PARIS, an optogenetic method for functionally mapping gap junctions

Abstract

10 Cell-cell communication via gap junctions regulates a wide range of 11 physiological processes by enabling the direct intercellular electrical and 12 chemical coupling. However, the in vivo distribution and function of gap 13 junctions remain poorly understood, partly due to the lack of non-invasive tools 14 with both cell-type specificity and high spatiotemporal resolution. Here we 15 developed PARIS (pairing actuators and receivers to optically isolate gap 16 junctions), a new fully genetically encoded tool for measuring the cell-specific 17 gap junctional coupling (GJC). PARIS successfully enabled monitoring of GJC 18 in several cultured cell lines under physiologically relevant conditions and in 19 distinct genetically defined neurons in Drosophila brain, with ~10-sec temporal 20 resolution and sub-cellular spatial resolution. These results demonstrate that 21 PARIS is a robust, highly sensitive tool for mapping functional gap junctions and 22 study their regulation in both health and disease.23 109 cell (Figure 1 -figure supplement 3). Specifically, the directly connected cell 110 had the strongest response, and the thirdly connected cell had the weakest 111 5 response (Figure 1 -figure supplement 3D). 112We then quantified the ArchT-induced pH change in the actuator cells using the 113 ratiometric pH indicator mTagBFP-pHluorinCAAX generated by fusing the pH-114 insensitive blue fluorescent protein mTagBFP (Subach et al., 2008) to the N-115 terminus of pHluorinCAAX and then calibrating the correlation between pH and 116 the ratio of GFP/BFP fluorescence (Figure 1 -figure supplement 4). Based on 117 a fit to the titration curve, we estimated that a 4-sec and 20-sec laser pulse 118 induces a transient increase of intracellular pH from 7.35 to 7.45 and 7.80 119 respectively in actuator cells (Figure 1 -figure supplement 4D-F), which allowed 120 us to repeatedly elicit a PARIS signal in specific cells as shown above. Together, 121these data provide proof-of-principle that PARIS is a robust tool for measuring 122 GJC between connected cells. 123 Electrophysiological validation of PARIS and its comparison with FRAP124 in HEK293T cells. 125 We have showed that PARIS could detect GJC in a photostimulation dependent 126 way and sensitive to CBX (Figure 2A, D1 & Figure 1). Next, we further validated 127 PARIS by patch-clamping the receiver cell in order to record the gap junction-128 mediated current induced by activating the actuator cell using a laser pulse 129 ( Figure 2B1). Applying increasingly stronger light pulses to the actuator cell 130 yielded time-locked currents in the receiver cell that were blocked by CBX 131 ( Figure 2B2,D2). In the same cells, voltage steps on the actuator cell also 132 elicited non-rectifying and CBX sensitive currents in the receiver cell ( Figure 133 2C,D3). Quantification of the group data showed that the CBX inhibition of GJC 134 was independent from the approaches used to activate the actuator cell (by 135 light or voltage) and from the signals measured in the receiver c...

An optogenetic tool to raise intracellular pH in single cells and drive localized membrane dynamics

“…In our first validation experiment, we tested whether we could achieve spatiotemporal pHi increases in single human retinal epithelial (RPE) cells expressing ArchT (Figure 1A). We observed good membrane localization of ArchT fused to blue fluorescent protein (BFP2) with an ER export signal (ArchT-BFP2-TSERex) 25 (Figure S1A), and co-transfected a genetically encoded pH biosensor (mCherry-pHluorin) 27 to monitor pHi changes in real time. Using a digital micromirror device (DMD), we can spatially restrict 561 nm photoactivation of ArchT to just a single cell (Figure 1A).…”

“…24 Second, ArchT was recently used as a high-resolution spatiotemporal tool to generate proton fluxes to measure gap junction connectivity in mammalian cells and the developing Drosophila brain. 25 Based on these foundational studies, we hypothesized that ArchT could be adapted to reversibly and robustly manipulate intracellular pHi in mammalian cells on the minutes timescale.…”

Neuroscience: The Hidden Diversity of Electrical Synapses

“…Which proteins are associated with electrical synapses in invertebrates and how is the cell-specific expression of these innexins regulated to create synaptic diversity? The description of the electrical connectome in C. elegans provides a preview of future studies of the functional diversity of electrical synapses and its underlying molecular determinants, which will be facilitated by the unparalleled amenability of this organism to genetic and cellular analysis, in combination with recently available functional approaches [16].…”

Approaches to Study Gap Junctional Coupling

“…There are many different techniques available to study gap junctions. These include but are not limited to whole-cell patch-clamp (paired recordings, analysis of isopotentiality, tracer injection), genetic approaches (FRAP, PARIS, StarTrack, transgenic mice), imaging of ion-sensitive dyes (e.g., SBFI), and expression studies (immunohistochemistry, western blotting) ( Abbaci et al, 2008 ; Giaume and Theis, 2010 ; Bedner et al, 2012 ; Langer et al, 2012 ; Griemsmann et al, 2015 ; Droguerre et al, 2019 ; Eitelmann et al, 2019 ; Gutierrez et al, 2019 ; Wu et al, 2019 ; Du et al, 2020 ; McCutcheon et al, 2020 ). Here, we focus on approaches to analyze gap junctional communication that can be implemented easily in most electrophysiology and imaging laboratories.…”