Distribution of ezrin and its variants in sub-cellular fraction.

<p>Cellular fractions were prepared from OK cells and equal amounts of proteins (50 μg/lane) from a membrane-enriched fraction (P, 100,000 x g pellet) and cytosol (S, 100,000 x g supernatant) were blotted and probed with anti-total ezrin. <b>A:</b> One of the immunoblots is shown. GFP = transfected mEGFP tagged ezrin (Ezrin-GFP) and Endo = endogenous ezrin. <b>B:</b> Statistical analysis of signal intensity ratios relative to the immuno-detected band in S and P is reported. The signal of total ezrin in S plus P fractions was defined as equal to 100% for each condition and results are expressed as percentage of total ezrin in the P vs. S fraction. Antigen signals were normalized to β-actin. Bars and error bars represent the means ± standard errors (SE), respectively (4 experiments per condition). *P<0.05/** P<0.01 ANOVA, the membrane fraction of tagged ezrin in the presented groups compared to the membrane fraction of tagged ezrin in cells transfected with Ezrin-WT. ^■P<0.05 ANOVA, membrane fraction of endogenous ezrin in the presented groups compared to the membrane fraction of endogenous ezrin in cells transfected with Ezrin-WT. ^$$P<0.01 ANOVA membrane fraction of tagged ezrin in Ezrin-PIP2^- compared to Ezrin-PIP2^- T/D transfected cells.</p

The Na⁺/H⁺ Exchanger-3 (NHE3) Activity Requires Ezrin Binding to Phosphoinositide and Its Phosphorylation

<div><p>Na⁺/H⁺ exchanger-3 (NHE3) plays an essential role in maintaining sodium and fluid homeostasis in the intestine and kidney epithelium. Thus, NHE3 is highly regulated and its function depends on binding to multiple regulatory proteins. Ezrin complexed with NHE3 affects its activity via not well-defined mechanisms. This study investigates mechanisms by which ezrin regulates NHE3 activity in epithelial Opossum Kidney cells. Ezrin is activated sequentially by phosphatidylinositol-4,5-bisphosphate (PIP2) binding and phosphorylation of threonine 567. Expression of ezrin lacking PIP2 binding sites inhibited NHE3 activity (-40%) indicating that ezrin binding to PIP2 is required for preserving NHE3 activity. Expression of a phosphomimetic ezrin mutated at the PIP2 binding region was sufficient not only to reverse NHE3 activity to control levels but also to increase its activity (+80%) similar to that of the expression of ezrin carrying the phosphomimetic mutation alone. Calcineurin Homologous Protein-1 (CHP1) is part, with ezrin, of the NHE3 regulatory complex. CHP1-mediated activation of NHE3 activity was blocked by expression of an ezrin variant that could not be phosphorylated but not by an ezrin variant unable to bind PIP2. Thus, for NHE3 activity under baseline conditions not only ezrin phosphorylation, but also ezrin spatial-temporal targeting on the plasma membrane via PIP2 binding is required; however, phosphorylation of ezrin appears to overcome the control of NHE3 transport. CHP1 action on NHE3 activity is not contingent on ezrin binding to PIP2 but rather on ezrin phosphorylation. These findings are important in understanding the interrelation and dynamics of a CHP1-ezrin-NHE3 regulatory complex.</p></div

Effect of CHP1 expression on Ezrin-WT or Ezrin-PIP2^-distribution in sub-cellular fraction.

<p>Cellular fractions were prepared as described in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129306#pone.0129306.g002" target="_blank">Fig 2</a>. <b>A:</b> One of the immunoblots is shown. GFP = transfected mEGFP tagged ezrin (Ezrin-GFP) and Endo = endogenous ezrin. <b>B:</b> Statistical analysis of signal intensity ratios relative to the immuno-detected band in S and P is reported. The signal of total ezrin in S plus P fractions was defined as equal to 100% for each condition and results are expressed as percentage of total ezrin in the P vs. S fraction. Antigen signals were normalized to β-actin. Bars and error bars represent the means ± SE, respectively (4 experiments per condition). *P<0.05/**P < 0.01 ANOVA, membrane fraction of tagged ezrin in the presented groups compared to the membrane fraction of tagged ezrin in cells transfected with Ezrin-WT. ^■P<0.05 ANOVA, membrane fraction of endogenous ezrin in the presented groups compared to the membrane fraction of endogenous ezrin in cells transfected with Ezrin-WT. ^$P<0.05 ANOVA, membrane fraction of tagged ezrin in Ezrin-PIP2^- compared to Ezrin-PIP2^- + CHP1 transfected cells.</p

Effect of CHP1 expression on the ezrin-mediated control of NHE3 activity.

<p>NHE3 activity was measured as described in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129306#pone.0129306.g003" target="_blank">Fig 3</a>. Bars and error bars represent the means and SE, respectively (8 experiments per condition). **P < 0.01 ANOVA compare to control (empty-vector-transfected cells), NS: Statistically insignificant compare to control. ^$$$P<0.001 ANOVA, CHP1 expression compared to CHP1 and Ezrin-T/A co-expression. Effect of expression of <b>A.</b> Ezrin-WT, Ezrin-PIP2^-, CHP1, CHP1 + Ezrin-WT or CHP1 + Ezrin-PIP2^-. <b>B.</b> Ezrin-T/A, Ezrin-PIP2^- T/A, CHP1, CHP1 + Ezrin-T/A or CHP1 + Ezrin-PIP2^- T/A on NHE3 activity.</p

List of ezrin variants and their localization in OK cells.

<p>Six ezrin constructs were used: <b>A.</b> A full length wild-type ezrin (Ezrin-WT); <b>B.</b> An ezrin in which the binding to PIP2 was abolished by mutation of two double lysines to asparagines, K253/254N and K262/263N, (Ezrin-PIP2^- [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129306#pone.0129306.ref006" target="_blank">6</a>]); <b>C.</b> An ezrin with a threonine to aspartic acid point mutation at position 567 (Ezrin-T/D—phosphothreonine mimic, constitutive active [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129306#pone.0129306.ref041" target="_blank">41</a>]); <b>D.</b> An ezrin with T567D mutation in the PIP2^- ezrin mutant (Ezrin-PIP2^- T/D [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129306#pone.0129306.ref006" target="_blank">6</a>]); <b>E.</b> An ezrin with a threonine to alanine point mutation at position 567 (Ezrin-T/A—non phosphorylable that binds inefficiently to the actin cytoskeleton [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129306#pone.0129306.ref041" target="_blank">41</a>]); <b>F.</b> An ezrin with T567A mutation in the PIP2^- ezrin mutant (Ezrin-PIP2^- T/A [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129306#pone.0129306.ref006" target="_blank">6</a>]). The six ezrin constructs were mEGFP. Localization of ezrin and variants was visualized by indirect immunofluorescence (4 experiments per condition). Highlighted in the figure are XZ cross-sections (plus zoom) and XY face views. Apical localization is indicated by an arrow. Scale bar 2 μm.</p

Effect of ezrin variants in PIP2 binding and 567 phosphorylation sites on NHE3 activity.

<p>NHE3 activity was measured as the rate of Na⁺-dependent intracellular pH recovery. Results are expressed as percentage of change in NHE3 activity. Bars and error bars represent the means and SE, respectively (4 experiments per condition). **P < 0.01 ANOVA compare to control (empty-vector-transfected cells). NS: Statistically insignificant compare to control. Effect of expression of <b>A.</b> Ezrin-WT, Ezrin-PIP2^-, Ezrin-T/D and Ezrin-PIP2^- T/D. <b>B.</b> Ezrin-WT, Ezrin-PIP2^-, Ezrin-T/A and Ezrin-PIP2^- T/A on NHE3 activity.</p

Analysis of ezrin and p-(T567) ezrin expression and localization in human normal and breast cancer.

<p>(A) representative immunolocalization (IHC) of total ezrin and p-ezrin in a normal breast and in breast tumor sections of increasing Grade from left to right panels, bar equals 10µm. The increased ezrin and p-ezrin expression in the large disorganized, breast tumor lobules (t) displays diffuse distribution in the cells of the lobule while normal, organized breast lobules (n) have a strictly apical distribution. (B) Histogram showing that total ezrin in tumor samples increases its relative membrane localization as Grade increases. (C) Histogram showing that relative p-ezrin expression in tumor samples increases as Grade increases. (D) Immunofluorescence was directly performed for HER2 (green) and p-ezrin (red) in a breast tumor section. The merge and the magnification of the area in the white square shows that there was a high amount of co-localization of the two proteins in the membrane. bar = 10µm.</p

Ezrin phosphorylation and binding to PIP2 are necessary for invasion.

<p>To examine the role of ezrin T567 phosphorylation and/or binding to PIP2 in invasive capacity, MDA-MB-231 cells were transfected with empty cDNA (Control) or plasmids containing ezrin cDNA mutated in the T567 site to be either phosphodead (pd) or phosphomimic (pmim) or mutated in the ezrin PIP2 binding site such that it no longer can bind PIP2 (PIP2-). Two days after transfection, a quantitative measure of the degree of <i>in </i><i>vitro</i> invasion of MDA-MB-231 cells was measured as the ability to traverse a 8 µm polycarbonate membrane coated with 5 µg matrigel (Chemicon Int., Livermore, CA) as previously described (Cardone et al., 2005). The fluorescent samples were read in a fluorescence plate reader at 480/520 nm (Cary Eclipse Fluorescence Spectrophotometer, Varian). Mean ± S.E., n=5, ***p<0.001 and n.s. is not significant.</p

NHE1 and p-ezrin are localized in the same lipid raft fraction in the invadopodia but not in the membrane compartments and lipid raft cholesterol is necessary for invadopodia function.

<p>(A) Raft (pellet) and nonraft (supernatant, Supernat) fractions were separated from the above membrane and invadopodia fractions by incubating the fractions (100 µg protein) at 4°C with lysis buffer containing 1% of Lubrol. Insoluble material was collected into pellets (centrifugation at 100,000 X <i>g</i> for 1 hour), and equal amounts of the resuspended pellet (P) and the supernatant (SN) were analyzed by Western Blotting for NHE1, p-ezrin, total ezrin, EGFR and ß1-integrin. Quality of separation was determined by immunoblotting for flotillin-1, a marker for lipid rafts. Representative blots are shown (n = 3). To determine the effect of cholesterol depletion on invadopodia function, MDA-MB-231 cells were incubated for 30 minutes at 37°C in the presence (MβCD-treated) or absence (untreated) of 0.5% MβCD as described in Methods and invadopodia-dependent ECM digestion was analyzed in confocal microscopy for a series of individual cells as described in Methods; (B) typical images of digestion for 6 hrs and overnight in cells treated or not. (C) histogram of a series of experiments performed as in (B). Mean ± S.E.M., n=3, ***p<0.001 for focal proteolysis compared to the control cells. (D) NHE1 immunofluorescence of vehicle or MβCD treated cells demonstrating a change in transporter distribution but not total expression. bar = 10 µm.</p

Ezrin phosphorylation and binding to PIP2 and NHE1 binding to both ezrin and PIP2 are necessary for invadopodia proteolytic activity.

<div><p>(A) To examine the role of ezrin T567 phosphorylation and/or binding to PIP2 in invadopodial-dependent focal digestion of the ECM, MDA-MB-231 cells were (A) transfected with empty cDNA (Control) or plasmids contain ezrin cDNA mutated in the T567 site to be either phosphodead (pd) or phosphomimic (pmim) or mutated in the ezrin PIP2 binding site such that it no longer can bind PIP2 (PIP2-) or treated with 1µM of the inhibitor of ezrin T567 phosphorylation, NSC668394 (NSC). (B) To examine the role of NHE1 binding to either ezrin or PIP2 in invadopodia proteolysis, MDA-MB-231 cells were transfected with an NHE1 mutant lacking the ability to bind to ezrin (KR/A 556-564-NHE1-HA) or to bind to PIP2 (KR/A 513-520-NHE1-HA).</p> <p>Two days after transfection, cells were plated on Matrigel with DQ-Green BSA and, 24 hr later, ECM digestion was analyzed in confocal microscopy for a series of individual cells as described in Methods. Mean ± S.E.M., n=4, ***p<0.001 for focal proteolysis compared to the control cells.</p></div