Mapping cellular cytosolic pH in vivo under physiological and pathophysiological conditions

Cytosolic pH (pHc) is one of the most essential intracellular microenvironments profoundly affecting charges, conformations and therefore functions of macromolecules. It remains challenging to quantitatively measure pHc at single-cell resolution in vivo, and how pHc of a specific cell type dynamically responds to physiological or pathophysiological fluctuations remains elusive. Smad5 is a pHc sensor with accelerated nuclear export upon pHc alkalization and ideally the nucleocytoplasmic ratio of Smad5 would be a potential indicator of pHc. Here, we revealed that the constitutive eGFP-Smad5 expression transgenic mice could serve as pHc reporter for quantitative detection of cellular pHc in vivo. We mapped pHc of major cell types in the reporter mice and found robust pHc heterogeneity in individual cell types or different cell types adjacently located within a tissue. By acute systemic acidification or alkalization challenges, we also characterized pHc-stable and pHc-sensitive cell types. In pathological microenvironments related to type 2 diabetes mellitus or tumors, pHc showed responsive or adaptive changes in some specific cell types, highlighting pathophysiological roles of pHc in disease onset and progression or plasticity of cells under chronic disease states. Our study establishes a previously unachievable method for mapping of cellular pHc in vivo under both physiological and pathophysiological conditions.