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Development 138 (18): 3907-3914

Hippo pathway regulation by cell morphology and stress fibers

Ken-Ichi Wada1,2, Kazuyoshi Itoga3, Teruo Okano3, Shigenobu Yonemura4, and Hiroshi Sasaki1,2,*

1 Department of Cell Fate Control, Institute of Molecular Embryology and Genetics, Kumamoto University, 2-2-1 Honjo, Kumamoto 860-0811, Japan.
2 Laboratory of Embryonic Induction, RIKEN Center for Developmental Biology, 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 560-0047, Japan.
3 Institute of Advanced Biomedical Engineering and Science, Tokyo Women's Medical University, 8-1 Kawada-cho, Shinjuku, Tokyo 162-8666, Japan.
4 Electron Microscopy Laboratory, RIKEN Center for Developmental Biology, 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 560-0047, Japan.

Figure 1
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Fig. 1. Cell morphology regulates Yap. (A-C) Relationship between cell area and subcellular Yap distribution in normal cell cultures. (A) Phase-contrast and fluorescent images of NIH3T3 cells. Yap distribution patterns were classified as mainly nuclear (Nuc), diffuse (nucleus and cytoplasm; N/C) and mainly cytoplasmic (Cyt). Representative cells showing each pattern are indicated by arrowheads with abbreviations. (B) Changes in cell area during cell culture. Values are means ± standard deviations (s.d.). (C) Relationship between the Yap distribution pattern and cell area. (D-F) Relationship between cell area and subcellular Yap distribution in microdomain cell culture. (D) Microdomain cell culture system. Upper panels: schematic illustration of microdomain production. Lower panels: examples of fabricated microdomains (left) and the morphology of cells cultured on microdomains (right). (E) Confocal images of cells on microdomains. F-actin (green) was used to visualize cell morphology. Dotted lines indicate the microdomain area. The top panels are confocal z-sectional views. Other panels are single xy-sections. (F) Relationship between domain size and Yap distribution pattern. Cells did not cover the 120x120 µm domain area. Lo is normal low-density culture. Data were collected from three independent experiments, analyzing ≥20 cells for each domain size. Values are means ± s.d. (G) Dynamics of Yap regulation by cell morphology. Yap is still present in the nuclei of suspended cells immediately after detachment (center, arrowheads). In A,E and G Nuc in blue indicates the staining of the nuclei not the location of Yap.


Figure 2
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Fig. 2. Stress fiber promotes nuclear Yap downstream of cell morphology. (A) F-actin distribution in NIH3T3 cells. The panel labeled Suspension (right) shows cells detached from culture dishes for 10 minutes. (B) F-actin distribution in NIH3T3 cells cultured on different sized microdomains. Stress fibers increase in proportion to the cell area increase. Note that the punctate signals and the signals at the cell edges are not from stress fibers. The graph shows the relationship between domain sizes and stress fiber lengths per unit area. Values are means ± s.d. (C-E) F-actin is required for nuclear Yap accumulation. (C) Images showing the effect of actin and microtubule inhibiting drugs on the cytoskeleton and nuclear Yap localization. CytoD, cytocalasin D; LatA, latrunculin A; Noco, nocodazole. Merged images of Yap and nuclear staining are shown in Fig. S2 in the supplementary material. (D) Graph summarizing the effects of various drugs on nuclear Yap localization. Percentage of cells with nuclear Yap (Yap-Nuc) are shown. Blebb, blebbistatin. The concentrations of the reagents are indicated in parentheses. The duration of reagent treatment is indicated by the color of the bars (see key). Values are means ± s.d. from three independent experiments with ≥20 cells in each experiment. n.a., not analyzed. (E) Effects of CytoD on NIH3T3 cells cultured on 50x50 µm domains. Yap distribution was altered by treatment with CytoD without changing the cell area. The graph summarizes the distribution of Yap patterns in control (DMSO) and CytoD-treated cells. The abbreviations for Yap distribution patterns are as in Fig. 1. (F) Reduction of transcriptional activity of endogenous Tead proteins by CytoD treatment. Schematic presentation of the Tead-reporter and the control plasmids used in this study is shown in the upper panel. GTIIC is a Tead-binding motif. Results of the luciferase assay are shown in the lower panel. Values are means ± s.d. from two independent experiments.


Figure 3
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Fig. 3. F-actin regulates Yap through the Hippo pathway. (A-C) Lats is epistatic to F-actin. (A) Representative cells showing the effects of dominant-negative Lats2 (Lats2-KD) and Lats2 on Yap in CytoD-treated cells. p-Yap is Yap phosphorylated at S112. The abbreviations for Yap distribution patterns are as in Fig. 1. and are shown in the corner of the panels. (B) Graph summarizing the effects on Yap distribution. The Yap distribution pattern was classified and abbreviated as shown in Fig. 1A. (C) Graph showing the relative changes in p-Yap signal levels expressed as the ratio of the p-Yap signal to total Yap signal. The average value of non-transfected cells was set at 1.0. Values are means ± s.d. from >18 cells for each cell type. *P<0.05; **P<0.01; ***P<0.001. (D,E) Requirement for pS112 in actin-dependent Yap regulation. (D) Representative cells showing the effects of F-actin disruption on Yap-S112A regulation. The Yap distribution pattern is given in the corner of the panels. (E) Graph summarizing the distribution of exogenously expressed Yap and Yap-S112A in CytoD-treated cells.


Figure 4
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Fig. 4. Phosphorylation at positions other than S112 in Yap is required for Yap exclusion from the nucleus. (A,B) p-Yap (phosphorylated at S112) is localized in the nucleus. (A) Distribution patterns of Yap and p-Yap in NIH3T3 cells and low-density MTD-1A cells under various conditions. Signals for p-Yap were not detected in cells pre-treated with lambda protein phosphatase (PPase +) before antibody reaction, demonstrating the specificity of the antibody. Arrowheads indicate nuclear p-Yap localization. (B) Graphs summarizing distribution patterns of Yap and p-Yap at low and high cell densities (left) and in CytoD-treated cells (right). To simultaneously show the distribution of Yap and p-Yap in a single graph, the following criteria were applied. Cells were first classified according to the Yap distribution pattern as described in Fig. 1A, and the percentage of each class was shown in a bar graph. Cells showing each class of Yap distribution pattern (i.e. each bar) were further classified according to the p-Yap distribution pattern. Based on the p-Yap classification, each bar, representing each Yap class, was subdivided according to the p-Yap class shown in the right panel (see key). (C) Cell morphology and F-actin regulate the phosphorylation level of Yap. Phos-tag-PAGE showing the phosphorylation level of Yap. PPase, samples pre-treated with lambda protein phosphatase; Low, low-density cells; High, high-density cells; DMSO, low-density cells treated with 0.05% DMSO for 1 hour; CytoD, low-density cells treated with 1 µM CytoD for 1 hour; Sus, low-density cells in suspension culture for 10 minutes. Conventional SDS-PAGE is also shown (AAm). Arrowheads indicate the position of non-phosphorylated Yap. (D) Model of density-dependent Yap regulation (see text for details).


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