Localization of Solo at the sites of traction force generation and a model for the role of Solo in hemidesmosome remodeling.

<p>(A) Schematic illustration of the side view of the cell on silicone substrates. Wrinkles appear on the substrate depending on the forces exerted by the cells. (B) Wrinkle formation assay. MCF10A cells were transfected with YFP or YFP-Solo, seeded on a thin Matrigel-coated silicone substrate, and cultured for 24 h. Ventral images of YFP (green) and phase-contrast images were acquired with a confocal microscopy. Red arrows indicate ventral localization of Solo, particularly along the wrinkles. Scale bar, 20 μm. (C) A model for Solo-mediated HD remodeling. Solo localizes at the site of force generation on the ventral surface of epithelial cells and promotes HD formation by activating RhoA signaling and reorganizing keratin networks.</p

ROCK inhibitor suppresses hemidesmosome formation.

<p>(A) Ventral images of endogenous β4, their binary images, and bright field images of Y-27632-treated or untreated MCF10A cells. Cells were cultured as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195124#pone.0195124.g002" target="_blank">Fig 2</a> and treated with 10 μM of Y-27632 or left untreated for 24 h. The red dotted lines indicate the total adhesion area defined by the bright field images. Scale bar, 20 μm. (B) Quantitative analysis of the effect of Y-27632 on HD formation. The ratio of HD area to total adhesion area was calculated, as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195124#pone.0195124.g002" target="_blank">Fig 2</a>. Data represent the means ± SD of 4 independent experiments (at least 7 images per experiment). **<i>P</i> < 0.01 (two-tailed paired <i>t</i>-test).</p

Solo binds to β4-integrin.

<p>(A) Co-immunoprecipitation assays. YFP-Solo was expressed in MCF10A cells and the cell lysates were immunoprecipitated (IP) with an anti-GFP antibody and analyzed by immunoblotting with anti-GFP and anti-β4 antibodies. (B–E) Mapping of the binding regions of Solo and β4. (B) Schematic domain structure of β4 and its deletion mutants used in this study. Numbers denote amino acid residues flanking each region. The binding ability of each fragment to FLAG-Solo is indicated in the right column. Conserved domains are denoted as: vWFA, von Willebrand factor type A; EGF, EGF-like; Calx, Calx-beta; FNIII, fibronectin type III; CS, connecting segment. (C) Co-immunoprecipitation assays of β4 fragments with Solo. YFP-tagged β4 fragment (β4-YFP) and FLAG-tagged Solo-WT were co-expressed in COS-7 cells, and the cell lysates were immunoprecipitated with an anti-FLAG antibody and analyzed by immunoblotting with anti-FLAG and anti-GFP antibodies. Arrowheads indicate the expected positions of YFP-tagged β4 fragments. (D) Schematic domain structure of Solo and its deletion mutants used in this study. The binding ability of each fragment to β4 (1451–1752)-YFP is indicated in the right column. Conserved domains are indicated as Solo, CRAL/TRIO, SPEC (spectrin repeats), DH, and PH domains. (E) Co-immunoprecipitation assays of Solo fragments with β4. FLAG-Solo or its fragments were co-expressed with β4 (1451–1752)-YFP in COS-7 cells, and the cell lysates were immunoprecipitated with an anti-FLAG antibody and analyzed by immunoblotting with anti-FLAG and anti-GFP antibodies. (A, C, and E) These experiments were repeated more than three times and reproducible results were obtained.</p

Knockdown of keratin-18 suppresses hemidesmosome formation.

<p>(A) Effects of K18-targeting siRNAs on K18 expression. MCF10A cells were transfected with control or K18-targeting siRNAs at the indicated concentrations of siRNAs and cultured for 48 h. Cell lysates were analyzed by immunoblotting with an anti-K18 antibody. (B) Ventral images of endogenous β4, their binary images, and bright field images of control and K18 knockdown MCF10A cells. Cells were seeded as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195124#pone.0195124.g002" target="_blank">Fig 2</a>, transfected with control or K18-targeting siRNAs, and cultured for 48 h. The red dotted lines indicate the total adhesion area defined by bright field images. Scale bar, 20 μm. (C) Quantitative analysis of the effect of K18 knockdown on HD formation. The ratio of HD area to total adhesion area was calculated, as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195124#pone.0195124.g002" target="_blank">Fig 2</a>. Data represent the means ± SD of 3 or 4 independent experiments (at least 7 images per experiment). ****<i>P</i> < 0.0001 (one-way ANOVA followed by Dunnett's test).</p

Knockdown of Solo impairs acinar development in 3D-cultured MCF10A cells.

<p>(A) Bright field images of MCF10A acini. MCF10A cells transfected with control of Solo-targeting siRNAs were seeded onto a solidified layer of Matrigel and allowed to grow in a medium containing 2% Matrigel for 4 and 12 days. Scale bar, 200 μm. (B) Size distributions of MCF10A cell clusters cultured for 4 days. Acini diameters were measured by ImageJ software. (C) Quantitative analysis of the effect of Solo knockdown on acinar growth. The percentage of cell clusters with a diameter above 20 μm was calculated. Data represent the means ± SD of 3 independent experiments. **<i>P</i> < 0.01 (one-way ANOVA followed by Dunnett's test). (D) Representative slice images of 3D-cultured MCF10A cells acquired with a confocal microscope. MCF10A cells were grown in a 3D culture system and cultured for 4, 8, or 12 days. Cells were fixed and stained with antibodies against β4 (green) and GM130 (magenta). GM130 was used as an apical polarity marker. The experiments were repeated more than three times for each time points and reproducible results were obtained. Scale bar, 20 μm.</p

Coordinated behaviors of the epithelial cells during tooth morphogenesis.

<p><b>(A)</b> Time-lapse sequences of the dental lamina (DL), enamel knot (EK) and growing apex of the epithelium (GAE) regions showing cell motility. The cells randomly expressed transgenes (YFP-Actin; green). The red, yellow and blue dots indicate individual cells in each region. The arrowhead indicates a membrane protrusion extending from the cell in the GAE. The scale bars represent 5 μm. <b>(B)</b> Distribution and measurement of mitotic spindle orientation in the developing tooth germ. The locations and orientations of the mitotic spindles of the dividing cells are shown in the wire frames. The mean distributions of the mitotic spindle angles (<i>θ</i>) in stellate reticulum (SR; upper) and growing apex of the epithelium (GAE; lower) are shown in the pie chart graph. The results are shown as the mean ± s.d. of three samples (<i>n</i> = 557, 627 and 626 cells [SR], <i>n</i> = 263, 183 and 234 cells [GAE]). *<i>P</i> < 0.01, * * 0.001 < <i>P</i> < 0.005, analyzed with <i>t</i>-tests. The scale bars represent 100 μm.</p

The regulation of the tooth morphogenesis via actin reorganization.

<p><b>(A)</b> The localizations of p-cofilin (green, left upper), cofilin (green, left lower), and F-actin (white, right) were detected by immunohistochemistry. The G0/G1 phase cells (red, center) are visualized with a Fucci probe. The lingual side is on the left in all panels. <b>(B)</b> Estimations of the p-cofilin/cofilin ratios in parts of the tooth germ epithelium (EK, enamel knot; DL, dental lamina; B and L, buccal side and lingual sides of the growing apex of the epithelium). The relative amounts of cofilin and p-cofilin in the regions of the epithelium were determined by immunoblotting (left). The intensities of the bands were calculated and are indicated in the bar graphs (right). <b>(C)</b> Measurements of actin dynamics in the epithelium using fluorescence recovery after photobleaching (FRAP). The right graph illustrates the best-fit curves of the normalized fluorescence intensity during the FRAP assay. The spots indicate the half-recovery times. <b>(D)</b> Gene expression of upstream molecules that regulate cofilin activity in the E14.5 tooth germ. The lingual side is on the left in all panels. The scale bars represent 100 μm. <b>(E)</b> Inhibition of cell proliferation by cofilin phosphorylation in a WST-8 assay. The results are presented as the mean ± s.d. of triplicate experiments. *<i>P</i> < 0.01, analyzed by <i>t</i>-test. LIMK WT, wild-type LIM-kinase (LIMK); LIMK D460A, dominant negative LIMK mutant; Rac V12, dominant active mutant of Rac1. <b>(F)</b> Inhibition of cell migration by cofilin phosphorylation in a wound healing assay. The bars indicate the migration distances of the epithelial cells. The results are presented as the mean ± s.d. of triplicate experiments. *<i>P</i> < 0.01, analyzed by <i>t</i>-test. <b>(G)</b> Schematic summarizing the observed results in this study.</p

The quantitative kinetic analysis of tooth morphogenesis.

<p><b>(A)</b> Trajectories of epithelial cells over 20 hours (h) are shown in fluorescent images (upper panel) and wire frames (lower panel) at each time point of the long-term live image. The contours of the epithelium before and after 20 hours are shown in blue and grey wire frames, respectively. The growth-arrested regions are shown in red wire frame. <b>(B)</b> Trajectories of epithelial cells in parts (EK, enamel knot; DL, dental lamina; GAE, growing apex of epithelium) of the tooth germ epithelium during tooth development. <b>(C)</b> The changes in the relative positions of cells in parts of the tooth germ epithelium. The red and gray spots indicate the center cells and surrounding cells, respectively. <b>(D)</b> Measurements of the relative distances of the epithelial cells in each region. The numbers of spots for the calculations in each region were as follow: <i>n</i> = 17 cells (DL), <i>n</i> = 16 cells (EK), <i>n</i> = 14 cells (GAE). The error bars indicate the standard errors. <b>(E)</b> Deformation analysis of the epithelial tissue for 5–10 hours. The upper and lower panels show the spatial patterns of the volume growth rate and anisotropic tissue stretching, respectively. In the lower panels, the colors indicate the degree of anisotropy, and the arrows indicate the major axes of tissue stretching. The numbers of spots used to estimate the deformation map for each time intervals were as follows: <i>n</i> = 425 cells (25–30 hours), <i>n</i> = 552 cells (50–55 hours), <i>n</i> = 532 cells (75–80 hours), <i>n</i> = 615 cells (100–110 hours). The scale bars represent 100 μm.</p

Tooth morphogenesis and spatial-temporal cell proliferation.

<p><b>(A)</b> Frontal sections of mandibular molar tooth germ derived from Fucci mice at embryonic day (E) 13.5–18.5. The lingual side is on the left in all panels. The scale bars represent 100 μm. <b>(B)</b> Three-dimensional volume rendering images of the molar epithelium shown from the jaw side. The scale bars represent 100 μm. <b>(C)</b> Three-dimensional reconstructions of frontal sections of Fucci tooth germ epithelia showing the cell proliferation pattern. The scale bars represent 100 μm. <b>(D)</b> Length and width measurements of each part of the molar tooth germ. The results are provided as the mean ± s.d. of 6 samples (E14), 6 samples (E15), 8 samples (E16), 7 samples (E18) and 4 samples (E20). The measurement parts are indicated in the left figure. The scale bars represent 100 μm. <b>(E)</b> <i>Ex vivo</i> imaging of four phases during tooth germ epithelium morphogenesis. Schematic (upper panels) and captured live images (lower panels) are provided. The lingual side is on the left in all panels. Red indicates the growth-arrested regions. DL, dental lamina; EK, enamel knot; GAE, growing apex of epithelium. The scale bars represent 100 μm.</p