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

Abstract

Intracellular pH (pHi) dynamics are critical for regulating normal cell physiology. For example, transient increases in pHi (7.2-7.6) regulate cell behaviors like cell polarization, actin cytoskeleton remodeling, and cell migration. Most studies on pH-dependent cell behaviors have been performed at the population level and use non-specific methods to manipulate pHi. The lack of tools to specifically manipulate pHi at the single-cell level has hindered investigation of the role of pHi dynamics in driving single cell behaviors. In this work, we show that Archaerhodopsin (ArchT), a light-driven outward proton pump, can be used to elicit robust and physiological pHi increases over the minutes timescale. We show that activation of ArchT is repeatable, enabling the maintenance of high pHi in single cells for approximately 45 minutes. We apply this spatiotemporal pHi manipulation tool to determine whether increased pHi is a sufficient driver of membrane ruffling in single cells. Using the ArchT tool, we show that increased pHi in single cells can drive localized membrane ruffling responses within seconds and 2 increased membrane dynamics (both protrusion and retraction events) compared to control cells. Overall, this tool allows us to directly investigate the relationship between increased pHi and cell behaviors such as membrane ruffling. This tool will be transformative in facilitating the experiments required to determine if increased pHi is a driver of these cell behaviors at the single-cell level.

Intracellular pH dynamics regulates intestinal stem cell lineage specification

“…In addition, our studies are consistent with current views about how crypt budding is regulated. Specifically, current views suggest that crypt budding is maintained by a constant pool of ISCs (van Es et al, 2012b; Tan et al, 2021), which is in line with our findings here, or by actin remodeling that drives a change in mechanical forces (Beuvery et al, 1986; Sumigray et al, 2018; Yang et al, 2021), which has been shown to be regulated by pHi in other contexts (Donahue et al, 2021; Frantz et al, 2008; Webb et al, 2016).…”

Structural characterization of proton-pumping rhodopsin lacking a cytoplasmic proton donor residue by X-ray crystallography

“…Their habitats are often alkalized, so that these bacteria use specialized pHhomeostasis systems to avoid intracellular alkalization (50). Although DTD/DTE-type proton pumping rhodopsins are efficient pumps, they continue to transport H + if the cells are illuminated under easily alkalizing conditions (51). In contrast, the H + pumping activity of DTG/DTS rhodopsins strongly depends on the intracellular pH (Fig.…”

Phototriggered Apoptotic Cell Death (PTA) Using the Light-Driven Outward Proton Pump Rhodopsin Archaerhodopsin-3

“…9 In fact, it has been reported that outward proton pump rhodopsins increase the intracellular pH of mammalian cells when exposed to light. 15,16 We heterologously expressed an outward proton pump rhodopsin, Archerhodopsin-3 (AR3 or Arch) 17 or an inward proton pump rhodopsin, Rubricoccus marinas xenorhodopsin (RmXeR) 18 constructs for efficient rhodopsin expression (Figure S2A). 19 The fluorescence signals from EYFP fused to the C-terminus of AR3 or RmXeR were predominantly observed at the periphery of the cells (Figure S2B).…”