Inhibition of PDZ2 binding blocks the NHERF1-dependent increase of NHE1 activity and invasion. To determine whether PDZ1 or PDZ2 are implicated in the NHERF1-dependent stimulation of NHE1 and invasion, the binding ability of these domains was blocked by either their truncation or mutation (HRF1 or HRF2), and the NHE1 activity (A and B), invasive ability (C), and cell morphology/NHERF1 distribution (D) were measured. NHE1 activity and invasive ability were measured as in Figure 5. Error bars are mean ± SE of 15–20 measurements for NHE1, ***p < 0.001 and **p < 0.01 in comparison with nondeprived empty pcDNA3.1 vector; †††p < 0.001 and ††p < 0.01 in comparison with deprived empty pcDNA3.1 vector. Error bars are mean ± SE of 20–25 measurements for invasive activity, **p < 0.001 and **p < 0.01 in comparison with nondeprived empty pcDNA3.1 vector, and the asterisks outside the lines refer to nondeprived empty pcDNA3.1 vector. Transfected NHERF1 expression was visualized by immunofluorescence microscopy with an anti-6His antibody. Bar in large field, 10 μm, and in the inset, 5 μm.

Analysis of NHERF1 and HIF-1α coexpression in human normal and breast cancer. Single (x-y) plane views from confocal 3D analysis of NHERF1 and HIF-1α localization in a normal (A) and a tumor (B) breast lobule. Normal, organized breast lobules display an apical distribution of NHERF1 that, in a disorganized tumor lobule, becomes redistributed diffusely in the cells of the lobule. HIF-1α is distributed in the cytosol in both normal and tumor lobules but is much more expressed in the tumor lobule. The third panel shows their colocalization as analyzed in all the 3D planes and displayed in VG. Cytofluorogram and ICA plots of colocalization using the JACoP image analysis plugin in ImageJ demonstrate a low colocalization in the normal lobule (ICQ = 0.226) and highly significant colocalization in the tumor lobule (ICQ = 0.381). The relative intensity scale is identical for both lobules. Bar, 10 μm.

NHERF1 is localized preferentially in the tip of invading cells and is colocalized with NHE1. (A) NHERF1 immunohistofluorescence in escaping cells of a tumor lobule of a patient. In the bottom panel, the entire lobule is shown in which NHERF1 staining is particularly strong in a group of cells at one border of the lobule. This section of the lobule is shown magnified in the top panel where a small group of cells can be seen to be escaping from the tumor lobule. Interestingly, a number of small pseudopodia extend from these cells and NHERF1 is strongly expressed along these pseudopodia and at their tips. Bar, 8 μm. (B) NHERF1 (red) and NHE1 (green) expression in a representative MDA-MB-435 tumor mass grown in 3D Matrigel culture in which a cell on one side of the mass is escaping and sending a pseudopodia that is invading the matrigel (top). Higher magnifications of the pseudopodia for NHERF1, NHE1 and their merge are shown in the bottom panels. Bars, 10 μm. (C) NHERF1 (red) and NHE1 (green) expression in a representative single MDA-MB-435 cell in Matrigel 3D culture, which has escaped from a mass and is actively invading the matrigel. Top, view from above the cell (x-y plane). Bottom, pseudopodia seen in the x-z plane. Higher magnification of the pseudopodial tip enclosed in the box is shown for both panels. Bar, 10 μm. The ICA graphs for each pseudopodial tip are shown next to the confocal expression images.

Inhibition of PDZ2 binding blocks the NHERF1-dependent signal transduction. To determine which PDZ domain is implicated in the NHERF1-dependent regulation of RhoA and p38 phosphorylation/activity, the binding ability of these domains was blocked by mutation. The phosphorylation state of RhoA (A) and p38 (B) were then measured in Western blot, and the histograms underneath each blot represent the value for the number of experiments indicated in each bar. (C) RhoA activity in the cell body and in the pseudopodia was measured by FRET analysis. In FRET measurements, a relative increase in the CFP emission compared with the YFP emission (EmCFP/EmYFP) denotes an increase in active RhoA. Error bars are mean ± SE of 30–45 measurements for RhoA activity, ***p < 0.001 and **p < 0.01 in comparison with nondeprived empty pcDNA3.1 vector. (D) Pseudocolor images of representative cells from each of the treatments in the FRET measurements. FRET images, obtained as described in Materials and Methods, are presented in pseudocolor IMD mode such that red indicated the highest EmCFP/EmYFP ratio (highest RhoA activity), and blue reflected the lowest EmCFP/EmYFP ratio (lowest RhoA activity). Bar, 10 μm.

Effect of exogenous NHERF1 overexpression on NHE1 activity and NHE1-dependent invasion, cellular morphology, and pseudopodial protrusion. (A) Transfection of wtNHERF1 (wt) in the pcDNA3.1hygro expression vector increased Na⁺/H⁺ exchanger activity in nonserum-deprived (solid bars) conditions and potentiated the serum deprivation-dependent stimulation (striped bars) of Na⁺/H⁺ exchanger activity. Both basal and NHERF1 overexpressed-dependent activity were >90% inhibited by the benzoylguanidine derivative cariporide (HOE642; 2 μM). (B) Transfection of wtNHERF1 increased invasive activity in nonserum-deprived conditions (solid bars) and potentiated the serum deprivation-dependent stimulation of invasion (striped bars). Both basal and NHERF1 overexpressed-dependent invasion were >90% inhibited by 2 μM cariporide, demonstrating that NHE1 is the principle mechanism underlying invasion. Error bars are mean ± SE from five independent experiments performed in quintuplicate. (C) Transfection of either wtNHERF1 or wtGFP-tagged NHERF1 elongated the cells to form a leading edge pseudopodia and redistributed NHERF1 expression to the tip of these pseudopodia. NHERF1 was visualized by immunofluorescence microscopy with anti-NHERF1 or by GFP fluorescence microscopy. Bars, 10 μm. (D) Light transmission images of wtNHERF1-transfected cells treated with 2 μM cariporide at time zero (t0, top) and after 60 min at 100×. Bar, 10 μm.

Analysis of NHERF1 expression and localization in human normal and breast cancer. (A) Representative Western blot of NHERF1 protein expression in the patients breast tumor (T) and in its contiguous, nontumor breast tissue (NT) for four patients and for an aliquot of total cell extract from MCF-7 cells. Biopsies and cells were extracted for total protein and NHERF1 and actin expression analyzed by Western blot as described in Materials and Methods. (B) Immunolocalization of NHERF1 in a breast tumor section at increasing signal intensity from left to right panels, 40× objective. Bar, 10 μm. The increased NHERF1 expression in the large disorganized, breast tumor lobules (t) displays diffuse distribution in the cells of the lobule, whereas normal, organized breast lobules (n) have a strictly apical distribution. (C) That NHERF1 is limited to the apical membrane region of cells in a normal lobule can be better observed in the micrograph showing the zone enclosed in the white box with the 100× objective. Bar, 10 μm. Arrow indicates NHERF1 in the apical membrane.

Hypoxia- and serum deprivation-induced NHERF1 overexpression at the pseudopodial tip stimulates RhoA phosphorylation, which is necessary for increased invasion. (A) MDA-MB-435 cells were treated for 24 h with serum deprivation (D, 24 h) or increasing times of hypoxia; and the expression of NHERF1, RhoA, and phospho-RhoA was determined by Western blot. (B) NHERF1, total RhoA, and phospho-RhoA expression in 35 μg of various cellular fractions of nondeprived (ND) or 24-h-serum–deprived (D) MDA-MB-435 cell monolayers. Control or 24-h-serum–deprived cell monolayers were fractionated and separated as described in Materials and Methods. (C) Cells were exposed to serum deprivation (SD), hypoxia (hyp), or transfected with wtNHERF1 and then exposed to these conditions. The homogenate was then immunoprecipitated with anti-NHERF1 and precipitated RIIβ subunit of PKA, phospho-RhoA, total RhoA, and NHERF1 were analyzed by Western blot. Protein input for each protein was measured by Western blot of the homogenate with the same antibodies and total input anti-β-actin antibody. Typical experiment of five replicates. (D) Confocal 3D immunofluorescence x-y plane image of NHERF1 (green) and phospho-RhoA (red) of a MDA-MB-435 cell in control conditions or treated with 2-h hypoxia. Inset in the hypoxic cell shows enlargement of NHERF1 and phospho-RhoA localization in the pseudopodial tip. Right, VG rendering of NHERF1 and phospho-RhoA colocalization. Bars, 5 μm. (E) NHERF1-dependent stimulation of invasion is reversed by treatment with the PKA inhibitor H89 or by transfection with RhoA mutated to alanine at serine 188 such that it can no longer be phosphorylated by PKA (pdRhoA). Mean ± SE, n = 5, ***p < 0.001 and n.s., not significant with respect to pcDNA3.1-transfected cells; ††p < 0.01 and †††p < 0.001 with respect to wtNHERF1-transfected cells.

Exposure to the tumor microenvironment increases NHERF1 protein expression in tumor cells and redistributes NHERF1 to the pseudopodial tip. Confluent cell monolayers of the human MCF-10A, MCF-7, or MDA-MB-435 breast cell lines were exposed to either serum deprivation (Dep, 24 h) (A) or hypoxia (Hyp, 2 h) (B), and NHERF1 expression was compared with nontreated monolayers (Con) by Western analysis. Histograms underneath each blot represent the trend of NHERF1 expression normalized to actin for five and four independent experiments for serum deprivation and hypoxia, respectively. (C) In MDA-MB-435 cells, NHERF1 changes localization upon stimulation with components of the tumor microenvironment. Cells were either not treated or subjected to serum deprivation for 24 h or hypoxia for 2 h and then probed for NHERF1 expression in 3D immunofluorescence as described in Materials and Methods. Top, typical 3D immunofluorescence views from above (x-y plane) and with the x-z and y-z plane profiles shown in the panels on the side. Hypoxia and serum deprivation elongated the cells to form a leading edge pseudopodia and NHERF1 is strongly redistributed to the pseudodial tip (arrowheads). Bar, 10 μm. Bottom, map plots of the relative intensity of NHERF1 expression in the representative x-y plane of the immunofluorescence image above. To analyze both this localization in the pseudopodial tip and to determine the relative pixel “concentration” of NHERF1 in the various parts of the cell we analyzed the pixel density at planes 1–30 starting from the bottom of the cell. We then took the “high map” feature of the AutoVisualize software (AutoQuant Software) to show the relative pixel density throughout the cell in which white is the highest pixel density. The relative intensity scale is identical for all three cells and increases from blue to white as indicated in the scale to the right. (D) Elongation factor of cells subjected to these treatments defined as the greatest length divided by the greatest width of each cell. Error bars are mean + SE, n = 100.