pH dependence of melanoma cell migration: protons extruded by NHE1 dominate protons of the bulk solution

Abstract

Migration and morphology of human melanoma cells (MV3) depend on extracellular pH (pHe) and the activity of the Na+/H+ exchanger NHE1. To distinguish effects of NHE1 activity per se from effects of pHe we compared an NHE1-deficient mutant with rescued and wild-type cells. Time lapse video microscopy was used to investigate migratory and morphological effects caused by pHe and NHE1 activity, and a membrane-bound fluorescein conjugate was employed for ratiometric pH measurements at the outer leaflet of the cell membrane. As long as NHE1 remained inactive due to deficiency or inhibition by cariporide (HOE642) neither migration nor morphology was affected by changes in pHe. Under these conditions pH at the outer leaflet of the plasma membrane was uniform all over the cell surface. The typical pH dependence of MV3 cell migration and morphology could be reconstituted by restoring NHE1 activity. At the same time the proton gradient at the outer leaflet of the plasma membrane with the higher proton concentration at the leading edge and the lower one at the cell rear was re-established as well. Hence, NHE1 activity generates a proton gradient at the cell surface accompanied by the cells' ability to respond to changes in pHe (bulk pH). We conclude that NHE1 activity contributes to the generation of a well-defined cell surface pH by creating a proton gradient at the outer leaflet of the plasma membrane that is needed for (i) the development of a variety of morphologies including a distinct polarity and (ii) migration. A missing proton gradient at the cell surface cannot be compensated for by varying pHe.

Figure 1. Na⁺-dependent recovery of intracellular pH (pH_i)

The Na⁺-dependent recovery of the intracellular pH (pH_i) of MV3 cells upon cytosolic acidification (by the NH₄⁺ technique) is significantly reduced in NHE1-deficient cells and can be rescued by transfecting NHE1-deficient cells with NHE1. A, exemplary recordings of single cells. Slopes, i.e. the rates of pH_i recovery plotted in B, were derived from the subsidiary lines. B, recovery of pH_i depending on the initial pH_i right after intracellular acidification (NHE1-deficient, filled circles; rescued, grey triangles; wild-type, open squares; n = at least 80 cells from at least 5 different trials for each cell line containing all 4 (wild-type) or 5 (deficient and rescued) mean values).

Figure 2. Cell migration depends on NHE1 activity and pH_e

A loss of NHE1 activity either by inhibition with cariporide (HOE642) or by mutagenesis impairs migration and reduces the cells' sensitivity to pH_e. In both HCO₃⁻ buffered medium (A and B) and Hepes-buffered Ringer solution (C and D) the migratory speed (A and C) and the cells' translocation over 5 h (B and D) could be restored for the most part by transfecting the NHE1-deficient cell clone with NHE1. n = at least 30 cells from at least 5 different trials in each case. For the purpose of comparison, data for wild-type cells (grey line, open squares) taken from Stock et al. (2005) were also plotted in A and B.

Figure 3. Cell morphology depends on NHE1 activity and pH_e

As shown by the structural index (SI), NHE1 deficiency or NHE1 inhibition with HOE642 prevent cells from spreading and make them stay rather spherical independently of pH_e. This effect can be countered by transfecting the cells with NHE1. n = 30 cells from 5 different trials. For the purpose of comparison data for wild-type cells (grey line, open squares) taken from Stock et al. (2005) were also plotted.

Figure 4. Morphology depending on NHE1 activity

The morphology of NHE1-deficient cells does not change upon NHE1 inhibition by HOE642 or NHE1 stimulation by cytosolic acidification (e–h). Wild-type and rescued cells react to NHE1 inhibition by getting spherical (b, d, k and m) and to NHE1 stimulation by branching out (c and l). pH_e was 7.0. Images represent observations based on at least five different experiments with 5–10 cells per experiment. Scale bar: 20 μm.

Figure 5. Intracellular pH (pH_i) depending on extracellular pH (pH_e) in wild-type, NHE1-deficient and rescued MV3 cells

pH_i of the three clones does not differ after a 3 h adaptation to different pH_e values. N per value is at least 40 cells from at least 4 independent experiments.

Figure 6. Proton gradient at the plasma membrane

A, wild-type (open squares) and NHE1-retransfected (grey triangles) cells show a proton gradient declining along the longitudinal axis from the leading edge to the rear end. B, NHE1-deficient cells (filled circles) and wild-type cells whose NHE1 is inibited by HOE642 (open squares) do not establish a proton gradient at the outer leaflet of the plasma membrane. n = 6–10 cells, 1 cell per experiment, at pH_e 6.84 in each case.

Figure 7. Typical immunolocalization of NHE1 underside of an NHE1-deficient (A), a rescued (B) and a wild-type (C) MV3 cell as observed in five independent experiments

Cells were seeded on collagen I. Leading edges are indicated by asterisks, localization of NHE1 by arrow heads; scale bars: 25 μm. See also movies 4-6.