Reduction of dendritic filopodia protrusion and changes in dendrite morphology in ZO-1-knockdown neurons.

<p>(<b>A</b>) Dendrite morphologies of the control and ZO-1-knockdown (ZO-1 KD) neurons. Cultured neurons were transfected with the control siRNA or the ZO-1 siRNA on 3 DIV, then transfected with EGFP on 5 DIV, and double-immunostained for EGFP and either ZO-1 or MAP2 on 7 DIV. (A1) EGFP and ZO-1; (A2a) EGFP and MAP2; (A2b) EGFP, ZO-1 and MAP2. <b>Upper </b><b>rows</b>, control neurons; <b>lower </b><b>rows</b>, ZO-1-knockdown neurons. <b>Bars</b>, (A1) 10 µm; (A2a) 20 µm; (A2b) 2.5 µm. (<b>B</b>) Time-lapse imaging of the control and ZO-1-knockdown (ZO-1 KD) neurons. Time-lapse images of the neurons expressing EGFP and either the control siRNA or the ZO-1 siRNA were acquired every 8 h from 5 to 6 DIV. (Ba) low magnification images; (Bb) high magnification images of the boxed areas in (Ba). <b>Upper </b><b>rows</b>, neurons expressing EGFP and the control siRNA; <b>lower </b><b>rows</b>, neurons expressing EGFP and the ZO-1 siRNA. <b>Bars</b>, (Ba) 10 µm; (Bb) 2.5 µm. Arrowheads indicate dendrites. (<b>C</b>) Quantitation of the dendrite morphologies of ZO-1-knockdown neurons. The average number of the shafts of the primary dendrites, the average length of the shafts of the primary dendrites, the average number of the shafts of the branches of the primary dendrites, the average density of the dendritic filopodia protruding from the primary dendrites and the median angle of the shafts of the primary dendrites in the neurons transfected with the control siRNA or the ZO-1 siRNA on 3 DIV were measured on 7 DIV. The data are presented as mean plus SEM (error bars) for each sample (n = 21 for the number of the shafts of the primary dendrites (Number of primary dendrite shafts); n = 60 for the length of the shafts of the primary dendrites (Length of primary dendrite shafts), the number of the shafts of the branches of the primary dendrites (Number of branch shafts) and the angles of the shafts of the primary dendrites (Angle of primary dendrite shafts); n = 33 for the density of the dendritic filopodia protruding from the primary dendrites (Density of dendritic filopodia)). Statistical analyses of the dendrite morphology of the ZO-1-knockdown neurons except for that of the angles of the shafts of each dendrite were performed using Student’s t-test. Statistical analysis of the angles of the shafts of each primary dendrite was performed using the Mann-Whitney U test. *, p value < 0.05; and **, p value < 0.01 (Student’s t-test) and *, p value <0.05 (Mann-Whitney U test).</p

Increased accumulation of the components of the nectin and cadherin systems at the dendritic filopodia-filopodia contact sites in ZO-1-overexpressing neurons.

<p>Cultured ZO-1-overexpressing hippocampal neurons on 7 DIV were prepared as described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076201#pone-0076201-g003" target="_blank">Figure <b>3A</b></a> and triple-stained for HA, EGFP and one of nectin-1, nectin-3, afadin, N-cadherin, β-catenin or αN-catenin. (A1) EGFP, HA and nectin-1; (A2) EGFP, HA and nectin-3; (A3) EGFP, HA and afadin; (B1) EGFP, HA and N-cadherin; (B2) EGFP, HA and β-catenin; (B3) EGFP, ZO-1 and αN-catenin. (<b>a</b>) low magnification images; (<b>b</b>) high magnification images of the boxed areas in (<b>a</b>). <b>Upper </b><b>rows</b>, control neurons; <b>lower </b><b>rows</b>, HA-tagged ZO-1-overexpressing neurons. <b>Bars</b>, <b>upper </b><b>rows</b> 10 µm; <b>lower </b><b>rows</b> 2.5 µm. Arrowheads indicate dendritic filopodia-filopodia contact sites. (<b>C</b>) Quantitation of the co-localization of the components of the nectin and cadherin systems with ZO-1 in ZO-1-overexpressing neurons. The data are presented as mean plus SEM (error bars) for each sample (n = 40 for nectin-1, afadin and N-cadherin; n= 50 for nectin-3 and β-catenin; n = 60 for αN-catenin). *, p value < 0.05; and **, p value < 0.01 (Student’s t-test) (AU) arbitrary unit.</p

Enhancement of dendritic filopodia protrusion and changes in dendrite morphology in ZO-1-overexpressing neurons.

<p>(<b>A</b>) Dendrite morphologies of the control and HA-tagged ZO-1-overexpressing neurons. Cultured neurons were transfected with EGFP and either the empty vector (MOCK) or the HA-tagged ZO-1 vector (ZO-1) on 0 DIV and triple-stained for EGFP, HA and MAP2 on 7 DIV. (Aa) low magnification images; (Ab) high magnification images of the boxed areas in (Aa). <b>Upper </b><b>rows</b>, control neurons<b>, lower </b><b>rows</b>, HA-tagged ZO-1-overexpressing neurons. <b>Bars</b>, (Aa) 10 µm; (Ab) 2.5 µm. Arrows and arrowheads indicate axons and dendritic filopodia-filopodia contact sites, respectively. (<b>B</b>) Time-lapse imaging of the control and HA-tagged ZO-1-overexpressing neurons. Time-lapse images of the neurons expressing EGFP and either the empty vector or the HA-tagged ZO-1 vector were acquired every 8 h from 5 to 6 DIV. (Ba) low magnification images; (Bb) high magnification images of the boxed areas in (Ba). <b>Upper </b><b>rows</b>, neurons expressing EGFP and the empty vector; <b>lower </b><b>rows</b>, neurons expressing EGFP and the HA-tagged ZO-1 vector. <b>Bars</b>, (Ba) 10 µm; (Bb) 2.5 µm. Arrowheads in (Ba) and (Bb) indicate dendrites and dendritic filopodia-filopdoia contact sites, respectively. (Bc) Quantitation of the dendritic filopodia-filopodia contact time. The data are presented as mean plus SEM (error bars) for each sample (n = 25). *, p value < 0.05 (Mann-Whitney U test). (<b>C</b>) Quantitation of the dendrite morphologies of HA-tagged ZO-1-overexpressing neurons. The average number of the shafts of the primary dendrites, the average length of the shafts of the primary dendrites, the average number of the shafts of the branches of the primary dendrites, and the average density of the dendritic filopodia protruding from the primary dendrites in the neurons transfected with the empty vector or the HA-tagged ZO-1 vector on 0 DIV were measured on 7 DIV. The data are presented as mean plus SEM (error bars) for each sample (n = 20 for the number of the shafts of the primary dendrites (Number of primary dendrite shafts); n = 64 for the length of the shafts of the primary dendrites (Length of primary dendrite shafts) and the number of the shafts of the branches of the primary dendrites (Number of branch shafts); n = 46 for the density of the dendritic filopodia protruding from the primary dendrites (Density of dendritic filopodia). *, p value < 0.05 (Student’s t-test)).</p

Transient interactions between dendritic filopodia in cultured hippocampal neurons.

<p>(<b>A</b>) Triple immunostaining for F-actin, β-catenin and either Tau-1 or MAP2 in cultured hippocampal neurons. Cultured neurons on 5 DIV were triple-stained for F-actin, β-catenin and either Tau-1 or MAP2. (A1) F-actin, β-catenin and Tau-1; (A2) F-actin, β-catenin and MAP2. <b>Upper </b><b>rows</b>, low magnification images; <b>lower </b><b>rows</b>, high magnification images of the boxed areas in the upper rows. <b>Bars, upper </b><b>rows</b> 10 µm; <b>lower </b><b>rows</b> 2.5 µm. Dendrites and axons were identified by the signals for MAP2 and Tau-1. Arrows indicate axons and arrowheads indicate dendritic filopodia-filopodia contact sites, which were identified by the signal for β-catenin. (<b>B</b>) Time-lapse imaging of EGFP-expressing neurons. Time-lapse images of EGFP-expressing neurons on 4 DIV were acquired every 30 min. <b>Bar</b>, 5 µm. Arrowheads indicate dendritic filopodia-filopodia contact sites. We identified neurites to be dendrites in (<b>B</b>) as follows: we identified MAP2-poitive and Tau-1-negative neurites to be dendrites and MAP2-negative and Tau-1-positive neurites to be axons; in cultured neurons on 4 DIV, axons were generally longer than dendrites; and the diameters of axons, which were 10-µm apart from the cell body, became smaller than those of dendrites at the same distance. We identified neurites to be dendrites by these characteristic morphologies.</p

Reduced accumulation of the components of the nectin and cadherin systems at the dendritic filopodia-filopodia contact sites in ZO-1-knockdown neurons.

<p>Cultured ZO-1-knockdown hippocampal neurons on 7 DIV were prepared as described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076201#pone-0076201-g004" target="_blank">Figure <b>4A</b></a> and double-stained for EGFP and one of nectin-1, nectin-3, afadin, N-cadherin, β-catenin or αN-catenin. (A1) EGFP and nectin-1; (A2) EGFP and nectin-3; (A3) EGFP and afadin; (B1) EGFP and N-cadherin; (B2) EGFP and β-catenin; (B3) EGFP and αN-catenin; (<b>a</b>) low magnification images; (<b>b</b>) high magnification images of the boxed areas in (<b>a</b>). <b>Upper </b><b>rows</b>, control neurons; <b>lower </b><b>rows</b>, ZO-1-knockdown neurons. <b>Bars</b>, <b>upper </b><b>rows</b> 10 µm; <b>lower </b><b>rows</b> 2.5 µm. Arrowheads indicate dendritic filopodia-filopodia contact sites. (<b>C</b>) Quantitation of the co-localization of the components of the nectin and cadherin systems with ZO-1 in ZO-1-knockdown neurons. The data are presented as mean plus SEM (error bars) for each sample (n = 30 for nectin-1, afadin, β-catenin and αN-catenin; n = 40 for nectin-3; n = 20 for N-cadherin) *, p value < 0.05; and **, p value < 0.01 (Student’s t-test) (AU) arbitrary unit. It is noted that the immunofluorescence signals for the components of the nectin and cadherin systems were still observed at some dendritic filopodia-filopodia contact sites in the ZO-1 knockdown neuronal cultures as shown in (<b>A</b>) <b>and </b>(<b>B</b>). We estimated that the transfection efficiency of the siRNA in cultured neurons was about 80%. Thus, ZO-1 might not be knocked-down in some neurons, in which these immunofluorescence signals were observed.</p

Model of the regulation of the phosphorylation and signaling of VEGFR2 by Necl-4 and PTPN13.

<p>The details are described in the Discussion. N13, PTPN13; P, phosphorylation.</p

Necl-4 enhances cellular responses in sparsely cultured ECs.

<p><b>A–C</b>, Reduced movement by Necl-4-knockdown. HUVECs transfected with control or Necl-4 siRNAs were subjected to wound-healing assays in the presence or absence of 50 ng/ml VEGF. Culture dishes were coated with collagen (<b>A and B</b>) or vitronectin (<b>C</b>) (<i>n</i> = 3). †<i>P</i><0.01 vs. control siRNA. <b>D</b>, Reduced proliferation by Necl-4-knockdown. HUVECs transfected with control or Necl-4 siRNAs were cultured on 96-well plates coated with type I collagen in EBM-2 plus 2% FBS in the absence or presence of 50 ng/ml VEGF. At the indicated time points, the numbers of the cells were quantified by crystal violet staining (<i>n</i> = 3). (<b>E and F</b>, Reduced tubulogenesis by Necl-4-knockdown. HUVECs, transfected with control or Necl-4 siRNAs were subjected to Matrigel network formation assays in the presence or absence of 50 ng/ml VEGF (<i>n</i> = 4). †<i>P</i><0.01 vs. control siRNA. <b>G, H, J, and K</b>, Restoration of the reduced movement and tubulogenesis of Necl-4-knockdown HUVECs by ROCK inhibitors. HUVECs, transfected with control or Necl-4 siRNAs and incubated with or without 10 μM Y-27632 or fasudil, were subjected to wound-healing assays (<b>G and H</b>) (<i>n</i> = 3) or Matrigel network formation assays (<b>J and K</b>) (<i>n</i> = 4) in the presence of 50 ng/ml VEGF. †<i>P</i><0.01 vs. VEGF. <b>I</b>, No effects of ROCK inhibitors on the reduced proliferation of Necl-4-knockdown HUVECs. HUVECs, transfected with control or Necl-4 siRNAs and incubated with or without 10μM Y-27632 or fasudil, were cultured on 24-well plates coated with collagen in EBM-2 plus 2% FBS in the presence of 50 ng/ml VEGF. After 48 h, the numbers of the cells were quantified by crystal violet staining (<i>n</i> = 3). <b>L–O</b>, Restoration of the reduced tubulogenesis and movement of Necl-4-knockdown HUVECs by additional knockdown of PTPN13. HUVECs, transfected with control, Necl-4, PTPN13, or Necl-4 plus PTPN13 siRNAs, were subjected to Matrigel network formation assays (<b>L and M</b>) (<i>n</i> = 3) or wound-healing assays (<b>N and O</b>) (<i>n</i> = 3) in the presence of 50 ng/ml VEGF. *<i>P</i><0.05; †<i>P</i><0.01; ns, not significant.</p

Necl-4 inhibits VEGFR2 activation, signaling, and cellular responses in confluently cultured ECs.

<p><b>A and B</b>, Enhanced phosphorylation of VEGFR2 by Necl-4-knockdown. HUVECs transfected with control or Necl-4 siRNAs were cultured under confluent conditions in the presence or absence of 50 ng/ml VEGF for the indicated periods of time. Cell lysates were subjected to Western blotting using the indicated antibodies (<i>n</i> = 4). †<i>P</i><0.01 vs. control siRNA. <b>C–H</b>, Reduced phosphorylation and signaling of VEGFR2 by Necl-4-overexpression. HUVECs transfected with FLAG or FLAG-Necl-4 were cultured under sparse conditions in the presence or absence of 50 ng/ml VEGF for the indicated periods of time. Cell lysates were subjected to Western blotting using the indicated antibodies or pull-down assays using GST-PAK-CRIB (<i>n</i> = 4). *<i>P</i><0.05; †<i>P</i><0.01 vs. FLAG. <b>I and J</b>, Reduced movement by Necl-4-overexpression. HUVECs transfected with FLAG or FLAG-Necl-4 were plated onto collagen-coated culture dishes and subjected to wound-healing assays in the presence or absence of 50 ng/ml VEGF (<i>n</i> = 4). †<i>P</i><0.01 vs. FLAG. <b>K</b>, Reduced VEGF-induced proliferation by Necl-4-overexpression. HUVECs transfected with FLAG or FLAG-Necl-4 were cultured on 24-well plates coated with collagen in EBM-2 plus 2% FBS in the presence of 50 ng/ml VEGF. At the indicated time points, HUVECs were detached and the number of the cells was counted (<i>n</i> = 3). *<i>P</i><0.05 vs. FLAG. <b>L and M</b>, Involvement of PTPN13 in the enhanced phosphorylation of VEGFR2 by Necl-4-knockdown. HUVECs transfected with control, Necl-4, PTPN13, or Necl-4 plus PTPN13 siRNAs were cultured under confluent conditions in the presence or absence of 50 ng/ml VEGF for 1 min and their lysates were subjected to Western blotting using the indicated antibodies (<i>n</i> = 3). †<i>P</i><0.01 vs. control siRNA.</p

Necl-4 interacts with VEGFR1 and VEGFR2 through their extracellular regions.

<p><b>A</b>, Interaction of Necl-4 with VEGFR1 and VEGFR2. HEK293 cells were transfected with FLAG-tagged Necl-4 and either VEGFR1 or VEGFR2. Cell lysates were subjected to co-immunoprecipitation assay using IgG as a control or the anti-FLAG mAb and samples were assessed by Western blotting using the indicated antibodies. <b>B</b>, Interaction of endogenous Necl-4 with endogenous VEGFR2 in ECs. Lysates of HUVECs cultured under sparse (S, 25% confluence) or confluent (C, 100% confluence) conditions were subjected to co-immunoprecipitation assays using IgG as a control or the anti-VEGFR2 pAb and samples were assessed by Western blotting using the indicated antibodies. <b>C and D</b>, Interaction of extracellular region of Necl-4 with VEGFR1 and VEGFR2. HEK293 cells were transfected with VEGFR1 (<b>C</b>) or VEGFR2 (<b>D</b>) and FLAG-tagged Necl-4, Necl-4-ΔCP, or Necl-4-ΔEC. Cell lysates were subjected to co-immunoprecipitation assay using IgG as a control or the anti-FLAG mAb. Samples were assessed by Western blotting using the indicated antibodies.</p

Necl-4 enhances the VEGFR2 signaling in sparsely cultured ECs.

<p><b>A</b>, No effect of Necl-4-knockdown on the phosphorylation of VEGFR2 under sparse conditions. Lysates of HUVECs, transfected with control or Necl-4 siRNAs and cultured in the presence or absence of 50 ng/ml VEGF for 1 min, were subjected to Western blotting using the indicated antibodies. <b>B and C</b>, Reduced activation of Rac1 by Necl-4-knockdown. Lysates of HUVECs, transfected with control or Necl-4 siRNAs and cultured in the presence or absence of 50 ng/ml VEGF for the indicated periods of time, were subjected to pull-down assays using GST-PAK-CRIB (<i>n</i> = 4). †<i>P</i><0.01 vs. control siRNA. <b>D–F</b>, Reduced phosphorylation of ERK by Necl-4-knockdown. Lysates of HUVECs, transfected with FLAG or FLAG-Necl-4 and cultured in the presence or absence of 50 ng/ml VEGF for the indicated periods of time, were subjected to Western blotting using the indicated antibodies (<i>n</i> = 4). *<i>P</i><0.05; †<i>P</i><0.01 vs. FLAG. <b>G and H</b>, Activation of ROCK by Necl-4-knockdown. Lysates of HUVECs transfected with control or Necl-4 siRNAs were subjected to Western blotting using the indicated antibodies. *<i>P</i><0.05; †<i>P</i><0.01 vs. control siRNA. <b>I and J</b>, Restoration of the reduced activation of Rac1 in Necl-4-knockdown HUVECs by ROCK inhibitors. Lysates of HUVECs, transfected with control or Necl-4 siRNAs, incubated with or without 10 μM Y-27632 or fasudil, and cultured in the presence or absence of 50 ng/ml VEGF for 5 min, were subjected to Western blotting using the indicated antibodies or pull-down assays using GST-PAK-CRIB (<i>n</i> = 3). *<i>P</i><0.05; †<i>P</i><0.01. <b>K and L</b>, Restoration of the activity of ROCK by additional knockdown of PTPN13. Lysates of HUVECs transfected with control, Necl-4, PTPN13, or Necl-4 plus PTPN13 siRNAs were subjected to Western blotting using the indicated antibodies (<i>n</i> = 3). †<i>P</i><0.01; ns, not significant. <b>M and N</b>, Restoration of the activity of Rac1 by additional knockdown of PTPN13. Lysates of HUVECs, transfected with control, Necl-4, PTPN13, or Necl-4 plus PTPN13 siRNAs and cultured in presence of 50 ng/ml VEGF for 5 min, were subjected to pull-down assays using GST-PAK-CRIB (<i>n</i> = 3). *<i>P</i><0.05; †<i>P</i><0.01; ns, not significant.</p