TAZ through Nucleocytoplasmic Shuttling of Merlin

Article

The Epithelial Circumferential Actin Belt Regulates YAP/TAZ through Nucleocytoplasmic Shuttling of Merlin Graphical Abstract

Authors Kana T. Furukawa, Kazunari Yamashita, Natsuki Sakurai, Shigeo Ohno

Correspondence [email protected]

In Brief Furukawa et al. demonstrate an actin cytoskeleton-mediated YAP/TAZregulatory mechanism by which circumferential actin belt contraction suppresses nuclear localization of YAP/ TAZ. They further show that YAP/TAZ physically interacts with Merlin, and YAP/ TAZ nucleocytoplasmic translocation is dependent on Merlin nuclear export sequences.

Highlights d

Circumferential actin belt contraction suppresses YAP/TAZ nuclear localization

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Merlin physically interacts with YAP/TAZ and suppresses its nuclear localization

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Merlin nuclear export signal domains are required for YAP/ TAZ translocation

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This actin belt-Merlin-YAP/TAZ axis acts independently of Hippo signaling

Furukawa et al., 2017, Cell Reports 20, 1435–1447 August 8, 2017 ª 2017 The Author(s). http://dx.doi.org/10.1016/j.celrep.2017.07.032

Cell Reports

Article The Epithelial Circumferential Actin Belt Regulates YAP/TAZ through Nucleocytoplasmic Shuttling of Merlin Kana T. Furukawa,1,2 Kazunari Yamashita,1,2 Natsuki Sakurai,1 and Shigeo Ohno1,3,* 1Department

of Molecular Biology, Graduate School of Medical Science, Yokohama City University, Yokohama 236-0004, Japan authors contributed equally 3Lead Contact *Correspondence: [email protected] http://dx.doi.org/10.1016/j.celrep.2017.07.032 2These

SUMMARY

Circumferential actin belts underlying the adherens junctions of columnar epithelial cell monolayers control intercellular surface tension and cell shape to maintain tissue integrity. Yes-associated protein (YAP) and its paralog TAZ are proliferation-activating transcriptional coactivators that shuttle between the nucleus and cytoplasm. Previous studies suggest the importance of stress fibers in the actin cytoskeleton for regulation of YAP nuclear localization; however, the role of the circumferential actin belt on YAP localization remains unclarified. By manipulating actin tension, we demonstrate that circumferential actin belt tension suppresses YAP/TAZ nuclear localization. This suppression requires Merlin, an F-actin binding protein associated with adherens junctions. Merlin physically interacts with YAP/TAZ, and nuclear export sequences of Merlin are required for suppression. Together, with the observation that the association between E-cadherin and Merlin was diminished by tension in circumferential actin belts, our results suggest that released Merlin undergoes nucleocytoplasmic shutting and mediates export of YAP/TAZ from the nucleus. INTRODUCTION A growing tissue will stop cell proliferation when it has achieved its correct size during development or regeneration. Studies on Drosophila imaginal disc epithelia suggest that mechanical compression concomitant with increased size counteracts growth factor-mediated induction of proliferation to limit further organ growth (Aegerter-Wilmsen et al., 2012; Hufnagel et al., 2007). Studies on cultured epithelial cells have also revealed the importance of mechanical and spatial constraints caused by cell crowding (i.e., space limitation due to the presence of neighboring cells) for the prevention of cell proliferation (Streichan et al., 2014). Collectively, these studies have established that cellular signaling pathways that transduce mechanical stresses exerted by the environment and neighboring cells are critical for the regulation of cell proliferation and organogenesis.

One of the most intensively studied signaling factors involved in mechanotransduction for regulation of cell proliferation is Yesassociated protein (YAP). YAP, its paralog TAZ (transcriptional co-activator with PDZ-binding motif, WWTR1), and the fly homolog Yorkie are transcriptional coactivators that shuttle between the cytoplasm and the nucleus where they associate with several transcription factors such as TEA domain family members (TEAD) to activate genes involved in cell proliferation (Huang et al., 2005; Moroishi et al., 2015; Vassilev et al., 2001). The signaling cascades that regulate YAP and TAZ include the Hippo signaling pathway, which is involved in organ size control in both Drosophila and mammals (Dong et al., 2007). The core component of the Hippo pathway includes the kinases MST (Mammalian STE20-like, Hippo in Drosophila) and LATS (large tumor suppressor homolog, Warts in Drosophila). MST1/2 phosphorylates LATS1/2, and activated LATS1/2, in turn, phosphorylates several serine residues on YAP and TAZ, resulting in suppression of YAP and TAZ nuclear localization (Dong et al., 2007; Pan, 2010). The upstream factors of the Hippo pathway include Merlin, KIBRA, and several other cell adhesion molecules and cell polarity-regulating proteins that can localize at cell-cell contacts, suggesting the importance of epithelial cell-cell junctions in growth-dependent control of proliferation (Genevet and Tapon, 2011; Hamaratoglu et al., 2006; Yu et al., 2010; Zhang et al., 2010). In addition, several Hippo-independent mechanisms of YAP/TAZ regulation have been reported. Both Amot and its family proteins physically bind YAP and sequester it in the cytoplasm (Wang et al., 2011; Zhao et al., 2011). Wnt signaling also regulates TAZ independently of the Hippo pathway by preventing proteasome-mediated degradation (Azzolin et al., 2012) and localization of YAP and TAZ by sequestration in the b-catenin destruction complex (Azzolin et al., 2014). The mechanical tension exerted by the environment and neighboring cells in epithelial tissues converge at two distinct cell adhesion structures, the focal adhesions that link cells to the extracellular matrix (cell-ECM) and cell-cell junctions that link adjacent cells (Chen et al., 2004). Tension on these structures alters the organization and contractile activity of cortical actin-myosin fibers, redistributes their intracellular forces, controls the maturation of adhesion structures, and initiates intracellular signaling cascades that ultimately influence many cellular behaviors, including proliferation. Recent studies have revealed the importance of YAP and TAZ in transduction of mechanical stress initiated by cell-ECM

Cell Reports 20, 1435–1447, August 8, 2017 ª 2017 The Author(s). 1435 This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

adhesion under specific conditions. For instance, ECM stiffness and Rho-mediated tension of actin stress fibers regulates YAP or TAZ nuclear localization and activation (Aragona et al., 2013; Dupont et al., 2011; Wada et al., 2011). However, the importance of mechanical stresses mediated by cortical actin-myosin networks underlying adherens junctions on YAP/TAZ activation remains poorly understood, although the functions of these networks in regulation of cell shape, tissue patterning, and morphogenesis are firmly established (Harris and Tepass, 2010; Hoffman and Yap, 2015). There are several types of cortical actin-myosin networks in columnar epithelial cell layers. These include circumferential actin belts underlying adherens junctions, apical cortical networks, lateral cortical networks, and stress fibers. Among them, circumferential actin belts exert strong tension and act as direct linkers between adjacent cells and intracellular signaling cascades (Wu et al., 2014) (see Figure 2A). A recent study demonstrated the importance of E-cadherin-dependent YAP activation in mechanical strain-induced proliferation of epithelial monolayer cells (Benham-Pyle et al., 2015) and another reported the involvement of E-cadherin in activation of the Hippo pathway in cancer cells (Kim et al., 2011). However, the roles of epithelia-specific cortical actinmyosin networks such as the circumferential actin belt on cell proliferation remains to be clarified. In this study, we evaluated the role of epithelia-specific cortical actin-myosin networks such as the circumferential actin belt on nuclear localization of YAP and TAZ in well-polarized columnar epithelial cells. We provide evidences suggesting that tension in circumferential actin belts suppresses YAP/TAZ nuclear localization and that this suppression involves nuclear export of Merlin. RESULTS Cell Density Regulates YAP/TAZ Nuclear Localization to Control Proliferation in MDCK Cells Cell density-dependent changes in the localization of YAP and TAZ have been reported in a variety of cells, including fibroblasts and epithelial cells. To evaluate the localization of YAP/TAZ, we used the antibody recognizing both YAP and TAZ for immunofluorescence (Figures 1A and 1D). In low-density MDCK epithelial cells, endogenous YAP/TAZ localized almost exclusively to the nucleus. However, YAP/TAZ nuclear localization was greatly suppressed at high cell density and YAP/TAZ distributed to both cytoplasm and nucleus (Figure 1A). Consistent with suppression of YAP/TAZ nuclear localization, expression levels of the YAP/TAZ target genes connective tissue growth factor (CTGF) and cysteine-rich angiogenic inducer 61 (Cyr61) (Hansen et al., 2015), were greatly downregulated at high cell density (Figure 1B). Further, small interfering RNA (siRNA)-mediated knockdown of YAP or TAZ and simultaneous knockdown of both YAP and TAZ suppressed cell proliferation (Figures 1C–1E). These results suggest that both YAP and TAZ are involved in density-dependent inhibition of MDCK cell proliferation. Western blot analysis revealed that phosphorylation of YAP on serine 127, a key target of the LATS1/2 kinase (Pan, 2010), was not significantly enhanced at high density, although YAP/ TAZ nuclear localization was greatly suppressed (Figures 1F

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and 1G). This suggests that mechanisms other than the Hippo pathway may be involved in the suppression of YAP/TAZ nuclear localization in polarized MDCK cells at high density. Localization and Function of YAP and TAZ Are Regulated by Contraction of Circumferential Actin Belts Detailed observation of medium-density MDCK monolayer cells revealed two types of cells, one showing nuclear localization of YAP/TAZ and the other with YAP/TAZ expressed in both cytoplasm and nucleus (whole cell localization hereafter) (Figure 2B). Cells with nuclear YAP/TAZ did not show cortical staining for myosin IIB or contracted circumferential actin belts as indicated by F-actin staining (Figure 2B). On the other hand, cells with whole cell YAP/TAZ localization showed both staining for myosin IIB and contracted circumferential actin belts at apical cell-cell junctions (Figures 2B and S1A). Because association of myosin II with F-actin reflects an increase in F-actin mechanical tension (Uyeda et al., 2011), the correlation between the loss of myosin II at cell-cell contacts and enhanced nuclear YAP/TAZ suggests that contraction of circumferential actin belts may suppress YAP/TAZ nuclear localization. To evaluate this hypothesis, we examined YAP/TAZ cell distribution in cells treated for 3 hr with blebbistatin, an inhibitor of myosin II that relieves F-actin tension. At low cell density, blebbistatin did not affect nuclear YAP/TAZ distribution, whereas it reduced the number of actin stress fibers (Figure 2C). At high cell density, however, where YAP/TAZ nuclear localization is suppressed, blebbistatin enhanced nuclear YAP/TAZ localization (Figures 2C and 2D). Blebbistatin treatment also increased whole cell perimeter length as evaluated by F-actin staining and decreased myosin IIB staining at cell-cell junctions, confirming that blebbistatin disrupted tension in circumferential actin belts (Figures 2E and S1B). On the other hand, cell shape was not substantially changed and staining for b-catenin, a major component of the adherens junction, was not affected (Figures S1C and S1D). All these results support the notion that mechanical tension in circumferential actin belts was reduced by blebbistatin treatment with minimal effects on other cell structures in this condition. Using TAZ-specific antibody, similar effect of blebbistatin was observed. In addition, blebbistatin treatment increased cell proliferation (Figures 2F and 2G) and upregulated CTGF and Cyr61 mRNA expression levels at high cell density (Figure 2H). Similar results were obtained using Y-27632, an inhibitor of ROCK that phosphorylates myosin light chain and activates myosin II, resulting in decreased F-actin tension (Figure S1E). Conversely, overexpression of a constitutively activated ROCK mutant (ROCKD3) (Ishizaki et al., 1997) in low density monolayers resulted in an ectopically organized and shrunken circumferential actin belt-like structure, as indicated by F-actin-staining, as well as suppression of YAP/TAZ nuclear localization (Figure 2I). Under this condition, expression of a TEAD-responsive element luciferase reporter (8xGTIIC-luc) was also suppressed (Figure 2J) (Ota and Sasaki, 2008). Blebbistatin treatment of high density cells did not change phosphorylation status (Figures S1F and S1G), suggesting that a mechanism other than the Hippo pathway inhibits YAP/TAZ nuclear localization at high cell density. Several studies reported that inhibition of stress fibers

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 mm. (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 3 105 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 densitydependent cell-cycle arrest. (G) Densitometry of the data in (F). Intensity of S127P-YAP was normalized to YAP (n = 3). Error bars represent SD.

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suppresses YAP/TAZ nuclear localization (Aragona et al., 2013; Dupont et al., 2011; Wada et al., 2011). In MDCK cells, however, inhibition of stress fibers did not affect YAP/TAZ nuclear localization in cells at low density, which develop many stress fibers, but did affect YAP/TAZ nuclear localization in cells at high density, which express organized epithelia-specific actomyosin networks in addition to stress fibers. These results suggest that tension within epithelia-specific actomyosin networks including circumferential actin belts may suppress YAP/TAZ nuclear localization, while stress fibers have little effect on YAP/TAZ localization in columnar epithelial cells. To test the universality of the above observation, we analyzed other columnar epithelial cell lines. Very similar results were obtained in Caco-2 and Eph4 cells; high cell density inhibited nuclear YAP/TAZ localization and blebbistatin rescued YAP/ TAZ nuclear localization (Figures S2A and S2B). On the other hand, blebbistatin inhibited nuclear localization of YAP/TAZ in low density DLD-1 cells (Figure S2C), suggesting that localization of YAP/TAZ is largely dependent on stress fiber-mediated regulation in this line, similarly to fibroblasts and mesenchymal stem cells (Dupont et al., 2011; Wada et al., 2011). Thus, the epithelia-specific actomyosin-mediated inhibition of YAP/TAZ nuclear localization is not a response specific to MDCK cells; however, among epithelial cells, there are subtypes in which stress fibers do strongly contribute to nuclear localization of YAP/TAZ. In these cell lines except MDCK cell, YAP was strongly detected when compared to TAZ by western blotting using YAP/TAZ-recognizing antibody (Figure S4J; data not shown), suggesting that YAP predominantly expresses in these cells. Together with the result obtained by TAZ-specific antibody (Figure 2F), these results suggest that both YAP and TAZ were similarly affected by blebbistatin treatment. The methods used to suppress or enhance tension of F-actin thus far target the entire actomyosin machinery in epithelial cell monolayers, including circumferential actin belts, stress fibers, and lateral F-actin networks. To analyze the specific effect of

circumferential actin belt contraction, we employed N-WASP and aPKC. N-WASP is an actin regulator that specifically localizes to the circumferential actin belt and is necessary for activation of Arp2/3 and exert tension in the actin belt (Wu et al., 2014). Depletion of N-WASP in high density-cultured MDCK cells resulted in translocation of YAP/TAZ into the nucleus with wider cell perimeter (Figures S2D and S2E), supporting that tension of circumferential actin belts is important for inhibition of YAP/TAZ nuclear localization. As the second approach to test the specificity to circumferential actin belts, we depleted aPKC (PKC l and z), cell polarity-regulating kinases (Suzuki and Ohno, 2006). ROCK and aPKC co-localize at the circumferential actin belt in polarized MDCK cells, where ROCK is phosphorylated by aPKC, resulting in inactivation of ROCK-dependent apical constriction (Ishiuchi and Takeichi, 2011). As expected, PKC l and z double knockdown in low-density MDCK cells resulted in contraction of the circumferential actin belt, an increase in myosin light chain (MLC) phosphorylation, inhibition of YAP/ TAZ nuclear localization (Figure S2F), decreased expression of CTGF (Figure S2G), and suppressed cell proliferation (Figure S2H) without affecting the phosphorylation level of YAP (Figure S2G). Further, blebbistatin rescued YAP/TAZ nuclear localization (Figure S2I). Collectively, these results strongly support the notion that contraction of the circumferential actin belt suppresses nuclear localization of YAP and TAZ. Mechanical Tension-Induced Suppression of YAP/TAZ Nuclear Localization Involves Nuclear Export To describe the molecular mechanisms of mechanical tensioninduced suppression of YAP nuclear localization in greater detail, we tested whether nuclear localization of the YAP 5SA mutant, in which five LATS1/2 phosphorylation sites were mutated, can also be suppressed by ROCKD3. Wild-type Flag-YAP was distributed to both the cytoplasm and nucleus. On the other hand, Flag-YAP 5SA localized to the nucleus (Figures 3A and 3B), consistent with a previous study (Zhao et al., 2007). Intriguingly, co-expression of ROCKD3, but not EGFP, attenuated

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 3 105/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 mM 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-ROCKD3 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 ROCKD3 significantly inhibited TEAD reporter activity (n = 3). Error bars represent SD. Scale bars represent 20 mm.

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nuclear localization of not only YAP WT but also YAP 5SA (Figures 3A and 3B). This supports the notion that the mechanism for inhibition of YAP nuclear localization by increased tension of circumferential actin belts involves mechanisms distinct from the Hippo pathway. We next compared the dynamics of suppressed YAP/TAZ nuclear localization caused by ROCKD3 with that caused by LATS2. Leptomycin B is an inhibitor of exportin 1 and can lead to nuclear accumulation of YAP/TAZ, presumably by inhibiting nuclear export (Dupont et al., 2011). As shown in Figure 3C, treatment of high-density MDCK cells with leptomycin B enhanced YAP/TAZ nuclear localization. In addition, treatment of ROCKD3-expressing low-density cells with leptomycin B enhanced YAP/TAZ nuclear localization, and this enhancement reached saturation within 30 min (Figures 3D, 3F, and 3G). In contrast, leptomycin B treatment of low-density cells expressing LATS2 enhanced YAP/TAZ nuclear localization very slowly compared to ROCKD3-expressing cells (Figures 3E–3G), suggesting that the mechanisms of YAP/TAZ nuclear localization inhibition by ROCKD3 and LATS2 are distinct. The very rapid rescue of ROCKD3-mediated YAP/TAZ nuclear localization suppression by leptomycin B suggests that mechanical tension of F-actin may enhance YAP/TAZ nuclear export by regulating an as yet unknown signaling pathway. On the other hand, LATS2 seems to affect YAP/TAZ nuclear import rather than export. Merlin Is Required for Inhibition of YAP/TAZ Nuclear Localization Mediated by F-Actin Contraction We next screened for intracellular factors that can transduce the contractile force of the circumferential actin belt into a signal controlling localization of YAP/TAZ. We assumed that candidate regulators would associate with actin filaments and also regulate YAP/TAZ nuclear localization. First, we focused on PTPN14, a YAP/TAZ regulator localized to cortical actin that increases in expression with cell density in MCF10A cells (Wang et al., 2012). However, PTPN14 protein level did not increase at high MDCK cell density (Figures S4A and S4B), suggesting that it is not involved in MDCK cells. Next, we focused on Amot, an F-actin-binding protein that also regulates YAP nuclear localization (Wang et al., 2011; Zhao et al., 2011). However, knockdown of AmotL2 (Angiomotin Like 2), the major protein of the Amot

family in MDCK cells, in ROCKD3 expressing low-density MDCK cells failed to rescue YAP/TAZ nuclear localization (Figures S4C and S4D). Merlin is an F-actin-binding protein implicated in densitydependent inhibition of cell proliferation through suppression of YAP nuclear localization (Scoles, 2008; Zhang et al., 2010). Knockdown of Merlin rescued suppression of YAP/TAZ nuclear localization caused by ROCKD3 without affecting contractility of the circumferential actin belt-like structure (Figures 4A–4C). This suggests that Merlin may transduce increased tension of circumferential actin belts into a signal suppressing YAP/TAZ nuclear localization. Consistent with this notion, YAP/TAZ was localized to the nucleus in Merlin-knockdown cells at high cell density, and CTGF was upregulated, although Merlin does not appear to modulate the Hippo pathway in MDCK cells (Figures 4D, 4E, and S4E–S4G). In addition, we overexpressed Merlin isoforms in low-density cultures and found that both isoform 1 and isoform 2 suppressed YAP/TAZ nuclear localization without affecting the Hippo pathway (Figures 4F, 4G, and S4H). Furthermore, a physical interaction of Merlin with YAP and TAZ was detected by immunoprecipitation using MDCK cells, and a physical interaction of Merlin with YAP using Caco-2 cells, respectively (Figures 4H, S4I, and S4J). The amount of Merlin also increased at high cell density, resulting in greater Merlin-YAP/TAZ complex accumulation. These observations are consistent with the notion that Merlin directly controls YAP/TAZ nuclear localization downstream of circumferential actin belt contraction. Nuclear Export Signal-Dependent Nucleocytoplasmic Shuttling of Merlin Is Necessary for the Suppression of YAP/TAZ Nuclear Localization The prompt rescue of ROCKD3-mediated suppression of YAP/ TAZ nuclear localization by leptomycin B suggests that nuclear export of YAP/TAZ is critical for controlling its cellular localization. In light of results showing that Merlin interacts with YAP/ TAZ and suppresses YAP/TAZ nuclear localization, we speculated that YAP/TAZ is exported to the cytoplasm by Merlin at high cell density. Indeed, leptomycin B rescued Merlin-mediated suppression of YAP/TAZ nuclear localization more rapidly than LATS2-mediated suppression (Figures 3F, 3G, and S3B). Merlin has a noncanonical nuclear localization signal (NLS) sequence

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-ROCKD3 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 ROCKD3-mediated suppression of YAP/TAZ nuclear localization. Episomally stable transformant MDCK cells were cultured at low density and induced to express ROCKD3 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 ROCKD3-, LATS2-, and Merlin-transfected cells (from F) (n = 3). Error bars represent SD. Scale bars represent 20 mm.

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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 ROCKD3. Cells were cultured at low density. Arrowheads indicate nuclei of ROCKD3-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.

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and a well characterized nuclear export signal (NES) sequence, and several groups reported that Merlin functions in the nucleus (Figure 5A) (Hikasa et al., 2016; Kressel and Schmucker, 2002; Li et al., 2014). Considering these attributes, the nuclear export of YAP/TAZ may be mediated by Merlin NES. To test this possibility, we first confirmed that Merlin shuttles between the cytoplasm and nucleus. Ectopically expressed Merlin mainly localized in cytoplasm (Figure 5B), but accumulated in the nucleus following leptomycin B treatment, indicating that Merlin is imported to nucleus and exported by an exportin 1-dependent mechanism (Figure S3B). Thus, we next mutated the C-terminal NES (NES3) reported previously (Kressel and Schmucker, 2002). However, mutation of NES3 did not significantly affect Merlin localization and only slightly reduced its capacity to export YAP/TAZ to the cytoplasm (Figures 5B–5D). However, we identified two additional regions that resembled the NES consensus sequence (Xu et al., 2012), termed NES1 and NES2 (Figure 5A). Mutation of all three candidate NESs abolished the export of Merlin to the cytoplasm, indicating that these sequences are genuine NESs (Figures 5B and 5C). More importantly, a Merlin mutant lacking all three NESs failed to suppress YAP/TAZ nuclear localization (Figures 5B and 5D). Further, ectopic expression of Merlin suppressed cell proliferation, while the Merlin mutant lacking all three NESs failed to suppress cell proliferation (Figures S5B and S5C). We also confirmed that Merlin NES mutants preserved interaction with YAP and KIBRA, other interacting proteins of Merlin (Zhang et al., 2010) (Figure S5D). Together, these results support the notion that Merlin NESs are involved in the export of YAP/TAZ from the nucleus to suppress its function. All three Merlin NESs are conserved from fruit fly to human, suggesting the importance of nuclear export to Merlin function across species (Figure S5A). To further confirm the importance of nucleocytoplasmic shuttling of Merlin, we used previously reported NLS mutant of Merlin, 24-27A (Li et al., 2014). As expected, the NLS mutant failed to inhibit YAP/TAZ nuclear localization and cell proliferation (Figures S5G–S5I), supporting that nuclear import is also required for this process. Together with the results obtained using Merlin NES mutants, these results suggest that nucleocytoplasmic shuttling, especially nuclear export of Merlin, is important for inhibition of YAP/TAZ nuclear localization. A previous report described a nuclear function of Merlin in mesothelioma cell line; Merlin inhibits YAP nuclear localization through inhibiting CRL4[VprBP/DCAF1] to prevent LATS1 protein from degradation and to activate LATS2 (Li et al., 2014). This mechanism appears to contradict our result that Merlin NES mutants, which preferentially accumulate in the nucleus, have weaker effect on inhibition of YAP/TAZ nuclear localization (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 mm.

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 HAMerlin-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 mm.

than wild-type Merlin. In addition, the NES1,2,3 mutant failed to interact with VprBP, implying that the dysfunction of this mutant might involve the lack of interaction with VprBP (Figure S5D). However, knockdown of VprBP failed to upregulate protein level and autophosphorylation of LATS1 (Chan et al., 2005) and also failed to inhibit nuclear localization of YAP/TAZ in MDCK cells (Figures S5E and S5F). These results suggest that the effect of Merlin on YAP/TAZ is not mediated through the Merlin-VprBP pathway but through nuclear export of Merlin in MDCK cells, polarized epithelial cells. Close inspection of endogenous Merlin subcellular distribution revealed that cytoplasmic and nuclear Merlin were moderately abundant in high-density MDCK cells but decreased following blebbistatin treatment (Figure 5E). The signal intensity ratio of Merlin in the nuclear and cytoplasmic region to Merlin at the cell-cell contact region was higher at high density than at

low density, and blebbistatin reversed this increase (Figure 5F). This result suggests that Merlin is released from cellcell contact regions into the cytoplasm and nucleus to function as an adaptor connecting YAP/TAZ to the nuclear export receptor following circumferential actin belt contraction. Importantly, Merlin strongly associated with E-cadherin in cells at low density as reported previously (Gladden et al., 2010), but this association was greatly diminished at high cell density when YAP/TAZ nuclear localization is inhibited and rescued by blebbistatin treatment (Figure 5G). These results also imply that high tension in circumferential actin belts disrupts the association between Merlin and E-cadherin. In other words, Merlin is released from the adherens junction into the cytoplasm and shuttles between the cytoplasm and the nucleus using its own NLS and NESs, resulting in the export of YAP/TAZ from the nucleus (Figure 6). DISCUSSION In the present study, we provide evidence that tension in the circumferential actin belt at high density epithelial cells

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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.

suppresses YAP/TAZ nuclear localization, thereby inhibiting cell proliferation. Our results apparently contradict reports that F-actin tension activates YAP/TAZ nuclear localization (Aragona et al., 2013; Dupont et al., 2011; Wada et al., 2011). However, the F-actin tension studied in previous reports was the tension in stress fibers induced by mechanical stress at ECM-cell junctions, and not the tension in circumferential actin belts. Mechanical stresses from ECM substrates such as substrate stiffness exert tension in stress fibers via focal adhesion, while circumferential actin belts exert tension between adjacent cells via adherens junctions. Consequently, these two pathways can be mechanically independent. Together with the previous reports, our results suggest the presence of multiple mechanotransduction pathways mediated by distinct actin cytoskeletal components regulating nuclear localization of YAP and TAZ. Several reports provide in vivo and in vitro evidence supporting our hypothesis that contraction of the actin cytoskeleton is involved in suppression of cell proliferation, although the mechanisms remain unclear. For example, Y-27632 promoted the proliferation of corneal and limbal epithelial cells in vivo and in vitro, respectively (Sun et al., 2015). Depletion of mDia, an actin nucleator, enhanced proliferation of mouse neuroepithelial cells (Thumkeo et al., 2011). Depletion of ArhGEF18 (p114RhoGEF), which regulates RhoA/ROCK and cortical F-actin, induced proliferation of neuroepithelial cells in Medaka fish (Herder et al., 2013). In addition, cell density- and F-actindependent inactivation of YAP occurred during caudal fin regeneration in Zebrafish (Mateus et al., 2015). YAP and TAZ shuttles between the nucleus and cytoplasm in a context-dependent manner, and several studies have provided insights into how YAP translocates into the nucleus. The homolog Yorkie has a non-canonical NLS that interacts with importin a1, and importin a1-mediated nuclear transport of Yorkie is Hippo pathway-dependent, consistent with our result that the Hippo pathway may inhibit the nuclear import of YAP/TAZ

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(Figures 3D–3G) (Wang et al., 2016). In addition, YAP interacts with ZO-2, and translocation of YAP to the nucleus is dependent on ZO-2 NLSs (Oka et al., 2010). In contrast to nuclear import, little is known about the mechanisms of YAP/TAZ nuclear export as YAP/TAZ lacks NES consensus sequences (data not shown). Thus, it is plausible to speculate that YAP/TAZ utilizes Merlin NESs for export. Merlin/NF2 is a bona fide tumor suppressor protein implicated in stable AJ formation by interacting with E-cadherin complex and F-actin, as well as in contact inhibition of cell proliferation (Gladden et al., 2010; Scoles, 2008). Merlin inhibits the RasMAPK pathway (Curto et al., 2007; Yi et al., 2011) and is an upstream mediator of the Hippo pathway. Further, several studies proposed that Merlin is a nuclear-cytoplasmic shuttling protein that functions in the nucleus (Hikasa et al., 2016; Kressel and Schmucker, 2002; Li et al., 2014). In the present study, we have identified two additional NESs and revealed their involvement in the suppression of nuclear localization of the MerlinYAP/TAZ complex, yet components and functions of the whole complex are not fully understood. Taken together, the pathway through which Merlin controls YAP/TAZ may differ among cell types and under distinct conditions. A great decrease in the association between Merlin and E-cadherin at high cell density is intriguing because it suggests that mechanical tension could induce remodeling of protein complexes at cell adhesion sites. A variety of proteins, including a-catenin, have been reported to associate with Merlin (Gladden et al., 2010). Other lines of evidence indicate that cortical Merlin is relocalized to the cytoplasm by pulling forces from actomyosin-based contractility during collective MDCK cell migration, suggesting that Merlin acts as mechanotransducer (Das et al., 2015). The association between Merlin and the protein complex containing E-cadherin may be controlled by tension-induced remodeling of mechanotransducing proteins at the adherens junction. One possible candidate molecule is a-catenin, in which

a conformational change induced by actomyosin-mediated stretching unmasks a binding site for vinculin, resulting in more robust actin binding (Hoffman and Yap, 2015; le Duc et al., 2010; Yonemura et al., 2010). Despite the dramatic decrease in the association between Merlin and E-cadherin under tension, Merlin did not completely disappear from cell-cell contact regions (Figure 5E). This suggests that associations between Merlin and other proteins localized to the cell cortex, such as CD44, ERM, and NHERF, are not sensitive to mechanical tension of the actin cytoskeleton (Scoles, 2008). One of the hallmarks of cancer is disruption of cell layers accompanied by deregulated cell proliferation. Disappearance of E-cadherin and deregulation of cell polarity are among the causes of such aberrant changes (Coopman and Djiane, 2016; Tanos and Rodriguez-Boulan, 2008; van Roy and Berx, 2008), in which adherens junctions and circumferential actin belts are also severely affected. The signaling axis from epithelia-specific actin-myosin network to YAP/TAZ suppression mediated by Merlin nuclear export (the circumferential actin belt-MerlinYAP/TAZ axis) suggested by the present study may explain one of the causes of cancer and the tumor suppressor mechanisms of Merlin. In support of this notion, mutations in the NESs of Merlin have been reported to occur in various cancer cells (Table S1). EXPERIMENTAL PROCEDURES Detailed methods can be found in the Supplemental Experimental Procedures. Cell Culture, Transfection, and Drug Treatment MDCKII, Caco-2, Eph4, and DLD-1 cells were cultured in Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum and 100 U/mL penicillin/streptomycin at 37 C in a humidified atmosphere containing 5% CO2. For experiments comparing low- and high-density monolayers, 3 3 104 (low) or 3 3 105 (high) MDCK cells were seeded per well in 12-well Transwell plates (Corning) and cultured for 48 hr. For transient transfection of low-density cultures, 5 3 104 cells were plated on coverslips (24-well size) and cultured for 24 hr. Cells were then transfected with the indicated plasmids using Lipofectamine 2000 (Invitrogen) or Lipofectamine LTX (Invitrogen) and cultured for an additional 48 hr. Prolonged constitutive expression of ROCKD3 resulted in excessive cell shrinkage followed by death (data not shown). To avoid this, we used the pOS-tet14 vector (Tet-On system) to control the timing of ROCKD3 expression (Mishima et al., 2004). To induce protein expression, 200 ng/mL doxycycline was added 6 hr before fixation. To efficiently introduce siRNAs, MDCK cells were transfected twice using Lipofectamine RNAi MAX (Invitrogen). Cells were treated with 50 mM (±)-blebbistatin (Calbiochem) or 20 mM Y-27632 (Calbiochem) for 3 hr or 6 hr. DMSO was used as a vehicle for blebbistatin and H2O as a vehicle for Y-27632. Cells were treated with 40 ng/mL leptomycin B (Calbiochem) for 2 hr with ethanol as a vehicle.

et al., 1999). Merlin isoforms 1 and 2 were subcloned into pCAG-GS with N-terminal V5 or HA tags. Merlin NES mutants were generated by PCR-mediated site-direct mutagenesis. The target sequences of siRNA are listed in the Supplemental Information. Antibodies Anti-YAP1 (H00010413-M01) was obtained from Abnova, anti-GAPDH (ab8245) and anti-CTGF (ab6992) from Abcam, and anti-GFP (GFP-1010) from Aves Labs. Anti-TAZ (560235), anti-p27Kip1 (610241), anti-BrdU (347580), and anti-E-cadherin (610181) were purchased from BD BioScience, and anti-LATS1/2 (A300-479A) from Bethyl Laboratories. Anti-MST1 (3682), anti-phospho-T183-MST1/T180-MST2 (3681), anti-LATS1 (3477), anti-phospho-S909-LATS1 (9157), anti-phospho-S19-Myosin Light Chain 2 (3675), anti-phospho-S127-YAP (4911), anti-myc tag (2272), anti-N-WASP (4848), and anti-NF2 (6995) were obtained from Cell Signaling Technology. AntiCTGF (sc-14939), anti-PKC z (sc-216), b-catenin (sc-7199), and normal mouse immunoglobulin G (sc-2025) were from Santa Cruz Biotechnology. Anti-FLAG (F-3165, M2), anti-NF2 (HPA003097), and myosin IIB (M7939) were from Sigma, anti-Ki67 (NCL-Ki67p) from Leica Biosystems, anti-HA (3F10) from Roche, anti-VprBP (11612-1-AP) from Proteintech, and anti-V5 (46-0705) from Life Technologies. Anti-KIBRA was described previously (Yoshihama et al., 2011). Immunofluorescence and Quantification of Fluorescent Signals Cells were fixed with 2% paraformaldehyde in PBS and permeabilized with 0.5% Triton X-100 in PBS. Images were obtained using an epifluorescent microscope (AxioImager; Carl Zeiss) or a confocal laser scanning microscope system (LSM700; Carl Zeiss). Real-Time qRT-PCR Total RNA was extracted from cells using the RNeasy Plus Mini Kit (QIAGEN). The SuperScript VILO cDNA Synthesis Kit (Invitrogen) was used for cDNA synthesis. Real-time qPCR analyses were conducted in triplicate using iQ SYBR Green Supermix (Bio-Rad) with retro-transcribed cDNAs and primers on a iCycler thermal cycler equipped with MyiQ (Bio-Rad). Statistics and sequences of primers are described in the Supplemental Information. BrdU Incorporation Assays Cells were cultured in 100 mM BrdU-containing medium for 3 hr before fixation followed by staining as described previously (Yamashita et al., 2015). Statistical Analysis The results are presented as mean ± SD. Differences were considered statistically significant if p < 0.05 by Student’s t test. Single asterisk and double asterisks denote p < 0.05 and p < 0.01, respectively. NS means no significance. To quantify immunofluorescence intensity, at least 30 cells in every sample were analyzed in one experiment unless otherwise indicated. SUPPLEMENTAL INFORMATION Supplemental Information includes Supplemental Experimental Procedures, five figures, and one table and can be found with this article online at http:// dx.doi.org/10.1016/j.celrep.2017.07.032. AUTHOR CONTRIBUTIONS

Expression Vectors and Small Interfering RNAs pcDNA-HA-LATS2 and pGL3-8 3 GTIIC-Luc were kind gifts from Dr. Hiroshi Sasaki (Ota and Sasaki, 2008). p2 3 Flag CMV2-YAP2 and pCMV-flag YAP2 5SA were obtained from Addgene (#19045 submitted by Dr. Marius Sudol [Oka et al., 2008], #27371 submitted by Dr. Kunliang Guan [Zhao et al., 2007]). pRL-TK was obtained from Promega. pCAG-myc-ROCK was a kind gift from Dr. Shuh Narumiya (Ishizaki et al., 1997). A partial sequence of ROCK (ROCKD3) was amplified by PCR and subcloned into the pOS-Tet14 vector with an N-terminal Myc tag sequence. EGFP was amplified from pEGFP-N1 (Clonetech) by PCR and subcloned into the pOS-Tet14 vector. pCAG-HA-Merlin isoform 2 was a gift from Dr. Sachiko Tsukita (Maeda

K.T.F. performed most of experiments. K.Y. and N.S. contributed to the experimental work. K.Y., K.T.F., and S.O. conceived and designed the study. K.Y., K.T.F., and S.O. wrote the manuscript. ACKNOWLEDGMENTS We thank Dr. H. Sasaki for providing materials. This work was supported in part by the grant for Creation of Innovation Centers for Advanced Interdisciplinary Research Areas Program from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to S.O.), JSPS KAKENHI

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(JP23112003, JP22247030 to S.O. and JP17K17991 to K.Y.), and the Yokohama Foundation for Advancement of Medical Science (K.Y.). Received: October 13, 2016 Revised: April 19, 2017 Accepted: July 10, 2017 Published: August 8, 2017 REFERENCES Aegerter-Wilmsen, T., Heimlicher, M.B., Smith, A.C., de Reuille, P.B., Smith, R.S., Aegerter, C.M., and Basler, K. (2012). Integrating force-sensing and signaling pathways in a model for the regulation of wing imaginal disc size. Development 139, 3221–3231. Aragona, M., Panciera, T., Manfrin, A., Giulitti, S., Michielin, F., Elvassore, N., Dupont, S., and Piccolo, S. (2013). A mechanical checkpoint controls multicellular growth through YAP/TAZ regulation by actin-processing factors. Cell 154, 1047–1059. Azzolin, L., Zanconato, F., Bresolin, S., Forcato, M., Basso, G., Bicciato, S., Cordenonsi, M., and Piccolo, S. (2012). Role of TAZ as mediator of Wnt signaling. Cell 151, 1443–1456. Azzolin, L., Panciera, T., Soligo, S., Enzo, E., Bicciato, S., Dupont, S., Bresolin, S., Frasson, C., Basso, G., Guzzardo, V., et al. (2014). YAP/TAZ incorporation in the b-catenin destruction complex orchestrates the Wnt response. Cell 158, 157–170. Benham-Pyle, B.W., Pruitt, B.L., and Nelson, W.J. (2015). Cell adhesion. Mechanical strain induces E-cadherin-dependent Yap1 and b-catenin activation to drive cell cycle entry. Science 348, 1024–1027. Chan, E.H., Nousiainen, M., Chalamalasetty, R.B., Scha¨fer, A., Nigg, E.A., and Sillje´, H.H. (2005). The Ste20-like kinase Mst2 activates the human large tumor suppressor kinase Lats1. Oncogene 24, 2076–2086. Chen, C.S., Tan, J., and Tien, J. (2004). Mechanotransduction at cell-matrix and cell-cell contacts. Annu. Rev. Biomed. Eng. 6, 275–302.

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