RESUMO
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 nonspecific 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 time scale. We show that activation of ArchT is repeatable, enabling the maintenance of high pHi in single cells for up to 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 increased membrane dynamics (both protrusion and retraction events) compared to unstimulated ArchT cells as well as control cells. Overall, this tool allows us to directly investigate the relationship between increased pHi and single cell behaviors such as membrane ruffling. This tool will be transformative in facilitating experiments that are required to determine roles for increased pHi in driving single cell behaviors.
