The Epithelial Circumferential Actin Belt Regulates YAP/TAZ through Nucleocytoplasmic Shuttling of Merlin  Kana T. Furukawa, Kazunari Yamashita, Natsuki Sakurai, Shigeo Ohno  Cell Reports  Volume 20, Issue 6, Pages 1435-1447 (August 2017) DOI: 10.1016/j.celrep.2017.07.032 Copyright © 2017 The Author(s) Terms and Conditions

Cell Reports 2017 20, 1435-1447DOI: (10.1016/j.celrep.2017.07.032) Copyright © 2017 The Author(s) Terms and Conditions

Figure 1 YAP/TAZ Is Regulated by Cell Density and Involved in Proliferation Regulation in MDCK Cells (A) MDCK cells were cultured at low density or high density in 12-well Transwell, followed by immunofluorescence staining. Scale bar represents 20 μm. (B) Gene expression levels of cells prepared as in (A) were analyzed by qRT-PCR (n = 3). Error bars represent SD. (C) MDCK cells (1 × 105 per well) were transfected with the indicated siRNAs and cultured in 12-well plates until the indicated times. Cells were then counted for assessment of proliferation. The averages of three independent experiments are plotted. Error bars represent SD. (D) Cells corresponding to day 2 of (C) were analyzed by western blotting. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control. Note that Anti-YAP1 (Abnova) recognizes both YAP and TAZ (upper and lower arrowhead, respectively). (E) Ki67-expressing cells were evaluated by immunofluorescence. Cells corresponding to day 2 of (C) were analyzed, and all cells in six photographs among two independent experiment were counted (n = 6). Error bars represent SD. (F) Total cell lysates from MDCK cells cultured at low density and high density were analyzed by western blotting. p27 was used as a marker for cell density-dependent cell-cycle arrest. (G) Densitometry of the data in (F). Intensity of S127P-YAP was normalized to YAP (n = 3). Error bars represent SD. Cell Reports 2017 20, 1435-1447DOI: (10.1016/j.celrep.2017.07.032) Copyright © 2017 The Author(s) Terms and Conditions

Figure 2 Contraction of Circumferential Actin Belts Suppresses YAP/TAZ Nuclear Localization (A) Graphical illustration of the relationship between cell density and actin cytoskeleton organization in columnar epithelial cells. Low-density epithelial cells display a fibroblast-like morphology and have many stress fibers and immature circumferential actin not associated with myosin II (left). In contrast, high-density epithelial cells display well polarized columnar morphology and have both stress fibers and a mature circumferential actin belt on the apical side and lateral F-actin beneath the lateral membrane. The circumferential actin belt associates with adherens junctions. (B) MDCK cells were cultured at 1 × 105/well in 12-well Transwell plates for 48 hr (medium density), followed by immunofluorescence staining. Asterisks indicate cells where YAP/TAZ is localized in the nucleus. (C) Low-density (top) and high-density (bottom) cultured MDCK cells were treated with vehicle or 50 μM blebbistatin for 3 hr and then subjected to immunofluorescence staining. Nuclear localization of YAP/TAZ was promoted by blebbistatin treatment in high-density cultures. Stress fibers were reduced by blebbistatin treatment in both low- and high-density cultures. (D) Cells displaying nuclear YAP/TAZ staining (N) and cells displaying both nuclear and cytoplasmic YAP/TAZ staining (N + C) were counted (n = 3). Error bars represent SD. (E) Perimeters of high-density cells were measured following vehicle and blebbistatin treatment (n = 3). Error bars represent SD. (F) Localization of TAZ and expression of Ki67 were investigated in low-density (top) and high-density (bottom) MDCK cell cultures following vehicle and blebbistatin treatment. (G) The proportions of Ki67-positive cells in blebbistatin-treated and vehicle-treated cultures (F) were counted (n = 3). Error bars represent SD. (H) Gene expression levels in high-density cultured MDCK cells were measured by qRT-PCR following vehicle and blebbistatin treatment for 6 hr (n = 3). Error bars represent SD. (I) Myc-ROCKΔ3 was induced in low-density cultures 6 hr before fixation (see the Experimental Procedures). (J) Luciferase assay using TEAD reporter (8xGTIIC-luc). Relative luciferase activity was normalized to the value of EGFP-expressing cells. Ectopic expression of ROCKΔ3 significantly inhibited TEAD reporter activity (n = 3). Error bars represent SD. Scale bars represent 20 μm. Cell Reports 2017 20, 1435-1447DOI: (10.1016/j.celrep.2017.07.032) Copyright © 2017 The Author(s) Terms and Conditions

Figure 3 ROCK-Induced Suppression of YAP/TAZ Nuclear Localization Involves Nuclear Export Independently of the Hippo Pathway (A) A Flag-YAP WT or Flag-YAP 5SA expression vector was co-transfected with an EGFP or Myc-ROCKΔ3 expression vector into low-density cultured MDCK cells. (B) Quantification of Flag staining intensity. Ratio of nuclear signal to total signal is plotted (n = 3). The precise methods are described in Figure S3A. Error bars represent SD. (C) MDCK cells cultured at low density (left) and at high density (right) were treated with 40 ng/mL leptomycin B (LMB) for 2 hr. LMB-dependent nuclear accumulation of YAP/TAZ was evident in high-density cells. (D) The effect of LMB on ROCKΔ3-mediated suppression of YAP/TAZ nuclear localization. Episomally stable transformant MDCK cells were cultured at low density and induced to express ROCKΔ3 by doxycycline. Cells were then treated with LMB for the indicated times. Rapid recovery of YAP/TAZ nuclear localization was observed (arrowheads). Reconstructed confocal z axis images are shown in the top panels. Dotted lines indicate the positions where z axis images are reconstructed. (E) The effect of LMB on LATS2-mediated suppression of YAP/TAZ nuclear localization. Low-density cultured MDCK cells transfected with HA-LATS2 were treated with LMB for the indicated times. Slow recovery of YAP/TAZ nuclear localization was observed (arrowheads). (F) Quantification of the intracellular YAP/TAZ distribution from (D), (E), and Figure S3B. YAP/TAZ signals in Myc- or HA-positive cells were quantified using ImageJ. EGFP transfection was used as a control. Ratios of nuclear signal to total signal are plotted (n = 3). Error bars represent SD. (G) Difference in the rate of YAP/TAZ nuclear relocalization from 0 to 15 min following LMB treatment in ROCKΔ3-, LATS2-, and Merlin-transfected cells (from F) (n = 3). Error bars represent SD. Scale bars represent 20 μm. Cell Reports 2017 20, 1435-1447DOI: (10.1016/j.celrep.2017.07.032) Copyright © 2017 The Author(s) Terms and Conditions

Figure 4 Merlin Is Involved in the Suppression of YAP/TAZ Nuclear Localization Induced by F-Actin Contraction (A) Episomally stable transformant MDCK cells were transfected with the indicated siRNAs and induced to express ROCKΔ3. Cells were cultured at low density. Arrowheads indicate nuclei of ROCKΔ3-expressing cells. (B) Western blots showing the Merlin-knockdown efficiency. (C) YAP/TAZ nuclear localization in Myc-positive cells (A) was quantified. Ratio of nuclear signal to total signal is plotted (n = 3). Error bars represent SD. (D) MDCK cell lines stably expressing non-silencing short hairpin RNA (shRNA) or shRNA for Merlin were established and cultured on Transwell at high density. Localization of YAP/TAZ was investigated by immunofluorescence. (E) High density-cultured control or Merlin-depleted cells were analyzed by western blotting. Asterisk indicates non-specific signal. (F) Low-density cultured MDCK cells were transfected with V5-Merlin isoform 1 or isoform 2. (G) YAP/TAZ nuclear localization in V5- and EGFP-positive cells (F) is quantified (n = 3). Error bars represent SD. (H) YAP/TAZ was immunoprecipitated from lysates of MDCK cells cultured at low density (lanes 1, 3, and 5) or high density (lanes 2, 4, and 6), and co-immunoprecipitation of Merlin was assayed. Scale bars represent 20 μm. Cell Reports 2017 20, 1435-1447DOI: (10.1016/j.celrep.2017.07.032) Copyright © 2017 The Author(s) Terms and Conditions

Figure 5 Nucleocytoplasmic Shuttling of Merlin Is Necessary for Suppression of YAP/TAZ Nuclear Localization and Is Controlled by Circumferential Actin Belt Tension (A) Schematic representation of Merlin protein structure. Sequences of all three NESs and their mutants are shown. (B) HA-Merlin and its NES mutants were overexpressed in low-density MDCK cells and effects on YAP/TAZ nuclear localization were evaluated. Arrowheads indicate nuclei of HA-Merlin-expressing cells. (C) Signal intensity of HA-Merlin staining (B) was quantified. Ratio of nuclear signal to total signal is plotted (n = 3). Error bars represent SD. (D) Quantification of Nuclear YAP/TAZ in HA-Merlin-expressing cells (B) (n = 3). Error bars represent SD. (E) Distribution of endogenous Merlin in MDCK cells cultured at low and high density. High-density cells treated with blebbistatin are also shown. Insets are magnifications of the region in the dotted rectangle. (F) Merlin staining intensities of the nuclear and cytoplasmic region (N + C) and the cell-cell contact region (cell-cell) in (E) were quantified using ImageJ. Ratio of N + C signal to cell-cell signal is plotted (n = 9). The image quantification procedure is described in Figure S5J. Error bars represent SD. (G) E-cadherin was immunoprecipitated from lysates of cells cultured at low (lanes 1 and 4) or high density (lanes 2, 5, and 7), or cells cultured at high density with blebbistatin (lanes 3 and 6). Scale bars represent 20 μm. Cell Reports 2017 20, 1435-1447DOI: (10.1016/j.celrep.2017.07.032) Copyright © 2017 The Author(s) Terms and Conditions

Figure 6 A Hypothetical View of the Mechanism by which Circumferential Actin Belt Tension Suppresses YAP/TAZ Nuclear Localization When cells are at low density, Merlin is associated with E-cadherin (left). When cells are at high density, contraction of the circumferential actin belt releases Merlin from E-cadherin. Merlin is then imported into the nucleus and acts to export nuclear YAP/TAZ into the cytoplasm. Cell Reports 2017 20, 1435-1447DOI: (10.1016/j.celrep.2017.07.032) Copyright © 2017 The Author(s) Terms and Conditions