
Voltage imaging in hiPSC-CMs
To examine the compatibility with excitable cell types that move we chose an in-vitro cardiomyocyte model. Then, we expressed LOTUS-V in cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs) via lentivirus gene expression system. During spontaneous contraction, a reciprocal change in the NanoLuc and Venus intensities was observed. The increase in the emission ratio value suggested a precise reflection of the action potential in cardiomyocytes (Fig. d).
Biouminescence imaging has been generally considered too dim for voltage imaging that requires a fast frame rate acquisition. To overcome this problem, we developed a bright bGEVI using NanoLuc, which is the brightest bioluminescent protein, and demonstrated that it was applicable for voltage imaging. As for in-vitro cardiomyocytes model, LOTUS-V mitigated the effect of motion artifact owing to ratiometric measurement and successfully captured the cardiac action potential. Collectively, bioluminescence imaging has several advantages over fluorescence imaging. LOTUS-V makes voltage imaging applicable to situations that are difficult to monitor with fluorescent GEVIs.
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