Application of an Optogenetic Tool to Increase Intracellular pH and Investigate pH-Dependent Cell Movement

Intracellular pH is tightly regulated (7.0-7.2) in normal cells, with transient changes in pHi dynamics (7.4 – 7.6) being linked to a various normal cell behaviors including cytoskeleton remodeling, changes in cell polarization, and directed cell migration. Studies linking increased pHi to these cell behaviors have been performed at the population level using non-specific pHi manipulation tools that often lack spatiotemporal control. In order to better understand the role of increased pHi on driving cell behaviors we need to develop better pHi manipulation tools. To that end, we developed and characterized Archaerhodopsin (ArchT) as an optogenetic tool to spatiotemporally increase pHi. We showed that we could increase pHi (0.1-0.47 pH units) in single cells with high spatial and temporal control and that this increase in pHi was robust, repeatable, and reversible. Additionally, this pHi increase is similar to physiologically relevant pHi changes (0.1-0.35) seen in single cells. We also showed that increased pHi could be used to drive localized membrane protrusion and membrane dynamics within single cells. Within these membrane dynamics, actin underwent pH-dependent remodeling while the microtubules were unaffected. Therefore, ArchT allows us to ask previously intractable questions regarding the role of increased pHi in driving cell behaviors such as cytoskeleton remodeling, changes in cell polarization, and single-cell migration. This tool has a broad implication in the field and can be applied to study both normal cell biology and disease related biology, allowing for the discovery of molecular mechanisms driving aberrant cell behaviors seen in cancer.