Engineering Photoactivatability in Genetically Encoded Voltage and pH Indicators.

Sungmoo Lee,Yoon-Kyu Song,Bradley J Baker

doi:10.3389/fncel.2019.00482

Abstract

Genetically-encoded indicators of neuronal activity enable the labeling of a genetically defined population of neurons to optically monitor their activities. However, researchers often find difficulties in identifying relevant signals from excessive background fluorescence. A photoactivatable version of a genetically encoded calcium indicator, sPA-GCaMP6f is a good example of circumventing such an obstacle by limiting the fluorescence to a region of interest defined by the user. Here, we apply this strategy to genetically encoded voltage (GEVI) and pH (GEPI) indicators. Three photoactivatable GEVI candidates were considered. The first one used a circularly-permuted fluorescent protein, the second design involved a Förster resonance energy transfer (FRET) pair, and the third approach employed a pH-sensitive variant of GFP, ecliptic pHluorin. The candidate with a variant of ecliptic pHluorin exhibited photoactivation and a voltage-dependent fluorescence change. This effort also yielded a pH-sensitive photoactivatable GFP that varies its brightness in response to intracellular pH changes.

Highlights

Encoded fluorescent sensors often suffer from extensive background fluorescence (Lin and Schnitzer, 2016; Bayguinov et al, 2017; Song et al, 2017; Nakajima and Baker, 2018)
The L64F and T65S mutations recovered the 390 nm absorbance peak that originally existed in wild-type GFP
Two photoactivatable voltage indicator candidates were designed based on the rationale described above (Figures 1A,C)

Summary

Introduction

Encoded fluorescent sensors often suffer from extensive background fluorescence (Lin and Schnitzer, 2016; Bayguinov et al, 2017; Song et al, 2017; Nakajima and Baker, 2018). Using a destabilized Cre recombinase was reported to induce sparse labeling in cortical layers 2/3 (Sando et al, 2013; Harris et al, 2014; Madisen et al, 2015). Utilizing this method to achieve a Förster resonance energy transfer (FRET) type genetically encoded voltage (GEVI) expression involved the generation of triple transgenic mice to acquire the desired level of sparseness in cortical pyramidal cells (Song et al, 2017; Quicke et al, 2019). Sparse labeling is a stochastic approach that is difficult to control. Approaches that empower the experimenter to define the circuits that optically respond would be a welcome addition to the imaging toolbox

Methods

Results

Conclusion