Question: 

Melanoma cell migration requires the formation and release of cell-matrix contacts mediated by integrins. Integrin avidity depends on pH and is increased by an acidic pH at the cell surface. Tumor cells, characterized by an increased net acid production, regulate their intracellular pH mainly via proton export through the Na⁺/H⁺-exchanger NHE1. NHE1 accumulates at the cell front and has been associated with focal adhesions. We hypothesize that NHE1 is the key element responsible for the adjustment of functional pH nanoenvironments in close vicinity to focal adhesions.

Methods: 

Human melanoma cells (MV3) were transfected with DsRed2-paxillin to visualize focal adhesions. Cells were seeded on collagen I. NHE1 expression was confirmed by immunofluorescence staining. At both focal adhesions and DsRed2-free regions, pH was measured intra- and extracellularly using the pH-sensitive, fluorescent dyes BCECF and WGA-fluorescein, respectively. Total Internal Reflection Fluorescence (TIRF) microscopy enabled us to selectively measure at the cell's basal plasma membrane. To elucidate whether possible pH nanoenvironments depend on NHE1, the specific NHE1 inhibitor cariporide or NHE1-deficient cells were used.

Results: 

Colocalizing with paxillin, NHE1 shows its highest expression levels at focal adhesions at the cell front. Accordingly, wild type cells exhibit the highest NHE1 activity, the most alkaline cytosolic and the most acidic cell surface pH values at these regions. Differences are abolished in NHE1-deficient cells.

Conclusions: 

Cells take advantage of the NHE1-driven, pH-regulating proton export at focal adhesions. Here, NHE1 generates pH nanoenvironments in the cytosol and at the cell surface, needed to establish cell-matrix contacts and to modulate their strength.