Extracellular pH (pHe) and activity of the Na+ /H+ exchanger (NHE1) are modulators of tumor cell migration. Yet, pHe at the outer surface of the cell membrane (pHem ), i.e. the pH nanoenvironment where cell/matrix interaction occurs and matrix metalloproteinases work has never been measured. Here, we present a method to determine this pH nanoenvironment at the single cell level by using proton-sensitive dyes to label the outer leaflet of the plasma membrane and the glycocalyx of human melanoma cells. We show that polarized cells generate an extracellular proton gradient at their surface that increases from the rear end to the leading edge of the lamellipodium along the direction of movement ([Delta]pH[cong]0.2). The presence of this proton gradient and the ability of the cells to migrate depend on NHE1 activity. NHE1 inhibition by cariporide (HOE642) causes the gradient to collapse and the cells to cease from migrating. NHE1 stimulation by intracellular acidification with propionic acid leads to an increase in the proton gradient. Acidified cells form numerous, thin lamellipodia which are very adhesive and frequently shed during cell migration. The shedding indicates that the lamellipodia are strongly attached to the extracellular matrix.

We conclude that a regulated NHE1 activity creates a pH nanoenvironment that promotes migration by facilitating cell adhesion at the cell front and the release of cell/matrix contacts at the cells' rear part.