An Optogenetic Tool to Raise Intracellular pH in Single Cells and Drive Localized Membrane Dynamics

Area of Science:

• Cell Biology
• Biophysics
• Physiology

Background:

• Intracellular pH (pHi) dynamics regulate crucial cell functions, including polarization, cytoskeleton remodeling, and migration.
• Previous studies on pH-dependent cell behaviors were limited by population-level analysis and non-specific pHi manipulation methods.
• A lack of single-cell resolution tools has impeded understanding of pHi's role in individual cell behaviors.

Purpose of the Study:

• To develop and validate a novel tool for precise, single-cell manipulation of intracellular pH (pHi).
• To investigate whether elevated pHi is sufficient to induce membrane ruffling in single cells.
• To explore the spatiotemporal relationship between pHi dynamics and single-cell behaviors.

Main Methods:

• Utilized Archaerhodopsin (ArchT), a light-driven outward proton pump, for targeted pHi increases in single cells.
• Demonstrated repeatable activation of ArchT for sustained pHi elevation (up to 45 minutes).
• Applied the ArchT tool to observe and quantify membrane ruffling and dynamics in response to controlled pHi changes.

Main Results:

• ArchT effectively induced robust and physiological increases in pHi on a minute timescale.
• Single-cell activation of ArchT rapidly drove localized membrane ruffling within seconds.
• Elevated pHi significantly increased membrane dynamics, including protrusion and retraction events, compared to controls.

Conclusions:

• Archaerhodopsin (ArchT) serves as a powerful tool for spatiotemporal control of single-cell pHi.
• Increased intracellular pH is a sufficient driver for initiating membrane ruffling and altering cell membrane dynamics.
• This technique enables direct investigation of pHi's role in various single-cell behaviors, advancing cell physiology research.