P120-Catenin Regulates Early Trafficking Stages of the N-Cadherin Precursor Complex.

It is well established that binding of p120 catenin to the cytoplasmic domain of surface cadherin prevents cadherin endocytosis and degradation, contributing to cell-cell adhesion. In the present work we show that p120 catenin bound to the N-cadherin precursor, contributes to its anterograde movement from the endoplasmic reticulum (ER) to the Golgi complex. In HeLa cells, depletion of p120 expression, or blocking its binding to N-cadherin, increased the accumulation of the precursor in the ER, while it decreased the localization of mature N-cadherin at intercellular junctions. Reconstitution experiments in p120-deficient SW48 cells with all three major isoforms of p120 (1, 3 and 4) had similar capacity to promote the processing of the N-cadherin precursor to the mature form, and its localization at cell-cell junctions. P120 catenin and protein tyrosine phosphatase PTP1B facilitated the recruitment of the N-ethylmaleimide sensitive factor (NSF), an ATPase involved in vesicular trafficking, to the N-cadherin precursor complex. Dominant negative NSF E329Q impaired N-cadherin trafficking, maturation and localization at cell-cell junctions. Our results uncover a new role for p120 catenin bound to the N-cadherin precursor ensuring its trafficking through the biosynthetic pathway towards the cell surface

Combinatorial analysis of p120 and NSF in N-cadherin processing.

<p>(A) SW48 cells expressing pro-WT in combination with empty vector (pcDNA) or p120-1, and NSF-WT (WT) or NSF-E329Q (E329Q). Shown is a representative blot probed with anti-N-cadherin to detect the precursor and mature form of N-cadherin, anti-p120, anti-NSF, and anti-tubulin. (B, C) Quantification of N-cadherin precursor/ mature ratio (B) or p120/ NSF ratio (C) from blot scans expressed in arbitrary units (A. U.). Graph bars represent means ± S.E.M. from three independent experiments. Statistical significance (p < 0.05) was determined using one-way ANOVA, followed by the Dunnett´s multiple comparison <i>post hoc</i> test (B), or a two-tailed Student´s <i>t</i>-test (C). Different letters in (B) indicate statistically different means.</p

Function of NSF in N-cadherin trafficking.

<p>(A) Western blotting analysis of HeLa cells co-transfected with pro-WT, and empty vector or vector encoding NSF-WT or NSF-E329Q. Precursor and mature forms of pro-WT were detected with anti-N-cadherin. The lower size of the endogenous N-cadherin is not depicted. Visualization of ectopic NSF-WT and NSF-E329Q signals under non-saturating conditions did not allow detection of endogenous NSF (first lane). At least three independent experiments were used for quantification of precursor and mature forms of pro-WT (right graph). Bars represent means ± S.E.M. of precursor/mature ratios normalized to tubulin. Statistical significance (p < 0.05) was determined using one-way ANOVA, followed by the Dunnett´s multiple comparison <i>post hoc</i> test. Different letters indicate statistically different means. (B-G) Distribution of N-cadherin-GFP and ectopic NSF constructs. HeLa cells were co-transfected with N-cadherin-GFP and either NSF-WT (B-D) or NSF-E329Q (E-G). (H-M) Colocalization of N-cadherin precursor (HA, red label) and the ER marker calnexin (green label) in cells co-transfected with pro-WT2 and NSF-WT (H-J) or NSF-E329Q (K-M). The expression of exogenous NSF constructs was verified in triple label samples (not shown). (N) Manders colocalization coefficients of the HA/calnexin signal overlapping in NSF-WT (n = 15 cells) and NSF-E329Q (n = 23 cells) conditions. Bars represent means ± S.E.M. Statistical significance was determined by a two-tailed Mann-Whitney test. Cells were analyzed by confocal microscopy and representative images projected along the z-axis are shown. Enlarged views (4x) from selected regions (yellow arrowheads) are shown as insets. Scale bar in (E), 35 μm. (O) HeLa cells were co-transfected with proWT and non-targeting siRNA duplexes (Control) or NSF siRNA duplexes (NSF siRNA). After 48 h, precursor and mature forms of the proWT construct, NSF and tubulin were analyzed by Western blotting. Quantification of NSF levels (NSF siRNA values relativized to control siRNA) and precursor/mature ratios in control and NSF siRNA conditions is shown at the right. Bars represent means ± S.E.M. from three independent experiments. Statistical significance (asterisks, p < 0.05) was determined by a two-tailed Student´s <i>t</i>-test.</p

N-cadherin distribution in SW48 colon carcinoma cells reconstituted with p120.

<p>(A) Cells were co-transfected with pro-WT and p120 isoforms 1, 3 or 4. Soluble protein extracts from parental (P) and reconstituted cells (1, 3 and 4) were analyzed in Western blotting probed with anti-N-cadherin to recognize the precursor and mature forms of pro-WT, the p120 isoforms (clone 15D2), and tubulin. The graph shows quantification of precursor/mature ratios normalized to tubulin. (B-H) Confocal analysis of representative cells reconstituted with isoform 1 (B-D), 3 (E-G) and 4 (H). Cells were labeled for p120 (B, E, H) and F-actin (C, F). Arrows point intercellular junctions. "<i>nt</i>" in (B) indicates non-transfected cells. (I-L) Distribution of precursor and mature forms of N-cadherin in parental cells expressing pro-WT and empty vector (I), or vectors encoding p120-1 (J), p120-3 (K) or p120-4 (L). The distribution of ectopic N-cadherin precursor (red label) and total N-cadherin (green label) was detected by anti-HA and GFP fluorescence, respectively. (M-P) Colocalization of the N-cadherin precursor with ER and Golgi markers. Cells expressing pro-WT2 in parental (M) and reconstituted with p120-1 (O) conditions were double-labeled for HA (red signal) and calnexin (green signal). The expression of p120-1 construct was verified in triple label samples (not shown). Note the redistribution of the HA signal to the perinuclear compartment in p120-1 reconstituted cells. The Golgi apparatus was revealed by GalNacT-DsRed transfection in parental (N) and p120-1 reconstituted (P) cells. Note the tight overlapping of the HA label (shown in red) with GalNacT-DsRed (shown in green) in the Golgi. (Q) Manders colocalization coefficients calculated from HA and calnexin (n = 13 cells), and HA and GalNacT (n = 13 cells) signals. Bars represent means ± S.E.M. Statistical significance was determined by a two-tailed Mann-Whitney test. All cell images represent equatorial confocal planes and white arrows point intercellular junctions. Scale bar in (P), 14 μm.</p

Distribution of N-cadherin precursor in p120KD HeLa cells.

<p>HeLa cells were co-transfected with pro-WT2 and either control (A-C, G-I) or p120KD (D-F, J-L) shRNAi. Cells were analyzed by confocal microscopy and representative images projected along the z-axis are shown. (A-F) Colocalization of N-cadherin precursor (HA, red label) with the ER marker calnexin (green label), detected by immunofluorescence. (G-L) Colocalization of the N-cadherin precursor (HA, red label) with transfected GFP-ERGIC-53 (green signal). Enlarged views from selected regions (white dotted boxes) are shown at the right. The expression of the control or p120KD shRNAi constructions in the analyzed cells is revealed by the modified nuclear-targeted GFP encoded by the vector (green channel). Scale bar in (L), 35 μm. (M) Number of HA puncta per cell in control and p120KD conditions (n = 27 cells per condition). Horizontal lanes indicate the position of means (control = 87,8; p120KD = 162.1). (N) Manders colocalization coefficients of the HA puncta overlapping calnexin in peripheral regions (control, n = 9 cells, total 422 puncta; p120KD, n = 7 cells, total 617 puncta). (O, P) Manders colocalization coefficients of the HA label overlapping GFP-ERGIC-53; independent analysis of the punctate (O) and clustered perinuclear distribution (P) of the HA label was performed (n = 24 cells per condition). Bars represent means ± S.E.M. Statistical significance was determined by a two-tailed Mann-Whitney test.</p

NSF association with the N-cadherin precursor.

<p>(A, B) Soluble protein extracts from HeLa cells transfected with pro-WT were sequentially immunoprecipitated with anti-HA and anti-N-cadherin, to isolate the precursor (A) and the mature (B) N-cadherin pools. Mock immunoprecipitations omitted the primary antibody. The presence of NSF in the complexes was analyzed in Western blotting probed with anti-NSF. (C) Immunoprecipitation of N-cadherin precursor from cells transfected with pro-WT or pro-3A constructs, and analysis of NSF in the complexes. (D) Cells co-transfected with pro-WT and either control or p120shRNAi. N-cadherin precursor was isolated by HA-immunoprecipitation and the presence of NSF in the complex was detected in blots. The graphs in (C) and (D) represent quantifications of NSF bands, normalized to the HA signal. (E) Murine PTP1B KO and WT cell lines transfected with pro-WT were immunoprecipitated with anti-HA, and the protein complexes were analyzed by Western blotting probed with antibodies against HA, NSF, phosphotyrosine and tubulin. Numbers under the panels indicate the pY-NSF/NSF ratio. The graph at the right shows the quantification of NSF bands normalized to HA. In all cases, at least three independent experiments were used for quantifications. Data represent means ± S.E.M. Statistical significance was determined by two-tailed Student´s <i>t</i>-test.</p

Evaluation of clinical specimens using LAMP assay.

<p>A. Visualization by Naked Eye: 1. positive control; 2. CCD6 Chronic Chagas Disease (case 6); 3. NI8: non-infected patient, (case 8); 4. AI-TxRID 2: acute infection of transplanted recipient from infected donor (case 2); 5. RCD 1: reactivated Chagas disease (case 1); 6. CCD1: chronic Chagas disease 1 (case 1); 7. CI 4: congenital Chagas disease (case 4); 8. negative control. B. Detection of LAMP reaction using Genie III Fluorimeter. 1: positive control; 2 to 7: clinical specimens indicated in A; 7: Negative control. The Y Axis denotes Fluorescence and X axis denotes Tt (time when fluorescence passes the threshold).</p

Analytical sensitivity of LAMP assay in spiked EDTA blood samples.

<p>EDTA blood samples spiked with different quantities of purified <i>T</i>. <i>cruzi</i> DNA were processed using RAS columns or the Boil & Spin method. (A) EDTA blood processed using RAS columns. (B): EDTA blood processed by Boil & Spin method. Upper panels: LAMP reaction products analyzed by electrophoresis in 1.2% agarose gels and stained with ethidium bromide. Bottom panels: pictures of LAMP reaction products visualized by the naked eye.</p

Clinical specimens results from LAMP test and qPCR as a comparator.

<p>Clinical specimens results from LAMP test and qPCR as a comparator.</p

Analytical sensitivity of LAMP assay.

<p>Top panel: Fluorescence observed with the naked eye from serial dilutions obtained from 3 different aliquots of DNA from <i>CL</i> Brener stock (DTU VI). Bottom panel: Fluorescence observed with the naked eye from serial dilutions obtained from 3 different aliquots of DNA from Sylvio X10 stock.The aliquots were expressed in fg/μL. A: 0; B: 1.0 x 10⁻³; C: 1.0 x 10⁻²; D: 1.0 x 10⁻¹, E: 1. NC: Non template control.</p