Fig 1.
(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.
Fig 2.
Knockdown of Solo suppresses hemidesmosome formation.
(A) Effects of Solo-targeting siRNAs on Solo expression. MCF10A cells were transfected with control or Solo-targeting siRNAs at the indicated concentrations of siRNAs and cultured for 48 h. Cell lysates were analyzed by immunoblotting with an anti-Solo antibody. (B) Ventral images of endogenous β4, their binary images, and bright field images of control and Solo knockdown MCF10A cells. Cells were seeded on a thin Matrigel-coated coverslip, transfected with control or Solo-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 Solo knockdown on HD formation. The ratio of HD area (measured from the binary image of β4) to total adhesion area (measured from the bright field image) was calculated. Data represent the means ± SD of 4 independent experiments (at least 5 images per experiment). **P < 0.01 (one-way ANOVA followed by Dunnett's test).
Fig 3.
ROCK inhibitor suppresses hemidesmosome formation.
(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 Fig 2 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 Fig 2. Data represent the means ± SD of 4 independent experiments (at least 7 images per experiment). **P < 0.01 (two-tailed paired t-test).
Fig 4.
Knockdown of keratin-18 suppresses hemidesmosome formation.
(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 Fig 2, 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 Fig 2. Data represent the means ± SD of 3 or 4 independent experiments (at least 7 images per experiment). ****P < 0.0001 (one-way ANOVA followed by Dunnett's test).
Fig 5.
Knockdown of Solo impairs acinar development in 3D-cultured MCF10A cells.
(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. **P < 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.
Fig 6.
Localization of Solo at the sites of traction force generation and a model for the role of Solo in hemidesmosome remodeling.
(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.