Developmental Cell Article

PTK7-Src Signaling at Epithelial Cell Contacts Mediates Spatial Organization of Actomyosin and Planar Cell Polarity

Anna Andreeva,1,4 Jianyi Lee,1,4 Madhura Lohia,2 Xiaoji Wu,3,5 Ian G. Macara,2,6 and Xiaowei Lu1,* 1Department of Cell Biology, P.O. Box 800732, University of Virginia, Charlottesville, VA 22908, USA 2Department of Microbiology, Center for Cell Signaling, University of Virginia, Charlottesville, VA 22908, USA 3School of Life Sciences, Peking University, Beijing, China, 100871 4Co-first authors 5Present address: PhD Program in Biological and Biomedical Sciences, Harvard Medical School, Boston, MA 02115, USA 6Present address: Department of Cell and Developmental Biology, Vanderbilt University Medical Center, Nashville, TN 37232, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.devcel.2014.02.008

SUMMARY of the cell apex, results in bending and invagination of an epithe- lial cell sheet (Martin et al., 2009; Sawyer et al., 2010). During Actomyosin contractility plays a key role in tissue embryonic axis elongation in Drosophila and Xenopus, aniso- morphogenesis. During mammalian development, tropic contractile forces mediate directional cell intercalation PTK7 regulates epithelial morphogenesis and planar and convergent extension (Blankenship et al., 2006; Rauzi cell polarity (PCP) through modulation of actomyosin et al., 2010; Skoglund et al., 2008). However, mechanisms contractility, but the underlying mechanism is un- underlying precise spatial and temporal control of actomyosin known. Here, we show that PTK7 interacts with the assembly on a tissue scale remain poorly understood. Emerging evidence indicates that planar cell polarity (PCP) tyrosine kinase Src and stimulates Src signaling signaling plays an important role in spatial regulation of actomy- along cell-cell contacts. We further identify ROCK2 osin contractility during vertebrate tissue morphogenesis. First as a target of junctional PTK7-Src signaling. PTK7 discovered in Drosophila, where it regulates polarity within knockdown in cultured epithelial cells reduced the the plane of the wing epithelial cell sheet, an evolutionarily level of active Src at cell-cell contacts, resulting in conserved core PCP pathway regulates morphogenesis of delocalization of ROCK2 from cell-cell contacts and both epithelial and nonepithelial tissues in vertebrates, including decreased junctional contractility, with a concomi- convergent extension, neural tube closure, and PCP in the audi- tant increase in actomyosin on the basal surface. tory sensory epithelium (Goodrich and Strutt, 2011). The core Moreover, we present in vivo evidence that Src family PCP pathway signals through the small GTPase RhoA and its kinase (SFK) activity is critical for PCP regulation in downstream effector Rho-associated kinases (ROCK), which the auditory sensory epithelium and that PTK7-SFK phosphorylates myosin RLC to stimulate actomyosin contrac- tility (Goodrich and Strutt, 2011). signaling regulates tyrosine phosphorylation of junc- In addition to the core PCP pathway, both invertebrates and tional ROCK2. Together, these results delineate a vertebrates employ alternative mechanisms for spatial regula- PTK7-Src signaling module for spatial regulation of tion of actomyosin contractility to drive planar polarized cell ROCK activity, actomyosin contractility, and epithe- behavior. In Drosophila, convergent extension, or germband lial PCP. extension, is driven by anisotropic junctional contractility inde- pendently of the core PCP pathway and is likely mediated by cadherin-mediated mechanotransduction and junctional remod- INTRODUCTION eling (Blankenship et al., 2006; Rauzi et al., 2010; Zallen and Wie- schaus, 2004). In the mouse, genetic evidence suggests that a Actomyosin contractility in nonmuscle cells is a central regulator protein tyrosine kinase 7 (Ptk7)-mediated pathway acts in of cell shape change and tissue morphogenesis (Lecuit et al., concert with the core PCP pathway to differentially regulate 2011). Actin filaments and the myosin II motor, which consists actomyosin contractility and orient PCP in the auditory sensory of two heavy chains, two essential light chains, and two regula- epithelium (Lee et al., 2012). tory light chains (RLC), assemble into contractile subcellular Ptk7 encodes a conserved receptor-tyrosine kinase (RTK)-like structures and supracellular networks to drive diverse physiolog- molecule, which is predicted to lack endogenous kinase activity ical processes, including cell division, cell migration, and tissue because the invariant ‘‘DFG’’ motif essential for correct posi- morphogenesis (Vicente-Manzanares et al., 2009). During devel- tioning of ATP is replaced with ‘‘ALG.’’ In the mouse, Ptk7 and opment, dynamic actomyosin networks have been shown to the core PCP genes are similarly required for a multitude of mediate the collective behavior of an interacting network of cells. developmental processes, including convergent extension, neu- For example, during Drosophila gastrulation and vertebrate neu- ral tube closure, PCP in the auditory sensory epithelium, and ral tube closure, coordinated apical constriction, or contraction heart and lung morphogenesis (Lu et al., 2004; Paudyal et al.,

20 Developmental Cell 29, 20–33, April 14, 2014 ª2014 Elsevier Inc. Developmental Cell PTK7-Src Signaling Regulates ROCK2 and PCP

Figure 1. Ptk7 Knockdown in MDCK Cells Causes Defects in Cell Shape and Actomyosin Organization (A) Confocal images of luciferase knockdown control (Luc KD) or Ptk7 KD cells immunostained for PTK7 and E-cadherin. Z-profile views show reduced height of Ptk7 KD cells. (B) Confocal images of myosin IIB and F-actin staining show actomyosin organization defects along both the lateral and basal membrane domains in Ptk7 KD cells. (C–E) Quantification of cell heights (C) and of myosin IIB localization along cell-cell contacts (D) and on the basal surface (E) in Luc KD and Ptk7 KD cell monolayers. Data are represented as mean ± SEM. (D) Perijunctional versus collapsed junctional staining of myosin IIB is defined using line scan analysis. If a line scan of stanining intensity shows two peaks flanking the cell junctions, it is scored ‘‘perijunctional,’’ whereas a single peak centered on cell junctions is scored ‘‘collapsed’’ (see Figure S1E). (F) Immunoblotting of Luc KD and Ptk7 KD cell lysates with the indicated antibodies. Total levels of E-cadherin, myosin IIB and IIA are unchanged, whereas pRLC levels are increased in Ptk7 KD cells. GAPDH served as loading control. Scale bars, 10 mm. See also Figure S1.

2010; Yen et al., 2009). Interestingly, studies in Xenopus and RESULTS zebrafish implicate vertebrate Ptk7 orthologs in both PCP and Wnt signaling and suggest different and sometimes conflicting Ptk7 Knockdown in MDCK Cells Results in Defects in functions of Ptk7 in regulating PCP and Wnt signaling (Bin-Nun Cell Shape and Actomyosin Organization et al., 2014; Hayes et al., 2013; Shnitsar and Borchers, 2008; MDCK II cells express PTK7 endogenously, which colocalizes Wehner et al., 2011). with E-cadherin along cell-cell contacts (Figure 1A). When Ptk7 Our recent work suggests that Ptk7 regulates PCP through expression was knocked down (KD) using short hairpin RNAs modulation of junctional contractility, but the underlying mecha- (shRNAs), cells displayed flattened morphology with increased nism is unknown. Here, we used cultured Madin-Darby canine apical surface area and reduced height (Figures 1A and 1C). kidney (MDCK) epithelial cells in vitro and the mouse auditory The cell-shape defect was not a secondary effect of decreased sensory epithelium in vivo to shed light on the mechanisms by cell density in confluent monolayers, as Ptk7 KD cell islands which Ptk7 regulates actomyosin contractility during mamma- also displayed flattened morphology and occupied larger surface lian epithelial morphogenesis. We show that in MDCK cells, area compared to control islands with equal numbers of cells PTK7 stimulates Src kinase signaling at cell-cell contacts and (Figure S1A available online). The localizations of E-cadherin, that Src signaling levels are critical for junctional ROCK2 b-catenin, plakoglobin, and the tight junction protein ZO-1 were localization. We then present in vivo evidence that Src family not significantly altered, indicating that epithelial organization of kinase (SFK) signaling at intercellular junctions regulates PCP Ptk7 KD cells was largely intact (Figures 1A, 1F, and S1B–S1D). in the mouse auditory sensory epithelium and that PTK7- In vivo evidence points to a role of PTK7 in myosin II regulation SFK signaling mediates tyrosine phosphorylation of junctional (Lee et al., 2012). We therefore examined the actomyosin orga- ROCK2. nization in Ptk7 KD cells. Control cells had robust cortical actin

Developmental Cell 29, 20–33, April 14, 2014 ª2014 Elsevier Inc. 21 Developmental Cell PTK7-Src Signaling Regulates ROCK2 and PCP

Figure 2. PTK7 Regulates ROCK2 Localization and Junctional Contractility in MDCK Cells (A) Confocal images of ROCK2 and F-actin staining. Perijunctional staining of ROCK2 was greatly reduced in Ptk7 KD cells. (B) Y-27632 treatment of control cells causes cell shape defects similar to those of Ptk7 KD cells. (C) Western blot analysis of MYPT1 phosphorylation and ROCK expression. Quantification of protein levels is shown on the right. Ptk7 KD cells show increased ROCK activity and decreased ROCK protein expression. Data are represented as mean ± SEM. (D) Confocal images of pRLC and F-actin staining on the basal surface of Luc KD or Ptk7 KD cells with the indicated treatment. Y-27632 treatment of Ptk7 KD cells reversed the increase in RLC phosphorylation and formation of the aberrant actin cables. (E) Impaired apical constriction in Ptk7 KD cells. Shroom-transfected cells are marked by GFP (cyan). Cell boundaries are marked by E-cadherin immunostaining (red). Confocal images show the apical and basal regions of the cells. Apical constriction is quantified as the ratio of the basal surface area to the apical surface area of transfected cells. Bars indicate the median. One outlier Ptk7 KD cell showed greatly increased apical constriction (arrow), likely a result of incomplete knockdown. * The p value is 0.425 when including the outlier (arrow) and is 0.011 when excluding the outlier. (F) In hanging drop assays, the size of Ptk7 KD cell aggregates is smaller on average, suggesting weakened cell-cell adhesion. The areas occupied by individual cell aggregates were quantified and binned. p value was calculated using the Kolmogorov-Smirnov test. Scale bars, 10 mm (A, B, D, and E) and 200 mm (F). See also Figure S2. and uniformly distributed basal stress fibers (Figure 1B). By tacts was strong for myosin IIB but weak for myosin IIA and contrast, F-actin staining along the lateral cortex was reduced phosphorylated RLC (pRLC), and all three were detected at in Ptk7 KD cells, whereas aberrant clusters of thick actin cables low levels on basal stress fibers (Figures 1B, 2D, and S1H). Inter- were observed near cell-cell contacts on the basal surface (Fig- estingly, myosin IIB was often organized into two parallel cables ure 1B). F-actin defects were accompanied by defects in myosin flanking the cell junctions between neighboring cells (termed II localization. In control monolayers, staining along cell-cell con- ‘‘perijunctional;’’ Figures 1B, 1D, and S1E–S1G). By contrast,

22 Developmental Cell 29, 20–33, April 14, 2014 ª2014 Elsevier Inc. Developmental Cell PTK7-Src Signaling Regulates ROCK2 and PCP

myosin IIB staining in Ptk7 KD monolayers frequently appeared undergo apical constriction, the shrinking of the apical surface as a thin line that largely overlapped with cell junctions (termed driven by junctional myosin II contractility (Hildebrand, 2005; ‘‘collapsed;’’ Figures 1B and S1E–S1G). On the basal surface, Nakajima and Tanoue, 2011). Ectopic expression of the actin- the abnormal actin cables showed increased staining of myosin binding protein Shroom induces robust apical constriction in IIB, myosin IIA, and pRLC (Figures 1B, 2D, and S1H). Of note, normal MDCK II cells, whereas it induced apical constriction pRLC levels were increased by 2-fold in Ptk7 KD cells, whereas much less efficiently in Ptk7 KD cells (Figure 2E). Thus, Ptk7 myosin IIB and IIA levels were unchanged (Figure 1F). Thus, Ptk7 KD cells had impaired ability to undergo apical constriction, a is required for normal epithelial cell shape and actomyosin cell shape change critical for neural tube closure. organization. Junctional contractility also regulates cadherin-mediated adhesion (Smutny et al., 2010). To determine whether cell-cell Delocalization of ROCK2 from Cell-Cell Contacts in Ptk7 adhesion was affected, we first examined the levels of the deter- KD Cells gent-insoluble pool of E-cadherin, which contains E-cadherin To determine the basis for the actomyosin defects in Ptk7 KD stabilized in junctional complexes by cortical actin (Nagafuchi cells, we next examined the localization of the two Rho kinase and Takeichi, 1988). No significant changes in E-cadherin deter- family members. ROCK positively regulates myosin II activity gent solubility were detected in Ptk7 KD cells, suggesting that through direct phosphorylation of RLC and inhibition of the cytoskeletal association of the E-cadherin adhesion complex RLC phosphatase by phosphorylating the myosin phosphatase was not affected (Figures S2E and S2F). Next, we performed a target subunit 1 (MYPT1) (Vicente-Manzanares et al., 2009). In hanging drop cell aggregation assay (Qin et al., 2005). Whereas both control and Ptk7 KD cells, ROCK1 was diffusely localized control cells were able to form large aggregates, Ptk7 KD cells in the cytoplasm (Figure S2A). By contrast, ROCK2 in control formed smaller aggregates compared to control cells, suggest- cells was localized both in the cytoplasm and flanking the cell ing weakened cell-cell adhesion (Figure 2F). Together, these junctions. Strikingly, the perijunctional, but not cytoplasmic, observations suggest that PTK7 regulates adhesive strength ROCK2 localization was severely disrupted in Ptk7 KD cells without significant impact on overall distribution of E-cadherin. (Figures 2A, S2B, and S2C). To test whether decreased junc- tional ROCK activity may be responsible for the cell-shape de- PTK7 Regulates Actomyosin and Cell Shape via Its fects in Ptk7 KD cells, we treated MDCK cells with Y-27632, a Cytoplasmic Domain specific ROCK inhibitor. Similar to Ptk7 KD cells, Y-27632- To determine the domains of PTK7 important for actomyosin and treated control cells had decreased height and increased apical cell shape regulation, we next performed knockdown and rescue surface area (Figure 2B). Moreover, Y-27632 treatment did not assays using bicistronic lentiviruses that express both shPtk7 further exacerbate the cell-shape defects of Ptk7 KD cells and GFP/Venus cDNA, either by itself or fused to shRNA-resis- (Figure S2D). Together, these results suggest that PTK7 regu- tant murine PTK7 cDNAs. Stable knockdown of Ptk7 resulted lates junctional ROCK activity, which is critical for epithelial in identical phenotypes compared to transient knockdown (Fig- cell shape. ures 3A–3F). Importantly, expression of Venus fusions of wild- type PTK7 (PTK7WT-Venus), but not of PTK7 that lacks the Increased ROCK Activity Contributes to the Basal cytoplasmic domain (PTK7Dcyt-Venus), significantly rescued Actomyosin Defects in Ptk7 KD Cells the defects in cell height and junctional and basal myosin IIB To further assess the effect of Ptk7 KD on ROCK, we measured localization and partially restored perijunctional ROCK2 localiza- total ROCK activity in Ptk7 KD cells using phospho-MYPT1 as tion (Figures 3A–3F). Of note, PTK7WT-Venus localized correctly readout, as RLC is not an exclusive substrate. ROCK phosphor- to cell-cell contacts, whereas PTK7Dcyt-Venus was largely mis- ylates MYPT1 at threonines (T) 696 and 853, with T853 being a localized to the apical membranes, suggesting that localization specific target (Garton et al., 2008; Grassie et al., 2011). Although of PTK7 to cell-cell contacts is important for its function and re- phospho-T696 levels were unchanged, phospho-T853 levels quires the cytoplasmic domain (Figure 3C). Together, these re- were increased in Ptk7 KD cells (Figure 2C). Moreover, total sults indicate that PTK7 regulates actomyosin and cell shape levels of ROCK1 and ROCK2 were both decreased by 30% via its cytoplasmic domain. (Figure 2C). These data suggest that ROCK activity was in- creased by Ptk7 KD. The PTK7 Kinase Domain Binds Directly to and Is We next asked whether the basal actomyosin defects in Phosphorylated by Src Ptk7 KD cells resulted from increased ROCK activity. Indeed, Next, we sought to identify PTK7 cytoplasmic domain-interact- Y-27632 treatment of Ptk7 KD cells reversed both the increase ing proteins. The nonreceptor tyrosine kinase Src was tested in RLC phosphorylation and formation of the aberrant actin as a candidate because it participates in RTK signaling (Parsons cables (Figure 2D). Taken together, these results suggest that and Parsons, 2004). Coimmunoprecipitation (coIP) experiments delocalization of ROCK2 from cell-cell contacts resulted in demonstrated that endogenous Src and PTK7 formed a com- increased total ROCK activity that contributed to the basal acto- plex, and this interaction was dependent on the PTK7 cyto- myosin defects in Ptk7 KD cells. plasmic domain (Figures 3G and S3A). To test whether the interaction was direct, we performed Impaired Apical Constriction and Weakened Cell-Cell glutathione S-transferase (GST) pull-down assays. Purified Adhesion in Ptk7 KD Cells PTK7 kinase domain was incubated with purified GST fusions To investigate the effect of decreased junctional contractility on of different domains of Src. Direct binding of the PTK7 kinase cell shape change in Ptk7 KD cells, we examined their ability to domain to the Src SH3 domain-containing fragments was

Developmental Cell 29, 20–33, April 14, 2014 ª2014 Elsevier Inc. 23 Developmental Cell PTK7-Src Signaling Regulates ROCK2 and PCP

Figure 3. PTK7 Regulates Myosin II and Junctional Src Signaling through Its Cytoplasmic Domain (A) PTK7WT-Venus, but not PTK7Dcyt-Venus, rescued cell shape and myosin IIB localization defects in Ptk7 KD cells. Four stable cell lines expressing the indicated constructs were stained for myosin IIB and F-actin. (B) PTK7WT-Venus, but not PTK7Dcyt-Venus, partially restored perijunctional ROCK2 localization in Ptk7 KD cells. (C) GFP or PTK7-Venus localization in the indicated cell lines. PTK7Dcyt-Venus was mislocalized to the apical membranes. (D–F) Quantification of cell height (D), of perijunctional (E), and of basal myosin IIB localization (F) in the indicated cell lines. (G) coIP of endogenous Src with PTK7WT-Venus, but not PTK7Dcyt-Venus. Venus fusion proteins were immunoprecipitated from stable cell lines using GFP-Trap beads and bound fractions analyzed by Src and PTK7 immunoblotting. (H) Confocal images of Luc KD or Ptk7 KD cells immunostained for total Src, pY416-Src, and pY527-Src.

(legend continued on next page) 24 Developmental Cell 29, 20–33, April 14, 2014 ª2014 Elsevier Inc. Developmental Cell PTK7-Src Signaling Regulates ROCK2 and PCP

detected, whereas it bound only weakly to the Src kinase domain weakened cell-cell adhesion, leads to decreased junctional and not significantly to the SH2 domain (Figure S3B). Moreover, pY416-Src localization. binding to an inactive SH3 mutant (W118K) was greatly reduced, Lastly, to determine whether Ptk7 KD also affected Src activity indicating a specific interaction (Figure S3C). We next deter- at focal adhesions, we examined the phosphorylation levels of mined whether Src phosphorylates PTK7 in vitro. Indeed, several Src substrates in focal adhesions, including FAK, whereas the PTK7 kinase domain did not phosphorylate itself, p130Cas, and paxillin using phospho-specific antibodies (Par- tyrosine phosphorylation of the purified PTK7 kinase domain sons and Parsons, 2004). We found no significant change in their was detected in the presence of purified active Src (Figure S3D). phosphorylation levels in Ptk7 KD cells (Figure S3O). Taken Because the Src SH2 domain can bind to phosphorylated tyro- together, these results suggest that PTK7 positively and specif- sine residues, we next asked whether phosphorylated PTK7 ki- ically regulates Src activity along cell-cell contacts through its nase domain can bind directly to the Src SH2 domain. To test cytoplasmic domain. this, the PTK7 kinase domain was first subjected to in vitro kinase reactions with immobilized EGFP or Src-EGFP fusion pro- Src-EGFP Expression Significantly Rescues Ptk7 teins (Figures S3E and S3F) and then incubated with GST- Knockdown Phenotypes tagged wild-type Src SH2 (SH2 WT) or inactive mutant SH2 If defective Src signaling caused the actomyosin defects in Ptk7 (R175L). Direct binding of tyrosine phosphorylated PTK7 kinase KD cells, then Src inhibition should result in similar defects. To domain to Src SH2 domain was detected, and importantly, bind- test this idea, we expressed Src251-EGFP, a dominant-negative ing to the Src SH2 mutant was reduced to background levels Src-EGFP fusion construct that lacks the kinase domain, in con- (Figure S3G). Thus, the PTK7 kinase domain is a substrate of trol MDCK cells. Indeed, expression of Src251-EGFP resulted in Src and interacts directly with the SH3 and SH2 domains of Src. lateral and basal actomyosin defects similar to those observed in Ptk7 KD cells (Figure S4A). Importantly, perijunctional ROCK2 localization was also disrupted, indicating a requirement of Src PTK7 Positively Regulates Src Activity at Cell-Cell signaling for ROCK2 localization (Figure S4A). These results Contacts suggest that Src is a potential signaling component in PTK7- To probe the functional significance of PTK7-Src interaction, we mediated actomyosin regulation. examined the localization of Src using pan-Src antibodies, as To test this, we sought to rescue the Ptk7 KD phenotypes by well as active Src phosphorylated at tyrosine 416 using pY416- ectopic expression of Src. We reasoned that if PTK7 positively specific Src antibodies. In control cells, both total Src and regulates junctional Src activity, it might be possible to express pY416-Src were detected along cell-cell contacts. In Ptk7 KD a modified version of Src that can restore junctional Src cells, whereas junctional localization and levels of total Src signaling in the absence of PTK7. To this end, we expressed were unchanged, pY416-Src staining along cell-cell contacts EGFP fusions to wild-type Src (SrcWT-EGFP) or a constitutively was significantly reduced (Figures 3H and 3I). Average levels of active form of Src (SrcCA-EGFP) carrying the Y527F mutation pY416-Src were 80% of control levels (Figure 3J). Comple- (Sandilands et al., 2004). We opted to use a truncated, minimal mentary to decreased levels of active Src in Ptk7 KD cells, junc- CMV promoter to drive low-level expression of these con- tional staining and levels of pY527-Src, phosphorylated at a structs, as overexpression of SrcCA induces epithelial-to- conserved C-terminal tyrosine critical for Src auto-inhibition, mesenchymal transition (McLachlan et al., 2007). Interestingly, were increased, suggesting increased levels of inactive Src in SrcCA-EGFP expression in control cells also caused defects in the absence of Ptk7 (Figures 3H–3J). Conversely, in cells overex- cell shape and actomyosin organization and disrupted perijunc- pressing PTK7WT-Venus, pY416-Src staining along cell-cell tional ROCK2 localization (Figure S4B). Likewise, SrcCA-EGFP contacts was increased compared to control cells, further sup- expression did not rescue Ptk7 KD phenotypes (data not shown). porting a positive role of PTK7 in junctional Src regulation (Fig- Thus, both decreased and increased Src signaling causes ures S3I and S3J). deleterious effects on actomyosin organization and junctional We wondered whether weakened cell-cell adhesion second- ROCK2 localization. arily caused reduced active Src localization along cell-cell con- In contrast to SrcCA-EGFP, expression of Src WT-EGFP tacts in Ptk7 KD cells, because cadherin-mediated adhesion moderately increased pY416-Src junctional staining (Fig- can stimulate Src signaling (McLachlan et al., 2007). To test ure S4C). Remarkably, it restored pY416-Src localization at this, we knocked down E-cadherin and examined the localiza- cell-cell contacts and significantly rescued defects in cell tion of pY416-Src. E-cadherin KD indeed reduced junctional height and myosin IIB organization and partially restored peri- pY416-Src staining, but to a lesser extent compared to Ptk7 junctional ROCK2 localization in Ptk7 KD cells (Figures 4A–4F). KD (Figures S3L and S3M). Importantly, junctional PTK7 staining By contrast, expression of untagged wild-type Src did not restore was also reduced, whereas total levels of PTK7 were unchanged pY416-Src localization or rescue Ptk7 KD phenotypes (Fig- (Figures S3L–S3N). Thus, we conclude that junctional pY416- ure S4D), suggesting that Src WT-EGFP is partially constitutively Src levels positively correlate with those of PTK7. Reducing active. Importantly, a kinase-inactive Src mutant (K295R) fused junctional PTK7 levels, either through direct knockdown or to EGFP also failed to rescue, indicating that Src kinase activity

(I) Quantifications show that junctional staining intensity was unchanged for total Src, decreased for pY416-Src, and increased for pY527-Src in Ptk7 KD cells. (J) Western blot analysis showing reduced levels pY416-Src and increased levels of pY527-Src in Ptk7 KD cells. Total Src levels were slightly increased. In (D)–(F), (I), and (J), data are represented as mean ± SEM. Scale bars, 10 mm. See also Figure S3.

Developmental Cell 29, 20–33, April 14, 2014 ª2014 Elsevier Inc. 25 Developmental Cell PTK7-Src Signaling Regulates ROCK2 and PCP

Figure 4. Expression of Src-EGFP Partially Rescues the Ptk7 KD Phenotypes (A–C) Expression of SrcWT-EGFP, but not of a kinase-dead mutant, significantly restored junctional pY416-Src (A), perijunctional myosin IIB (B), and, to a lesser degree, perijunctional ROCK2 localization (C) in Ptk7 KD cells. Transfected Luc KD cells are shown as controls. (D–F) Quantification of cell height (D), of perijunctional (E), and of basal myosin IIB localization (F) in Ptk7 KD cells transfected by the indicated constructs. (G) Western blot analysis showing levels of Src-EGFP expression, pY416-Src, and PTK7 in cells expressing the indicated constructs. Endo Src, endogenous Src. (H) Quantifications of pY416-Src levels in cells expressing the indicated constructs by immunoblotting. The activity of SrcWT-GFP is higher than untagged Src in both control and Ptk7 KD cells. In (D)–(F) and (H), data are represented as mean ± SEM. Scale bars, 10 mm. See also Figure S4.

26 Developmental Cell 29, 20–33, April 14, 2014 ª2014 Elsevier Inc. Developmental Cell PTK7-Src Signaling Regulates ROCK2 and PCP

is required for its function in actomyosin regulation (Figures 4A– We have shown previously that PTK7 regulates myosin II- 4F). Taken together, these results demonstrate a crucial role of mediated contractile tension in the OC to orient PCP (Lee a PTK7-Src signaling module in regulating junctional contractility. et al., 2012). To identify targets of PTK7-Src signaling important for myosin II regulation in the OC, we examined the localization ROCK2 Is a Target of Src Signaling at Cell-Cell Contacts and phosphorylation of ROCK2, a likely target of junctional To determine the mechanisms by which Src signaling regulates PTK7-Src signaling in MDCK cells. In control OC, ROCK2 was ROCK2 localization, we tested whether ROCK2 is a direct target localized to cell junctions in a punctate pattern (Figure 5I). In of Src at cell-cell contacts. Previously, Src has been shown to contrast to Ptk7 KD MDCK cells, ROCK2 was still localized to phosphorylate ROCK2 at tyrosine 722 (Y722), and this event is cell junctions in Ptk7À/À OC (Figure 5J). Overall levels of important for regulation of focal adhesion dynamics (Lee et al., ROCK2 were decreased by 40% in Ptk7À/À cochleae, whereas 2010). Using a phospho-Y722 specific antibody (Lee and Chang, ROCK1 levels were comparable to the control (Figures 5T and 2008), we found that the steady-state level of Y722 phosphory- 5U). Remarkably, in control OC, pY722-ROCK2 was also local- lation is very low in normal MDCK cells (data not shown). This ized to cell junctions and enriched along the medial boundaries may be a result of rapid dephosphorylation of Y722 by tyrosine between outer hair cells and supporting cells (Figures 5K–5M phosphatases, such as Shp2 (Lee and Chang, 2008). Consistent and 5Q). This junctional localization was likewise greatly reduced with this idea, although normally undetectable by immunostain- in Ptk7À/À OC (Figures 5N–5P and 5Q). Thus, PTK7 mediates ing, pY722-ROCK2 staining was observed at cell-cell contacts ROCK2 tyrosine phosphorylation at cell-cell contacts in vivo. upon expression of SrcWT-EGFP (Figure S4E). This result sug- gests that Src can indeed stimulate ROCK2 phosphorylation at Src Inhibition and Hyperactivation Both Result in PCP cell-cell contacts. Defects in the OC In addition to ROCK2 phosphorylation, Src may regulate Disrupted localization of active Src in Ptk7À/À cochleae suggests ROCK2 localization along cell-cell contacts through other tar- that Src signaling is required for PCP in the OC. In the mouse, gets. Specifically, we tested whether PI3K is involved. Src sig- Src, Yes and Fyn kinases are ubiquitously expressed and func- naling activates PI3K, which phosphorylates PIP2 to generate tion redundantly (Thomas et al., 1995). Therefore, to determine PIP3. PIP3 has been shown to stimulate ROCK2 membrane local- the role of SFK signaling in the OC, we took a pharmacological ization and activity (Yoneda et al., 2005). Consistent with this, 1 hr approach and treated cochlear explants with the SFK inhibitor treatment with the PI3K inhibitor wortmannin reduced ROCK2 SU6656 (Blake et al., 2000). Vehicle-treated explants had normal localization to cell-cell contacts (Figure S4F). However, using PCP: the axonemal kinocilium at the vertex of V-shaped hair the PH domain of Akt fused to GFP (PH-Akt-GFP) as a probe bundles was positioned near the lateral pole of hair cells (Figures for PIP3 (Gassama-Diagne et al., 2006), we found that PIP3 6A and 6C). By contrast, many hair bundles in SU6656-treated was enriched at cell-cell contacts in both control and Ptk7 KD explants were misoriented relative to the medial-lateral axis of cells (Figure S4G). Together, these findings suggest that PTK7 the cochlea (Figures 6B, arrows, and 6D). Immunoblotting of is unlikely to mediate ROCK2 localization through PI3K. treated cochlear tissues confirmed that Src inhibitors effectively reduced pY416-Src levels (Figure 6E). Thus, we conclude that PTK7 Positively Regulates Junctional Src Signaling and SFKs and/or structurally related kinases are required for PCP ROCK2 Phosphorylation in the Mouse Auditory Sensory in the OC. Epithelium The asymmetric junctional pY416-Src localization suggests Having established the role of junctional PTK7-Src signaling in that spatial regulation of Src signaling is important for PCP in MDCK cells, we next investigated whether Src signaling is rele- the OC. To test this idea, we uniformly elevated levels of Src vant for Ptk7-mediated epithelial PCP regulation in vivo. The signaling in the cochlea using a conditional allele of C-terminal mouse auditory sensory epithelium, or the organ of Corti (OC), Src kinase (Cskfl)(Schmedt et al., 1998). Csk is a key negative located in the cochlea is a well-established model for PCP regulator of SFK activity by phosphorylating a conserved C-ter- signaling. The V-shaped hair bundles atop auditory hair cells minal tyrosine critical for autoinhibition (Y527 in Src). Csk and their uniform orientation serve as a robust readout for knockout mice exhibited hyperactivation of SFKs, resulting in PCP. To determine whether Ptk7 regulates active Src localiza- neural tube defects and lethality around E10.5 (Imamoto and tion in the OC, we first examined Src and pY416-Src localization Soriano, 1993). In CskcKO mutants, where Csk was deleted in at embryonic day (E) 16.5. Similar to MDCK cells, both Src and the cochlea using a Pax2-Cre driver (Ohyama and Groves, pY416-Src was localized to cell junctions in control OC (Figures 2004), two classes of PCP defects were observed. In the basal 5A–5H). Interestingly, whereas Src was uniformly distributed region of the cochlea, misorientation of the hair bundle and the around cell junctions, pY416-Src staining was enriched along kinocilium were observed, often accompanied by hair bundle the medial boundaries between outer hair cells and supporting structural defects (Figures 6G and 6I, arrows). In the apical region cells, with an average medial/lateral intensity ratio of 1.7 (Figures of the cochlea, supernumerary rows of outer hair cells were 5A, 5C–5E, and 5Q). By contrast, junctional staining of pY416- observed (Figures 6J and 6K), likely due to defects in medial- Src was significantly reduced in Ptk7À/À OC, whereas total Src lateral cell intercalations. Overall pY416-Src levels in CskcKO localization was unchanged (Figures 5B, 5F–5H, and 5Q). Overall cochleae were increased by 2-fold (Figure 6L). These results levels of pY416-Src were decreased by 40% in Ptk7À/À suggest that the level of Src signaling needs to be tightly regu- cochleae, with a slight decrease in total Src levels (Figures 5R lated for PCP establishment. and 5S). Taken together, these results indicate that PTK7 posi- To determine whether altered Src signaling affects the core tively regulates junctional Src signaling in the OC. PCP pathway, we examined the asymmetric localization of

Developmental Cell 29, 20–33, April 14, 2014 ª2014 Elsevier Inc. 27 Developmental Cell PTK7-Src Signaling Regulates ROCK2 and PCP

Figure 5. PTK7 Regulates Junctional Src Signaling and ROCK2 Phosphorylation in the Mouse OC (A and B) Src was uniformly distributed around cell junctions in both control (A) and Ptk7À/À OC (B) at E16.5. (C–H) E16.5 cochleae stained for pY416-Src (red) and phalloidin (green). (C–E) pY416-Src was en- riched on the medial boundaries of hair cells in the control. (F–H) Junctional pY416-Src staining was significantly reduced in Ptk7À/À OC. (E and H) Higher magnifications of the hair cell indicated by asterisks in (D) and (G), respectively. (I and J) ROCK2 showed punctate staining around cell junctions in both control (I) and Ptk7À/À OC (J) at E16.5. (K–P) E16.5 cochleae stained for pY722-ROCK2 (red) and phalloidin (green). (K–M) In control OC, pY722-ROCK2 staining was also enriched on the medial boundaries of hair cells. (N–P) Junctional pY722-ROCK2 staining was significantly reduced in Ptk7À/À OC. (M and P) Higher magnification of the hair cell indicated by asterisks in (L) and (O), respectively. Images were taken from the mid- basal region of the cochlea (25% cochlear length). Arrowheads indicate the row of pillar cells. Brackets indicate OHC rows. Lateral is up in all micrographs. Scale bars, 2 mm (E, H, M, P) and 4 mm (all other panels). (Q) Quantification of medial to lateral (M:L) staining intensity ratios of junctional Src, pY416- Src, and pY722-ROCK2. Data are represented as mean ± SEM. (R–U) Total levels of pY416-Src, Src, ROCK2, and ROCK1 in E16.5 cochlear lysates. pY416-Src (R) and ROCK2 (T) levels were decreased in Ptk7À/À cochleae. Lysates from two cochleae of the same genotype were pooled and loaded in each lane. Numbers indicate percentage of normalized levels. the core PCP proteins Dvl2 and Fz3. To better match the develop- tinib treatment and Csk deletion disrupted the apical myosin IIB mental stage examined in different mutants, we inhibited foci in supporting cells, whereas junctional myosin IIB staining Src signaling systemically in vivo using Bosutinib, a bioavailable was relatively unchanged, suggesting that normal levels of Src dual Src/Abl inhibitor (Amsberg and Koschmieder, 2013). signaling is important for the assembly of the apical myosin Membrane recruitment and asymmetric localization of Dvl2 in network (Figures 7A–7F). Moreover, CskcKO OC was disorga- the OC was largely unchanged in both Bosutinib-treated nized and much wider along the medial-lateral axis, consistent and CskcKO OC (Figures S5A–S5F). Previous observations sug- with cell intercalation defects (Figures 7E and 7F). gest that Fz3 localization is sensitive to changes in cortical Importantly, asymmetric junctional pY416-Src localization cytoskeleton (Grimsley-Myers et al., 2009; Lee et al., 2012). In was disrupted by both conditions. Whereas Src inhibition greatly Bosutinib-treated and CskcKO OC, Fz3 was still asymmetric local- reduced junctional pY416-Src staining, in Csk mutants pY416- ized, albeit with reduced staining intensity, suggesting that altered Src became circumferentially localized around cell junctions Src signaling may affect the cortical cytoskeleton (Figures S5G– (Figures 7G–7L). Strikingly, whereas altered Src signaling did S5L). Together, these results suggest that the core PCP pathway not significantly impact ROCK2 localization to cell junctions (Fig- was still active when Src signaling is perturbed. ures 7M–7R), pY722-ROCK2 localization closely correlated with pY416-Src staining. It was greatly reduced by Src inhibition and Src Signaling Mediates Junctional ROCK2 became circumferentially localized around cell junctions in Phosphorylation in the OC CskcKO mutants (Figures 7S–7X). Together, these results identify Our data so far suggest that Src is a signaling component in ROCK2 as a target of junctional PTK7-Src signaling in vivo. PTK7-mediated actomyosin regulation. To further test this idea, we assayed the effects of Src inhibition and hyperactivation DISCUSSION on the localizations of myosin IIB, active Src, and pY722-ROCK2 in the OC, all of which are regulated by PTK7. At E16.5, PTK7 How actomyosin contractility is coordinately regulated in regulates both junctional myosin IIB and a network of apical groups of mechanically coupled cells to drive tissue morpho- myosin IIB foci in supporting cells (Lee et al., 2012). Both Bosu- genesis is a fundamental question in developmental biology.

28 Developmental Cell 29, 20–33, April 14, 2014 ª2014 Elsevier Inc. Developmental Cell PTK7-Src Signaling Regulates ROCK2 and PCP

Figure 6. Src Inhibition and Hyperactivation Both Result in PCP Defects in the OC (A and B) Phalloidin (green, labels the hair bundle) and acetylated-tubulin (red, labels the kinocilium) staining of Src-inhibited cochlear explants. Hair bundle orientation is disrupted in SU6656-treated cochlear explants (B, arrows). Hair bundle fragmentation is also observed (B, open arrow). (C and D) Quantification of kinocilium positions in vehicle (C) and SU6656-treated (D) explants. (E) SU6656- and Bosutinib-treatment reduced pY416-Src levels in cochlear explants. Numbers on the bottom indicate percentage of normalized levels. (F–K) Phalloidin (green) and acetylated-tubulin (red) staining of E18.5 CskcKO OC. (F–I) CskcKO hair cells have defects in hair bundle polarity and orientation (G and I). Images were taken from the midbasal region of the cochlea (25% cochlear length). (H and I) Higher magnification of the boxed OHCs in (F) and (G), respectively. Arrows indicate the kinocilium position. (J and K) CskcKO mutants (K) have an extra row of OHCs in the apical region of the cochlea (80% cochlear length). (L) pY416-Src levels are increased by 2-folds in CskcKO cochlear tissues. Arrowheads indicate the row of pillar cells. Brackets indicate OHC rows. Lateral is up in all micrographs. Scale bars, 6 mm (A and B), 5 mm (H and I), and 10 mm (all other panels).

In addition to the RhoA-ROCK signaling axis (Nakajima and Ta- and stabilize Src in its active conformation along cell-cell con- noue, 2011; Nishimura et al., 2012; Ratheesh et al., 2012; Terry tacts to control actomyosin contractility and adhesive strength. et al., 2011), our study now reveals another layer of actomyosin Mechanistically, our biochemical data are consistent with regulation at intercellular junctions. We present both in vitro PTK7 being an allosteric activator of Src. Through interactions and in vivo evidence that PTK7-Src signaling at cell-cell con- with Src SH3 and SH2 domains, PTK7 relieves Src autoinhibi- tacts spatially organizes the actomyosin cytoskeleton to regu- tion and reduces phosphorylation of Y527. Because PTK7 late junctional contractility, cell shape change, and PCP. More- lacks intrinsic kinase activitiy, we suggest a positive feedback over, we identify ROCK2 as one of the targets of PTK7-Src model in which pre-existing active Src phosphorylates PTK7, signaling at cell-cell contacts and implicate ROCK2 tyrosine enabling it to interact with the SH2 domain of additional Src phosphorylation at intercellular junctions in PCP regulation molecules to result in further activation. Of interest, expression in vivo. of Src-EGFP, but not untagged Src, restored junctional Src ac- Importantly, the Ptk7 KD phenotypes in cell culture have func- tivity in Ptk7 KD cells. As indicated by their respective pY416 tional relevance in vivo. Specifically, similar cell shape and basal levels, Src-EGFP is partially constitutively active compared to myosin IIB defects were observed in the developing neural tube untagged Src. We suggest that the GFP tag may interfere of Ptk7À/À mutants during axis elongation (Williams et al., 2014, with Src intramolecular interactions by steric hindrance, this issue of Developmental Cell). Thus, PTK7-mediated cell thereby partially relieving its autoinhibition. Owing to incom- shape change and spatial organization of actomyosin are crucial plete knockdown, we cannot rule out the possibility that the for epithelial morphogenesis in specific developmental contexts. rescuing activity is mediated through residual PTK7. Despite Our data support a model whereby PTK7, an evolutionarily the caveat, the most parsimonious interpretation of our data conserved transmembrane pseudokinase, acts to stimulate is that Src is a signaling effector of PTK7 in actomyosin

Developmental Cell 29, 20–33, April 14, 2014 ª2014 Elsevier Inc. 29 Developmental Cell PTK7-Src Signaling Regulates ROCK2 and PCP

Figure 7. Src Inhibition and Hyperactivation Have Opposing Effects on pY722-ROCK2 Localization in the OC (A–X) E16.5 cochleae stained for myosin IIB (A–F), pY416-Src (G–L), ROCK2 (M–R), and pY722-ROCK2 (S–X). Green, phalloidin staining. (A and B) In control OC, myosin IIB (red) is localized to cell junctions and to apical foci in supporting cells (arrows). (C–F) Apical myosin IIB foci are absent in Bosutinib-treated OC (C and D) and CskcKO OC (E and F). Junctional myosin IIB localization is unchanged. (G–H) In control OC, pY416-Src (red) is enriched along medial boundaries between hair cells and neighboring supporting cells. pY416-Src staining was significantly reduced in Bosutinib-treated OC (I and J) and became circumferentially localized along cell-cell contacts in CskcKO OC (K and L). (M–P) ROCK2 (red) was distributed along cell junctions in a punctate manner in control (M and N) and Bosutinib-treated OC (O and P). (Q and R) In CskcKO OC, ROCK2 is still localized to cell junctions. Of note, multicellular rosettes were frequently observed, where ROCK2 was enriched at their vertices (arrows). (S and T) In control OC, pY722-ROCK2 (red) staining shows similar asymmetric localization to thatof pY416-Src. pY722-ROCK2 staining was greatly reduced in Bosutinib-treated OC (U and V) and became circumferentially localized along cell-cell contacts in CskcKO OC (W and X). Images were taken from the midbasal region of the OC (25% cochlear length). Arrowheads indicate the row of pillar cells. Brackets indicate OHC rows. Lateral is up in all micrographs. Scale bars, 4 mm. regulation, although it remains possible that additional effec- epithelia (Bardet et al., 2013). By contrast, phospho-Y722 tors may be involved. ROCK2 was localized to cell junctions in wild-type OC but was We identify ROCK2 as one of the targets of PTK7-Src signaling greatly reduced in both Ptk7À/À and Src-inhibited OC. Together, at cell-cell contacts in both MDCK cells and the mouse OC. It is these results suggest that PTK7-Src signaling regulates junc- worth noting that Src signaling critically regulates both junctional tional contractility in part through ROCK2 phosphorylation. In ROCK2 localization and phosphorylation, which were differen- addition, Src phosphorylates numerous actin regulatory pro- tially regulated in the two epithelia examined. In MDCK cells, teins, some of which may also be regulated by PTK7-Src ROCK2 junctional localization depends on PTK7-Src signaling, signaling to influence actomyosin assembly. and intriguingly, ROCK2 localization at cell-cell contacts showed How ROCK2 tyrosine phosphorylation regulates junctional a biphasic response to Src signaling levels. Expression of domi- contractility remains to be determined. ROCK2 phosphorylation nant-negative and constitutively active Src both disrupted at Y722 has been shown to be required for focal adhesion turn- ROCK2 localization to cell-cell contacts. Because both con- over and to decrease its binding to RhoA-GTP (Lee and Chang, ditions decrease intercellular adhesion, we speculate that 2008; Lee et al., 2010). ROCK2 phosphorylation at cell junctions ROCK2 localization is regulated mechanically by intercellular may likewise promote the turnover of cadherin adhesions and adhesion strength. Furthermore, ROCK2 Y722 phosphorylation decrease ROCK2 activation by junctional RhoA-GTP. However, at cell junctions only became detectable when Src-EGFP was we did not detect any changes in overall E-cadherin surface dis- expressed. Thus, we suggest that PTK7-Src signaling regulates tribution or its association with the cytoskeleton in Ptk7-depleted junctional ROCK2 activity through both direct and indirect mech- MDCK cells. More sensitive assays will be necessary to detect anisms. In the mouse OC, on the other hand, ROCK2 was still cadherin adhesion dynamics in developing epithelia. In the localized to cell-cell contacts in Ptk7À/À mutants, suggesting mouse OC, pY722 ROCK2 is enriched along the medial bound- that there are additional mechanisms that regulate junctional aries of hair cells and supporting cells. We have previously ROCK localization in vivo, as observed in other developing proposed that junctional contractility is increased at these

30 Developmental Cell 29, 20–33, April 14, 2014 ª2014 Elsevier Inc. Developmental Cell PTK7-Src Signaling Regulates ROCK2 and PCP

boundaries, based on the polarized recruitment of vinculin (Lee To induce apical constriction, LucKD or Ptk7 KD cells were cotransfected et al., 2012). Thus, junctional ROCK2 tyrosine phosphorylation with 3 mg Shrm3 (Hildebrand, 2005) (a gift of Jeff Hildebrand) and 0.3 mg may be associated with increased contractile tension. GFP plasmids using GenJet (SignaGen Laboratories), cultured for another 24 hr and then processed for immunostaining. Curiously, despite decreased ROCK2 levels, Ptk7 loss led to 2 For E-Cadherin-cytoskeleton association analysis, cells grown on 3.8 cm increased levels of RLC phosphorylation, both in MDCK cells plastic wells were washed with Hank’s balanced salt solution supplemented and in the mouse cochlea. This is likely connected to altered with 2.5 mM CaCl2 and 2mM MgCl2 and extracted with actin extraction buffer ROCK2 localization and activity, as both ROCK2 KD and overex- (138 mM KCl, 3 mM MgCl2, 2 mM EGTA, 0.32 M sucrose, 10 mM MES [pH 6.1], pression of Y722F-ROCK2 are reported to increase RLC phos- 0.5% Tx100, and protease inhibitors [Roche]) for 10 min on ice before SDS phorylation and stress fiber formation (Lee and Chang, 2008; lysis. For hanging drop assays, 2 3 104 LucKD or Ptk7 KD cells were trypsinized Lee et al., 2010; Lock et al., 2012; Yoneda et al., 2005). Thus, and resuspended in 35 ml hanging drops of media. After overnight incubation, together with accumulating evidence from other cell types, our cell aggregates were triturated five times and imaged by phase-contrast data suggest that ROCK1 and ROCK2 may have nonoverlapping microscopy. and even antagonistic functions in spatial regulation of myosin II. GFP-positive stable clones of lentivirus-transduced MDCK cells were In the mouse OC, junctional Src signaling requires PTK7 and is isolated by single cell dilution and screened for efficient Ptk7 knockdown. crucial for PCP regulation. At present, it is unclear whether PTK7 Four stable cell lines were established by transducing MDCK II cells with the plays an instructive or permissive role in asymmetric junctional following bicistronic lentiviral vectors: (1) Luc KD and EGFP, (2) Ptk7 KD and EGFP, (3) Ptk7 KD and PTK7WT-Venus, and (4) Ptk7 KD and PTK7Dcyt-Venus. Src activity. Although PTK7 is expressed in both hair cells and Efficient knockdown of Ptk7 in the stable cell lines were confirmed by immuno- supporting cells, it is conceivable that its activity and/or localiza- staining using an anti-PTK7 antibody that recognizes the endogenous canine, tion are regulated by posttranslational mechanisms, such as pro- but not the murine PTK7 protein (a gift of David Lewis). teolysis (Golubkov et al., 2010) and tyrosine phosphorylation. For ROCK inhibition, cells were cultured for 32 hr and then changed into Indeed, reciprocal regulation between Src and RTKs has been media containing vehicle (DMSO) or 30 mM Y27632 (EMD Biosciences) widely observed (Parsons and Parsons, 2004). Src signaling in for 20 hr. For PI3K inhibition, cells were cultured for 32 hr and then changed into media the OC is likely regulated by multiple pathways. In other systems, containing vehicle (DMSO) or 2 mM wortmannin (EMD Biosciences) for 1 hr. Src can be rapidly activated by mechanical force (Wang et al., 2005) and cadherin adhesion (McLachlan et al., 2007). Because Immunoblotting and Immunoprecipitations hair cells and supporting cells are mechanically coupled through For analysis of total cell extracts, cells were scraped directly into Laemmli cadherin-based adhesions, we speculate that Src signaling is sample buffer. After SDS-PAGE, proteins were transferred to nitrocellulose cooperatively regulated by PTK7 and cadherin-mediated me- and detected by either chemiluminescence using the Immobilon Western chanotransduction (Gomez et al., 2011). The core PCP pathway HRP chemiluminescence substrate (Millipore) or, for quantification, with the Odyssey Infrared Imaging System (LI-COR). may participate in cadherin-mediated mechanotransduction, for GFP-Trap agarose beads (Allele Biotechnology) were used for immunopre- example, through regulation of E-cadherin surface expression cipatation of GFP/Venus fusion proteins. Briefly, cells were lysed in 20 mM (Chacon-Heszele et al., 2012; Warrington et al., 2013). Our Tris/Hcl (pH 7.6), 200 mM NaCl, 2.5 mM MgCl2, 1%NP-40, 10% Glycerol, data further suggest that the PTK7-Src signaling module and 1 mM dithiothreitol (DTT), 25 mM NaF, 1 mM Na3VO4, 5 mM Na4P2O7, and the core PCP pathway may converge on ROCK signaling to 1X Protease inhibitor cocktail (Roche). Lysates were incubated with GFP-  spatially coordinate contractile tension and PCP in the OC. Trap beads for 1 hr at 4 C. Beads were washed six times with 0.1% NP-40 lysis buffer, and bound fractions were analyzed by SDS-PAGE or used for in vitro How does junctional contractility affect stereociliary bundle kinase reactions. orientation? It has been established that the positioning of the For coIP of endogenous Src and PTK7, Luc KD and Ptk7 KD cell lysates hair cell basal body and the associated kinocilium plays an were incubated with 3 mg of Src 2-17 antibodies for 3 hr at 4C. Immunocom- instructive role in stereociliary bundle orientation. Our previous plexes were then precipitated with protein A/G-agarose preblocked with BSA. work suggests a model in which microtubule capture at the Following washing with 0.1% NP-40 lysis buffer, bound proteins were eluted hair cell cortex anchors the basal body at the lateral pole of with 2x Laemmli buffer and subjected to immunoblot analysis. hair cells through a positive feedback loop involving Rac-PAK signaling (Sipe et al., 2013; Sipe and Lu, 2011). We propose Immunofluorescence and Image Analysis MDCK II cells were plated on 12 mm glass coverslips (Fisher Scientific) coated that increased tension exerted on the medial boundary of hair with 5 mg/ml fibronectin (Sigma) at 5 3 105 cells/well and grown for 48 hr. Cells cells locally inhibit cortical capture of microtubules, thereby were fixed with 4% paraformaldehyde for 25 min at room temperature and favoring microtubule capture at the lateral pole to position the permeabilized with 0.1% Triton X-100 (Tx100). Alternatively, cells were fixed basal body. with ice-cold methanol. Fixed cells were incubated for 1 hr in PBS with 1% bovine serum albumin (BSA) and 5% heat-inactivated goat serum, stained with primary antibodies overnight at 4C, and stained with secondary anti- EXPERIMENTAL PROCEDURES bodies for 45 min at room temperature. Immunostaining of whole-mount cochleae were carried out as previously described (Lee et al., 2012). Cell Culture and Transfection Confocal images were collected on a Zeiss LSM700 Confocal Laser Scan- MDCK II cell lines were maintained in high glucose Dulbecco’s modified ning Microscope (Carl Zeiss) with Photo multiplier tubes using a 633 objective Eagle’s medium (GIBCO), supplemented with 10% FBS (GIBCO) and peni- (NA 1.4) at 0.4 mm intervals. Images were collected at 8-bit depth, 1,024 3  cillin/streptomycin at 37 C, 5% CO2. 1,024 pixels resolution with fixed laser settings, and detector gain for the For transient transfection, 2 3 106 MDCK II cells were electroporated with same experiment. Optical slices along the z axis were generated using the 6 mg control pSUPER-shLuciferase construct (LucKD) or a pSUPER construct Zen 2009 LE software (Carl Zeiss). Alternatively, z stacks of images were targeting the canine Ptk7 mRNA (Ptk7 KD) using the Amaxa Nucleofector collected using a Deltavision deconvolution microscope equipped with a System following the manufacturer’s instructions. For cell height quantifica- Plan-Apochromat N 60 3 /1.42 oil objective (Olympus) and a CoolSNAP tion, the lengths of the lateral cell-cell contacts were measured in ImageJ, HQ2 CCD camera (Photometrics) at 0.2 mm intervals using the Softworx soft- using F-actin staining to mark the apical and basal surfaces. ware package (GE Healthcare). Images were assembled in Adobe Photoshop

Developmental Cell 29, 20–33, April 14, 2014 ª2014 Elsevier Inc. 31 Developmental Cell PTK7-Src Signaling Regulates ROCK2 and PCP

(Adobe Systems). Identical imaging conditions were used for control and REFERENCES mutant samples. Fluorescence intensity measurements of junctional staining for various pro- Amsberg, G.K., and Koschmieder, S. (2013). Profile of bosutinib and its clinical teins in MDCK cells and the mouse OC were taken from single z plane images potential in the treatment of chronic myeloid leukemia. Onco Targets Ther. 6, using line scan analysis in ImageJ. 99–106. Bardet, P.L., Guirao, B., Paoletti, C., Serman, F., Le´ opold, V., Bosveld, F., Cochlear Explant Cultures and Src Inhibitor Treatment Goya, Y., Mirouse, V., Graner, F., and Bellaı¨che, Y. (2013). PTEN controls junc- Cochlear explants from E15.5 embryos were established as previously tion lengthening and stability during cell rearrangement in epithelial tissue. described (Lee et al., 2012). Explants were treated with either vehicle Dev. Cell 25, 534–546. (DMSO) or 5 mM SU6656 (Millipore) after 4 hr in vitro. After 48 hr of drug treat- Bin-Nun, N., Lichtig, H., Malyarova, A., Levy, M., Elias, S., and Frank, D. (2014). ment, media were replaced with drug-free media. Explants were maintained PTK7 modulates Wnt signaling activity via LRP6. Development 141, 410–421. for another 3 days in vitro and then processed for immunostaining. For in vivo Src inhibition, a single dose of Bosutinib (LC laboratories) was administered Blake, R.A., Broome, M.A., Liu, X., Wu, J., Gishizky, M., Sun, L., and intraperitoneally to wild-type dams on E16.5 or 17.5 at 30 mg/kg, and embryos Courtneidge, S.A. (2000). SU6656, a selective src family kinase inhibitor, were harvested 4 hr later and processed for immunostaining. Embryos from used to probe growth factor signaling. Mol. Cell. Biol. 20, 9018–9027. vehicle-injected dams were used as controls. Blankenship, J.T., Backovic, S.T., Sanny, J.S., Weitz, O., and Zallen, J.A. Western blot analysis of cochlear tissues was performed as previously (2006). Multicellular rosette formation links planar cell polarity to tissue described (Lee et al., 2012). Briefly, lysates from two cochleae were pooled morphogenesis. Dev. Cell 11, 459–470. and loaded in each lane. GAPDH served as loading control. To assay for Src Chacon-Heszele, M.F., Ren, D., Reynolds, A.B., Chi, F., and Chen, P. (2012). inhibition, cochlear explants were established on E16.5, treated with DMSO, Regulation of cochlear convergent extension by the vertebrate planar cell SU6656, or Bosutinib at 5 mM for 1 hr, lysed in SDS sample buffer, and polarity pathway is dependent on p120-catenin. Development 139, 968–978. analyzed by pY416-Src immunoblotting. Garton, A.J., Castaldo, L., and Pachter, J.A. (2008). Quantitative high- throughput cell-based assays for inhibitors of ROCK kinases. Methods Mice Enzymol. 439, 491–500. All mice were maintained in compliance with National Institutes of Health Gassama-Diagne, A., Yu, W., ter Beest, M., Martin-Belmonte, F., Kierbel, A., guidelines and the Animal Care and Use Committee at the University of Engel, J., and Mostov, K. (2006). Phosphatidylinositol-3,4,5-trisphosphate Virginia. Mice either were obtained from the referenced sources or were pro- regulates the formation of the basolateral plasma membrane in epithelial cells. duced in-house and maintained on a mixed genetic background. Nat. Cell Biol. 8, 963–970. Statistical Analysis Golubkov, V.S., Chekanov, A.V., Cieplak, P., Aleshin, A.E., Chernov, A.V., Zhu, When appropriate, data are represented as the mean ± SEM of a field of cells W., Radichev, I.A., Zhang, D., Dong, P.D., and Strongin, A.Y. (2010). The Wnt/ from at least three independent experiments. p values were calculated using planar cell polarity protein-tyrosine kinase-7 (PTK7) is a highly efficient proteo- two-tailed t tests unless indicated otherwise. lytic target of membrane type-1 matrix metalloproteinase: implications in can- Details of the constructs and primary antibodies used and other methods cer and embryogenesis. J. Biol. Chem. 285, 35740–35749. are described in the Supplemental Experimental Procedures. Gomez, G.A., McLachlan, R.W., and Yap, A.S. (2011). Productive tension: force-sensing and homeostasis of cell-cell junctions. Trends Cell Biol. 21, SUPPLEMENTAL INFORMATION 499–505. Goodrich, L.V., and Strutt, D. (2011). Principles of planar polarity in animal Supplemental Information includes Supplemental Experimental Procedures development. Development 138, 1877–1892. and five figures and can be found with this article online at http://dx.doi.org/ Grassie, M.E., Moffat, L.D., Walsh, M.P., and MacDonald, J.A. (2011). The 10.1016/j.devcel.2014.02.008. myosin phosphatase targeting protein (MYPT) family: a regulated mechanism for achieving substrate specificity of the catalytic subunit of protein phospha- AUTHOR CONTRIBUTIONS tase type 1d. Arch. Biochem. Biophys. 510, 147–159. Grimsley-Myers, C.M., Sipe, C.W., Ge´ le´ oc, G.S., and Lu, X. (2009). The small A.A. performed most of the experiments in MDCK cells. J.L. performed the GTPase Rac1 regulates auditory hair cell morphogenesis. J. Neurosci. 29, analysis of the mouse inner ear. Both A.A and J.L. participated in the writing 15859–15869. of the manuscript. Hayes, M., Naito, M., Daulat, A., Angers, S., and Ciruna, B. (2013). Ptk7 pro- ACKNOWLEDGMENTS motes non-canonical Wnt/PCP-mediated morphogenesis and inhibits Wnt/ b-catenin-dependent cell fate decisions during vertebrate development. We thank Drs. Amy Bouton, Jim Casanova, Barry Gumbiner, Rick Horwitz, and Development 140, 1807–1818. Sally Parsons (University of Virginia), Zee-Fen Chang and Hsiao-Hui Lee Hildebrand, J.D. (2005). Shroom regulates epithelial cell shape via the apical (National Taiwan University), Margaret Frame (Edinburgh Cancer Research positioning of an actomyosin network. J. Cell Sci. 118, 5191–5203. UK Centre), Jeff Hildebrand (University of Pittsburgh), David Lewis (Stanford Imamoto, A., and Soriano, P. (1993). Disruption of the csk gene, encoding a University), Alexander Tarakhovsky (Rockefeller University), and Gangjian negative regulator of Src family tyrosine kinases, leads to neural tube defects Qin (Northwestern University) for reagents; Dr. Darkhan Utepbergenov (Uni- and embryonic lethality in mice. Cell 73, 1117–1124. versity of Virginia) for assistance with baculovirus work; Dr. Luke McCaffrey Lecuit, T., Lenne, P.F., and Munro, E. (2011). Force generation, transmission, (McGill University) for advice on lentivirus work; and Drs. Jim Casanova and and integration during cell and tissue morphogenesis. Annu. Rev. Cell Dev. Barry Gumbiner (University of Virginia) and members of the Lu laboratory for Biol. 27, 157–184. helpful comments. This study was supported by Basil O’Connor Starter Lee, H.H., and Chang, Z.F. (2008). Regulation of RhoA-dependent ROCKII Scholar Research awards (#5-FY07-166) from the March of Dimes Foundation activation by Shp2. J. Cell Biol. 181, 999–1012. and by a National Institutes of Health (NIH) grant (DC009238) (to X.L.) and by a NIH grant (GM070902; to I.G.M.). Lee, H.H., Tien, S.C., Jou, T.S., Chang, Y.C., Jhong, J.G., and Chang, Z.F. (2010). Src-dependent phosphorylation of ROCK participates in regulation of Received: February 11, 2013 focal adhesion dynamics. J. Cell Sci. 123, 3368–3377. Revised: January 15, 2014 Lee, J., Andreeva, A., Sipe, C.W., Liu, L., Cheng, A., and Lu, X. (2012). PTK7 Accepted: February 12, 2014 regulates myosin II activity to orient planar polarity in the mammalian auditory Published: April 3, 2014 epithelium. Curr. Biol. 22, 956–966.

32 Developmental Cell 29, 20–33, April 14, 2014 ª2014 Elsevier Inc. Developmental Cell PTK7-Src Signaling Regulates ROCK2 and PCP

Lock, F.E., Ryan, K.R., Poulter, N.S., Parsons, M., and Hotchin, N.A. (2012). Shnitsar, I., and Borchers, A. (2008). PTK7 recruits dsh to regulate neural crest Differential regulation of adhesion complex turnover by ROCK1 and ROCK2. migration. Development 135, 4015–4024. PLoS ONE 7, e31423. Sipe, C.W., and Lu, X. (2011). Kif3a regulates planar polarization of auditory Lu, X., Borchers, A.G., Jolicoeur, C., Rayburn, H., Baker, J.C., and Tessier- hair cells through both ciliary and non-ciliary mechanisms. Development Lavigne, M. (2004). PTK7/CCK-4 is a novel regulator of planar cell polarity in 138, 3441–3449. vertebrates. Nature 430, 93–98. Sipe, C.W., Liu, L., Lee, J., Grimsley-Myers, C., and Lu, X. (2013). Lis1 medi- Martin, A.C., Kaschube, M., and Wieschaus, E.F. (2009). Pulsed contractions ates planar polarity of auditory hair cells through regulation of microtubule of an actin-myosin network drive apical constriction. Nature 457, 495–499. organization. Development 140, 1785–1795. McLachlan, R.W., Kraemer, A., Helwani, F.M., Kovacs, E.M., and Yap, A.S. Skoglund, P., Rolo, A., Chen, X., Gumbiner, B.M., and Keller, R. (2008). (2007). E-cadherin adhesion activates c-Src signaling at cell-cell contacts. Convergence and extension at gastrulation require a myosin IIB-dependent Mol. Biol. Cell 18, 3214–3223. cortical actin network. Development 135, 2435–2444. Nagafuchi, A., and Takeichi, M. (1988). Cell binding function of E-cadherin is Smutny, M., Cox, H.L., Leerberg, J.M., Kovacs, E.M., Conti, M.A., Ferguson, regulated by the cytoplasmic domain. EMBO J. 7, 3679–3684. C., Hamilton, N.A., Parton, R.G., Adelstein, R.S., and Yap, A.S. (2010). Nakajima, H., and Tanoue, T. (2011). Lulu2 regulates the circumferential acto- Myosin II isoforms identify distinct functional modules that support integrity myosin tensile system in epithelial cells through p114RhoGEF. J. Cell Biol. 195, of the epithelial zonula adherens. Nat. Cell Biol. 12, 696–702. 245–261. Terry, S.J., Zihni, C., Elbediwy, A., Vitiello, E., Leefa Chong San, I.V., Balda, Nishimura, T., Honda, H., and Takeichi, M. (2012). Planar cell polarity links axes M.S., and Matter, K. (2011). Spatially restricted activation of RhoA signalling of spatial dynamics in neural-tube closure. Cell 149, 1084–1097. at epithelial junctions by p114RhoGEF drives junction formation and morpho- Ohyama, T., and Groves, A.K. (2004). Generation of Pax2-Cre mice by modi- genesis. Nat. Cell Biol. 13, 159–166. fication of a Pax2 bacterial artificial chromosome. Genesis 38, 195–199. Thomas, S.M., Soriano, P., and Imamoto, A. (1995). Specific and redundant Parsons, S.J., and Parsons, J.T. (2004). Src family kinases, key regulators of roles of Src and Fyn in organizing the cytoskeleton. Nature 376, 267–271. signal transduction. Oncogene 23, 7906–7909. Vicente-Manzanares, M., Ma, X., Adelstein, R.S., and Horwitz, A.R. (2009). Paudyal, A., Damrau, C., Patterson, V.L., Ermakov, A., Formstone, C., Lalanne, Non-muscle myosin II takes centre stage in cell adhesion and migration. Z., Wells, S., Lu, X., Norris, D.P., Dean, C.H., et al. (2010). The novel mouse Nat. Rev. Mol. Cell Biol. 10, 778–790. mutant, chuzhoi, has disruption of Ptk7 protein and exhibits defects in neural Wang, Y., Botvinick, E.L., Zhao, Y., Berns, M.W., Usami, S., Tsien, R.Y., and tube, heart and lung development and abnormal planar cell polarity in the ear. Chien, S. (2005). Visualizing the mechanical activation of Src. Nature 434, BMC Dev. Biol. 10, 87. 1040–1045. Qin, Y., Capaldo, C., Gumbiner, B.M., and Macara, I.G. (2005). The mammalian Warrington, S.J., Strutt, H., and Strutt, D. (2013). The Frizzled-dependent Scribble polarity protein regulates epithelial cell adhesion and migration planar polarity pathway locally promotes E-cadherin turnover via recruitment through E-cadherin. J. Cell Biol. 171, 1061–1071. of RhoGEF2. Development 140, 1045–1054. Ratheesh, A., Gomez, G.A., Priya, R., Verma, S., Kovacs, E.M., Jiang, K., Brown, N.H., Akhmanova, A., Stehbens, S.J., and Yap, A.S. (2012). Wehner, P., Shnitsar, I., Urlaub, H., and Borchers, A. (2011). RACK1 is a novel Centralspindlin and a-catenin regulate Rho signalling at the epithelial zonula interaction partner of PTK7 that is required for neural tube closure. adherens. Nat. Cell Biol. 14, 818–828. Development 138, 1321–1327. Rauzi, M., Lenne, P.F., and Lecuit, T. (2010). Planar polarized actomyosin con- Williams, M., Yen, W.W., Lu, X., and Sutherland, A. (2014). Distinct apical and tractile flows control epithelial junction remodelling. Nature 468, 1110–1114. basolateral mechanisms drive PCP-dependent convergent extension of the mouse neural plate. Dev. Cell 29. Published online April 14, 2014. http://dx. Sandilands, E., Cans, C., Fincham, V.J., Brunton, V.G., Mellor, H., doi.org/10.1016/j.devcel.2014.02.007. Prendergast, G.C., Norman, J.C., Superti-Furga, G., and Frame, M.C. (2004). RhoB and actin polymerization coordinate Src activation with endo- Yen, W.W., Williams, M., Periasamy, A., Conaway, M., Burdsal, C., Keller, R., some-mediated delivery to the membrane. Dev. Cell 7, 855–869. Lu, X., and Sutherland, A. (2009). PTK7 is essential for polarized cell motility and convergent extension during mouse gastrulation. Development 136, Sawyer, J.M., Harrell, J.R., Shemer, G., Sullivan-Brown, J., Roh-Johnson, M., 2039–2048. and Goldstein, B. (2010). Apical constriction: a cell shape change that can drive morphogenesis. Dev. Biol. 341, 5–19. Yoneda, A., Multhaupt, H.A., and Couchman, J.R. (2005). The Rho kinases I Schmedt, C., Saijo, K., Niidome, T., Ku¨ hn, R., Aizawa, S., and Tarakhovsky, A. and II regulate different aspects of myosin II activity. J. Cell Biol. 170, 443–453. (1998). Csk controls antigen receptor-mediated development and selection of Zallen, J.A., and Wieschaus, E. (2004). Patterned gene expression directs T-lineage cells. Nature 394, 901–904. bipolar planar polarity in Drosophila. Dev. Cell 6, 343–355.

Developmental Cell 29, 20–33, April 14, 2014 ª2014 Elsevier Inc. 33