Alpha-catenin actin bundling experiments - EM images

Negative stain transmission electron micrographs of 2 micromolar F-actin in presence of 4 micromolar alpha-E-catenin homodimer, 4 micromolar alpha-T-catenin or 4 micromolar alpha-T-catenin V94D mutant

NHERF1^-/- depletion causes hydrocephalus.

<p>(A), Comparison of two 28-day old NHERF1^+/+ (left) and NHERF1^-/- (right) littermates. (B) Nissl-stained coronal sections from NHERF1^+/+ (left) and NHERF1^-/- mice at P28. (C) Depletion of <i>nherf1</i> in zebrafish embryos caused hydrocephalus (arrows) 2 days post-fertilization.</p

Vangl1 requires NHERF1 to traffic to the plasma membrane.

<p>(A) Vangl1 colocalizes with Fzd1 at the plasma membrane only in cells that express NHERF1. (B) A Vangl1 mutant in which the PDZ binding motif has been deleted does not traffic to the plasma membrane independently of the expression of NHERF1. Solid white bar: 10 μm.</p

NHERF1^-/- mice have altered Wnt signaling and mislocalization of Vangl2.

<p>(A) <b>Increased nuclear β-catenin levels in ependyma of NHERF1</b>^<b>-/-</b> <b>mice.</b> Brain slices from NHERF1^+/+ and NHERF1^-/- mice were fixed and stained with a monoclonal antibody that recognizes the activated form of β-catenin. (B) <b>Vangl2 mislocalization in NHERF1</b>^<b>-/-</b> <b>ependyma.</b> Slices showing the cerebral aqueduct of NHERF1^+/+ (upper panels) and NHERF1^-/- (lower panels) were stained with specific anti-NHERF1 and anti-Vangl2 antibodies. The second row of nuclei is absent in the NHERF1^-/- sample because of the dilation of the aqueduct as a consequence of hydrocephalus. (C) <b>Defective ciliogenesis is not accompanied by gross defects in the apical-basolateral polarization of the ependymal epithelium.</b> Brain sections showing the third ventricles of wild-type and knockout animals were stained with mucin-1 and α-tubulin antibodies. Notice normal expression of mucin-1 in the ependyma of NHERF1^-/- animals independently of the presence of cilia.</p

NHERF1 ablation alters cilia organization and function in the respiratory tract.

<p>(A) Reduced number of motile cilia on the surface of the respiratory epithelium of NHERF1^-/- mice. (B) Scanning electron micrographs of the tracheae of NHERF1^-/- mice shows patches of cells with no cilia and cells with vestigial cilia. (C), Ciliary function was determined in sagittal sections of the tracheae of NHERF1^+/+ and NHERF1^-/- animals. Fresh sagittal sections were extended on a microscope slide. After addition of medium and 5 μl of a suspension of fluorescent beads, a coverslip was placed on top of the sections and the movement of the beads was recorded by fluorescence microscopy using a 20X objective at 1-sec intervals. The figure shows the relative positions of the beads using a color code (blue: time = 0, green: time = +1 second, red: time = +2 seconds). (D) The average velocity of the microbeads was determined as described in (C). * denotes statistical significance (p<0.001, N = 3 independent experiments; at least 15 beads tracked per experiment).</p

NHERF1 promotes plasma membrane localization of Vangl2.

<p>(A) CHO-N10 cells expressing NHERF1 in a tetracycline-sensitive manner were transfected with HA-Fzd1 and EGFP-Vangl2. The cells were treated with 50 nM tetracycline or vehicle, decorated with anti-HA antibody followed by Alexa 594-labeled secondary antibody, and examined by confocal microscopy. (B) Same as A except that the cells were transfected with an EGFP-tagged Vangl2 construct in which the C-terminal PDZ binding motif has been mutated to preclude binding to PDZ domains (V512A-Vangl2). (C) Co-localization of HA-Fzd1 and EGFP-Vangl2. The co-localization was quantified using the Pearson correlation coefficient (R) measured using ImageJ. The correlation coefficients shown represent the average obtained from >50 cells for each set, examined in six separate experiments. The symbol (*) denotes statistically significant differences (p<0.001). (D) NHERF1-induced plasma membrane localization of Vangl2 requires an intact PDZ1. CHO cells co-transfected with Flag-tagged NHERF1 constructs and EGFP-Vangl2 were fixed, decorated with anti-Flag antibodies and examined with a confocal microscope. All NHERF1 constructs were present at the plasma membrane but only the wild-type and S2 NHERF1 constructs co-localized with Vangl2. (D) Quantitative analysis of the co-localization of Vangl2 and NHERF1 mutants. To determine co-localization, we measured the Pearson correlation coefficient of the red and green channels using an ImageJ plugin. The data show the results from >30 cells/set obtained from 4 different experiments. NS: not statistically significant. For all images: the solid white bar is10 μm long.</p

Model for the NHERF1-induced interaction of Fzd4 and Vangl2.

<p>(A) Experimental design of the studies described in Panels (B) and (C). We propose that Fzd4 and Vangl2 interact with the PDZ domains of NHERF1. Thus, in the absence of NHERF1, Fzd4 and Vangl2 are expected to interact only weakly; however, NHERF1 expression is predicted to stabilize the formation of Fzd4-Vangl2 complexes. (B) Cells transfected with HA-Fzd4 exposed to the extracellular milieu and EGFP-Vangl2 were treated with an Alexa594-tagged anti-HA antibody. Addition of cross-linking antibodies that recognize the HA tag on Fzd4 immobilizes Vangl2 only when NHERF1 is expressed. CHO-N10 cells transfected with HA-Fzd4 and EGFP-Vangl2 were induced with vehicle or 100 ng/ml tetracycline. Live cells were tagged with Alexa594-labeled anti-HA antibody (HA.11) followed by a secondary anti-mouse IgG cross-linking antibody. The mobility of EGFP-Vangl2 was determined by FRAP. (C) Cross-correlation analysis of the formation of Fzd4-Vangl2 complexes. CHO-N10 cells cotransfected with HA-Fzd4 and EGFP-Vangl2 were incubated with Alexa594-anti-HA antibody. The autocorrelation function of EGFP-Vangl2 and the cross-correlation function of the EGFP label to the Alexa594 label were determined as described [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153144#pone.0153144.ref018" target="_blank">18</a>]. The ratio of the cross-correlation (G_CC) to the autocorrelation amplitudes is directly proportional to the fraction of Vangl2-Fzd4 complex formed (see[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153144#pone.0153144.ref018" target="_blank">18</a>]). ETVV and ETSV denote the C-termini of Fzd4 and Vangl2, respectively. EB: Ezrin binding domain. MERM: a family of actin-binding protein adaptors that includes moesin, ezrin, radixin and merlin.</p

The ependymal cilia of NHERF1^-/- mice are dysfunctional.

<p>(A) Ciliary function was determined by measuring the motion of 1-μm fluorescein-labeled beads placed within the third ventricle of sagittal brain slices obtained from P28 wild-type and knockout mice. To illustrate the motion, 3 successive images (Δt = 0.1 s) were colored red, green and blue, respectively, and combined in ImageJ. The image on the left, characteristic of the wild-type brain slices, shows no superposition of the three colors, indicating rapid motion of the beads. The beads of the NHERF1^-/- slices appear white in the montage because all three colors (red, green and blue) coincide at all times, indicating lack of motion. The right side panel (Azide) shows the results obtained with a wild-type slice poisoned with azide. Refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153144#pone.0153144.s003" target="_blank">S1 Movie</a> for complete visualization of the experiment. (B) Tracking of individual beads in wild-type (WT) and knockout (KO) mice calculated as described. The right panel shows average velocities estimated from the tracking of individual beads. (*) denotes statistically significant differences (p<0.001; results obtained from three independent experiments).</p

Coordinated viral gene expression during MCV replication in 293 cells.

<p>One microgram of recircularized MCV genomes (wild-type MCV-HF or replication-defective MCV-Rep⁻) was transfected into 293 cells. Immunoblotting was performed to examine T antigen expression over 5 days (indicated by hollow arrows) and VP1 protein (indicated by solid arrow) using CM2B4 (LT, 57kT), CM8E6 (sT) and CM9B2 (VP1) antibodies, respectively. Alpha-tubulin detection was used as a protein loading control. LT protein is expressed equally at day 2 for both viruses but decreases for MCV-Rep⁻ on days 3–5. VP1 increases on days 3–5 only for MCV-HF, corresponding to viral DNA replication (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022468#pone-0022468-g003" target="_blank">Fig. 3D</a>). Other early proteins are also diminished (57kT) or absent (sT) in the replication deficient MCV-Rep⁻.</p

Fzd and Vangl2 bind NHERF1.

<p>(A) CHO–N10 cells were transfected with HA-Fzd4, and the formation of a Fzd4-NHERF1 complex was determined by coimmunoprecipitation of the HA-tag followed by Western blotting with specific anti-NHERF1 antibodies. NHERF1 expression was induced by the addition of 100 ng/ml tetracycline. (B) CHO-N10 cells were cotransfected with an EGFP-tagged Vangl2 construct and induced with either vehicle or 100 ng/ml tetracycline. After immunoprecipitation of NHERF1 using a specific antibody, the presence of the GFP label in the immunoprecipitated material was determined by Western blotting. (C) CHO-N10 cells were cotransfected with HA-Fzd4 and EGFP-Vangl2. After induction with either vehicle or 15 ng/ml tetracycline, the HA-tagged material was immunoprecipitated, and the presence of the GFP tag in the co-immunoprecipitate was determined by Western blotting. (D) Both PDZ domains of NHERF1 are required for the interaction of Fzd4 and Vangl2. HEK293S GnTI cells cotransfected with HA-Vangl2 and FLAG-tagged NHERF1 constructs (wt, S1, S2 and S1S2) were lysed and treated with anti-HA antibodies to immunoprecipitate Vangl2.</p