Jasplakinolide induces primary cilium formation through cell rounding and YAP inactivation

<div><p>Primary cilia are non-motile cilia that serve as cellular antennae for sensing and transducing extracellular signals. In general, primary cilia are generated by cell quiescence signals. Recent studies have shown that manipulations to increase actin assembly suppress quiescence-induced ciliogenesis. To further examine the role of actin dynamics in ciliogenesis, we analyzed the effect of jasplakinolide (Jasp), a potent inducer of actin polymerization, on ciliogenesis. Unexpectedly, Jasp treatment induced ciliogenesis in serum-fed cells cultured at low density. In contrast, Jasp had no apparent effect on ciliogenesis in cells cultured at higher densities. Jasp-induced ciliogenesis was correlated with a change in cell morphology from a flat and adherent shape to a round and weakly adherent one. Jasp treatment also induced the phosphorylation and cytoplasmic localization of the YAP transcriptional co-activator and suppressed cell proliferation in low density-cultured cells. Overexpression of an active form of YAP suppressed Jasp-induced ciliogenesis. These results suggest that Jasp induces ciliogenesis through cell rounding and cytoplasmic localization and inactivation of YAP. Knockdown of LATS1/2 only faintly suppressed Jasp-induced YAP phosphorylation, indicating that LATS1/2 are not primarily responsible for Jasp-induced YAP phosphorylation. Furthermore, overexpression of active Src kinase suppressed Jasp-induced cytoplasmic localization of YAP and ciliogenesis, suggesting that down-regulation of Src activity is involved in Jasp-induced YAP inactivation and ciliogenesis. Our data suggest that actin polymerization does not suppress ciliogenesis <i>per se</i> but rather that cell rounding and reduced cell adhesion are more crucially involved in Jasp-induced ciliogenesis.</p></div

Effects of LATS1/2 knockdown on Jasp-induced YAP phosphorylation and ciliogenesis.

<p>(A) Effects of LATS1/2 siRNAs on the expression of LATS1 (left) and LATS2 (right). RPE1 cells were transfected with control, LATS1, or LATS2 siRNAs and cultured for 48 h. Cell lysates were analyzed by immunoblotting with antibodies against LATS1, LATS2, and β-actin. (B) Effects of LATS1/2 knockdown on Jasp-induced YAP phosphorylation. RPE1 cells were transfected with control siRNA or LATS1/2 siRNAs, cultured for 24 h at low density in serum-containing medium, and then further incubated with 0.5 μM Jasp for 24 h. Cell lysates were subjected to Phos-tag-containing or conventional SDS-PAGE and analyzed by immunoblotting with anti-YAP and anti-β-actin antibodies. In lane 1, lysates were treated with λ phosphatase. Asterisks indicate the positions of partially dephosphorylated YAP. (C) Effects of LATS1/2 knockdown on Jasp-induced ciliogenesis. RPE1 cells were transfected with control siRNA or LATS1/2 siRNAs, cultured for 24 h at low density in serum-containing medium, further incubated with 0.5 μM Jasp for 24 h, and then fixed. The percentage of ciliated cells was counted based on staining for Ac-tubulin and Arl13b. Data are means ± SEM from four independent experiments. n.s., not significant.</p

Jasp treatment induces cell quiescence and the cytoplasmic localization and phosphorylation of YAP.

<p>(A) Effect of Jasp treatment on cell proliferation. RPE1 cells were cultured at low density in serum-containing medium, treated with 1 μM Jasp for the indicated lengths of time, and then fixed and stained with Alexa-488-conjugated anti-Ki-67 antibody (green). DNA was stained with DAPI (blue). Scale bar, 20 μm. (B) Jasp induces the cytoplasmic localization of YAP in cells at low density. RPE1 cells were cultured at low, medium, and high densities in serum-containing medium, treated with 0.5 μM Jasp for 24 h, and then fixed and stained with anti-YAP antibody (green). DNA was stained with DAPI (blue). DIC images are shown in the right panels. Scale bar, 20 μm. (C) Quantification of the effects of Jasp treatment on YAP localization. The percentage of cells with YAP localization in the nucleus (preferentially in the nucleus or equally in the nucleus and cytoplasm) was counted. Data are means ± SEM from three independent experiments. *<i>P</i> < 0.05; n.s., not significant. (D) Jasp promotes YAP phosphorylation. RPE1 cells were cultured at low density in serum-containing medium and treated with the indicated concentrations of Jasp for 24 h. Cell lysates were subjected to Phos-tag-containing and normal SDS-PAGE and analyzed by immunoblotting with anti-YAP and anti-β-actin antibodies, respectively.</p

Overexpression of LIMK1 suppresses serum starvation-induced ciliogenesis in adherent cells.

<p>(A) Effect of LIMK1 overexpression on serum starvation-induced ciliogenesis. RPE1 cells were transfected with GFP or Myc-tagged wild-type (WT) or kinase-dead (KD) LIMK1, cultured at medium density in serum-fed medium, and then subjected to serum starvation for 48 h. Cells were fixed and stained with anti-Ac-tubulin (red) and anti-Myc (green) antibodies and DAPI (blue). Cells were also imaged by GFP fluorescence (green). Actin filaments were stained with Alexa-633-phalloidin (right panels). Arrows indicate primary cilia. Scale bar, 20 μm. (B) Quantification of the frequency of ciliated cells. The percentage of ciliated cells was counted among GFP- or Myc-positive cells, based on staining for Ac-tubulin. Data are means ± SEM from three independent experiments. <i>*P</i> < 0.05.</p

Overexpression of Src suppresses Jasp-induced ciliogenesis and the cytoplasmic translocation of YAP.

<p>(A) Expression of Src-(Myc+His) or its mutants in Jasp-treated RPE1 cells. RPE1 cells were transfected with (Myc+His)-tagged Src(WT), or its active (ΔC) or kinase-dead (KD) mutant; cultured at low density in serum-containing medium; treated with 0.5 μM Jasp for 24 h. Cell lysates were subjected to SDS-PAGE and analyzed by immunoblotting with anti-Myc and anti-β-actin antibodies. (B) Effect of Src overexpression on Jasp-induced ciliogenesis. RPE1 cells were transfected with (Myc+His)-tagged Src or its mutants or control mCherry, cultured, and treated with Jasp, as in (A), and then fixed. The percentage of ciliated cells was counted based on staining for Ac-tubulin and Arl13b. (C) Effect of Src overexpression on Jasp-induced cytoplasmic translocation of YAP. RPE1 cells were transfected, cultured, treated with Jasp, and then fixed, as in (B). Cells were stained with anti-Myc (red) and anti-YAP (green) antibodies. In the first and second rows, cells were imaged by mCherry fluorescence (red). DNA was stained with DAPI (blue). DIC images are shown in the right panels. Arrows indicate the mCherry- or Myc-positive cells. Scale bar, 20 μm. (D) Quantification of the effect of Src overexpression on the localization of YAP. The percentage of cells with nuclear localization of YAP was analyzed, as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183030#pone.0183030.g002" target="_blank">Fig 2C</a>. In (B) and (D), data are means ± SEM from three independent experiments. <i>*P</i> < 0.05; n.s., not significant.</p

Overexpression of active YAP suppresses Jasp-induced ciliogenesis.

<p>(A) Expression of GFP or GFP-YAP(5SA) in Jasp-treated RPE1 cells. RPE1 cells transfected with GFP or GFP-YAP(5SA) were cultured at low density in serum-containing medium and treated with 0.5 μM Jasp for 24 h. Cell lysates were subjected to SDS-PAGE and analyzed by immunoblotting with anti-GFP and anti-β-actin antibodies. (B) Effect of YAP(5SA) overexpression on Jasp-induced ciliogenesis. RPE1 cells were transfected with GFP or GFP-YAP(5SA) and treated with Jasp as in (A), and then fixed and stained with anti-Arl13b antibody (red) and DAPI (blue). Cells were also imaged by GFP fluorescence (green). Arrowheads indicate primary cilia. Arrow indicates nuclear localization of GFP-YAP(5SA). Scale bar, 20 μm. (C) Quantification of the percentage of ciliated cells among GFP-positive cells. Data are means ± SEM from four independent experiments. <i>*P</i> < 0.05.</p

Jasplakinolide (Jasp) induces ciliogenesis and cell rounding in cells cultured at low density.

<p>(A) Effects of actin-modulating drugs on ciliogenesis. RPE1 cells were cultured at low density in serum-containing medium; treated with 0.5 μM CytoD, 1 μM LatB, or 1 μM Jasp for the indicated lengths of time; and then fixed. The percentage of ciliated cells was counted based on staining for Ac-tubulin and Arl13b. (B) Dose-dependent effect of Jasp on ciliogenesis. RPE1 cells were cultured at low density in serum-containing medium, treated with the indicated concentrations of Jasp for 24 h, and then fixed. The percentage of ciliated cells was analyzed as in (A). (C) Effects of Jasp on ciliogenesis and cell morphology in cells cultured at distinct cell densities. RPE1 cells were cultured at low (2.1 x 10³ cells/cm²), medium (1.0 x 10⁴ cells /cm²), or high (5.2 x 10⁴ cells /cm²) density in serum-containing medium, treated with 0.5 μM Jasp for 24 h, and then fixed. Cells were stained with anti-Ac-tubulin (red) and anti-Arl13b (green) antibodies. DNA was stained with DAPI (blue). Arrows indicate primary cilia. Scale bar, 20 μm. (D) Quantification of the frequency of ciliated cells in Jasp-treated or untreated cells cultured at distinct densities. In (A), (B), and (D), data are means ± SEM from three independent experiments. <i>*P</i> < 0.05; n.s., not significant.</p

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

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