PTK7 Localization and Protein Stability Is Affected by Canonical Wnt Ligands

RESEARCH ARTICLE PTK7 localization and protein stability is affected by canonical Wnt ligands Hanna Berger1, Marlen Breuer1,4, Hanna Peradziryi2, Martina Podleschny1, Ralf Jacob3,4 and Annette Borchers1,4,*

ABSTRACT Wnt binding results in membrane recruitment of a destruction β Protein tyrosine kinase 7 (PTK7) is an evolutionarily conserved complex, leading to the stabilization of -catenin and expression of β transmembrane receptor with important roles in embryonic -catenin-dependent target genes (MacDonald and He, 2012; β development and disease. Originally identified as a gene MacDonald et al., 2009). In contrast, non-canonical -catenin- upregulated in colon cancer, it was later shown to regulate planar independent Wnt signaling pathways use alternative co-receptors cell polarity (PCP) and directional cell movement. PTK7 is a Wnt like the receptor tyrosine kinase-like orphan receptor 2 (Ror2) or co-receptor; however, its role in Wnt signaling remains controversial. protein tyrosine kinase 7 (PTK7) (Lu et al., 2004; Nomachi et al., Here, we find evidence that places PTK7 at the intersection of 2008; Schambony and Wedlich, 2007). The best-characterized non- canonical and non-canonical Wnt signaling pathways. In presence of canonical Wnt signaling pathway is the planar cell polarity (PCP) canonical Wnt ligands PTK7 is subject to caveolin-mediated pathway, which determines the coordinated polarity of cells in endocytosis, while it is unaffected by non-canonical Wnt ligands. the plane of an epithelial tissue, thereby regulating diverse PTK7 endocytosis is dependent on the presence of the PTK7 developmental processes (Simons and Mlodzik, 2008; Vladar co-receptor Fz7 (also known as Fzd7) and results in lysosomal et al., 2009; Wallingford, 2012). Activation of PCP signaling degradation of PTK7. As we previously observed that PTK7 activates modifies the cytoskeleton and changes cell polarity, for example, by non-canonical PCP Wnt signaling but inhibits canonical Wnt activating small GTPases or JNK-dependent transcription factors signaling, our data suggest a mutual inhibition of canonical and (Anastas and Moon, 2013) like ATF2 and their respective target PTK7 Wnt signaling. PTK7 likely suppresses canonical Wnt signaling genes (Ohkawara and Niehrs, 2011; Schambony and Wedlich, by binding canonical Wnt ligands thereby preventing their interaction 2007). Thus, receptor context is crucial to determine signaling with Wnt receptors that would otherwise support canonical Wnt outcome. signaling. Conversely, if canonical Wnt proteins interact with the A Wnt co-receptor of particular interest is the evolutionarily PTK7 receptor, they induce its internalization and degradation. conserved transmembrane receptor PTK7. Originally identified as a gene upregulated in colon carcinomas and named colon-carcinoma KEY WORDS: Wnt signaling, PTK7, Planar cell polarity, Endocytosis, kinase 4 (CCK4) (Mossie et al., 1995), PTK7 was later shown to Caveolin function in a variety of developmental and physiological processes, including the determination of PCP, and the control of cell migration INTRODUCTION and invasion as well as regeneration (Berger et al., 2017; Dunn and Wnt proteins are important for embryonic development and adult Tolwinski, 2016; Peradziryi et al., 2012). PTK7 is a single-pass tissue homeostasis, and these distinct functions require a precise transmembrane receptor with extracellular immunoglobulin fine-tuning of downstream signaling events. The demonstration that domains and an intracellular evolutionarily conserved kinase a single Wnt ligand can activate distinct signaling pathways homology domain, which lacks catalytic activity (Kroiher et al., depending on its choice of receptor (Mikels and Nusse, 2006), 2001; Miller and Steele, 2000). In vertebrates, PTK7 plays a role in suggested that receptor context determines signaling output. It is the regulation of PCP, and its loss of function results in classical now acknowledged that combinatorial co-receptor complexes PCP phenotypes, including convergent extension defects, provide a molecular code by which Wnt ligands can cause distinct disruption of stereociliary bundle orientation and inhibition of cellular responses (Niehrs, 2012; van Amerongen et al., 2008). For neural crest cell migration (Hayes et al., 2013; Lu et al., 2004; example the canonical β-catenin-dependent Wnt signaling pathway Paudyal et al., 2010; Shnitsar and Borchers, 2008; Yen et al., 2009). is activated by binding of Wnt ligands to members of the Frizzled Consistent with a role in activation of PCP signaling, PTK7 has receptor family and the low-density lipoprotein receptor-related been shown to interact with Wnt ligands as well as the Frizzled 7 proteins (LRP5 or LRP6) (Tamai et al., 2000; Wehrli et al., 2000). (Fz7; also known as Fzd7) and Ror2 receptors (Linnemannstöns et al., 2014; Martinez et al., 2015; Peradziryi et al., 2012; Podleschny et al., 2015; Shnitsar and Borchers, 2008). 1Department of Biology, Molecular Embryology, Philipps-Universität Marburg, Marburg 35043, Germany. 2Institute for Clinical Research, Georg-August Furthermore, PTK7 has been reported to recruit Dishevelled UniversitätGöttingen, Göttingen 37075, Germany. 3Department of Cell Biology and (Dsh) proteins to the plasma membrane and to activate JNK and Cell Pathology, Philipps-Universität Marburg, Marburg 35037, Germany. 4DFG ATF2-dependent signaling (Martinez et al., 2015; Peradziryi et al., Research Training Group, Membrane Plasticity in Tissue Development and Remodeling, GRK 2213, Philipps-Universität Marburg, Marburg 35043, Germany. 2011; Shnitsar and Borchers, 2008). However, PTK7 function is likely not limited to the regulation of non-canonical Wnt PCP *Author for correspondence ([email protected]) signaling, because PTK7 has also been shown to interact with A.B., 0000-0002-8314-9024 components of the canonical Wnt pathway including Wnt ligands, the LRP6 receptor and β-catenin (Bin-Nun et al., 2014; Peradziryi

Received 12 October 2016; Accepted 7 April 2017 et al., 2011; Puppo et al., 2011). Recently, we demonstrated that Journal of Cell Science

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PTK7 interacts with canonical Wnt ligands Wnt3a and Wnt8, but treated with Wnt3a or Wnt5a. Consistent with our previous not non-canonical Wnt5a or Wnt11. Overexpression of PTK7 findings, the amount of cell surface PTK7–GFP and ΔkPTK7– blocked activation of canonical Wnt signaling by Wnt3a and Wnt8 GFP significantly decreased after Wnt3a treatment, but remained in Xenopus double axis and luciferase assays. Conversely, PTK7 unchanged after Wnt5a stimulation (Fig. 1C,D). loss of function activated canonical Wnt activity, suggesting that In order to perform live-cell imaging of the Wnt-mediated PTK7 PTK7 inhibits canonical Wnt signaling (Peradziryi et al., 2011). internalization, total internal reflection fluorescence (TIRF) These findings are also supported by PTK7 loss-of-function studies microscopy was used. TIRF microscopy has the advantage that in zebrafish (Hayes et al., 2013), but are in conflict to others the basal plasma membrane and the cytoplasmic regions reporting activation of canonical Wnt signaling by PTK7 (Bin-Nun immediately beneath the plasma membrane can be visualized, et al., 2014; Puppo et al., 2011). Thus, the specific role of PTK7 in allowing the analysis of PTK7 vesicle formation and internalization the regulation of Wnt signaling pathways is controversial. in response to Wnt3a or Wnt5a. MCF7 cells expressing PTK7–GFP A conundrum is that PTK7 regulates PCP signaling; however, it were imaged before and after the addition of canonical Wnt3a or also serves as a receptor for canonical Wnt ligands. Our previous non-canonical Wnt5a (Movies 1,2). While few PTK7-positive data indicated that PTK7 interacts with canonical Wnt ligands, vesicles with limited mobility were observed in PTK7–GFP- leading to inhibition of canonical Wnt signaling (Peradziryi et al., expressing cells, Wnt3a treatment caused a significant increase in 2011). Currently, it remains unclear how PTK7 is affected by the number of PTK7-positive vesicles (Movie 1; Fig. 2A). These binding to canonical Wnt ligands. Here, we further analyze this by vesicles appeared larger and more mobile compared to those of studying PTK7 protein localization and stability in the presence of untreated controls (Fig. 2B,C). Furthermore, we also observed a different Wnt ligands. We find that canonical Wnt ligands lead significant increase in the internalization of PTK7-positive vesicles to caveolin-mediated endocytosis and lysosomal degradation of (Fig. 2A). An example of the formation and internalization of a PTK7, while the localization of PTK7 is not affected by the PTK7-positive vesicle in presence of Wnt3a is shown in Fig. 2D. In presence of non-canonical Wnt ligands. Thus, our data suggest that contrast, cells treated with Wnt5a resembled untreated control cells PTK7 is removed from the cell membrane in presence of canonical (Movie 2; Fig. 2A–C). In summary, these data show that in the Wnt ligands. Thereby a Wnt morphogen gradient could direct the presence of canonical Wnt ligands membrane-localized PTK7 is cellular polarization of PTK7-expressing cells. Conversely, PTK7 internalized and accumulates in vesicle-like structures, while non- can also trap canonical Wnt ligands and accordingly inhibit their canonical Wnt ligands do not affect PTK7 localization. function. Thus, the interaction of canonical Wnt ligands and PTK7 can result in a mutual inhibition, and might be a mechanism to PTK7 colocalizes with canonical Wnt in intracellular define border regions of active canonical versus non-canonical vesicles signaling or for directed cellular polarization in the wider sense. As PTK7 enters the cell via a Wnt-mediated mechanism, we would expect that the Wnt ligand likely colocalizes with PTK7 in the RESULTS cytoplasm. To clarify this, MCF7 cells expressing PTK7–RFP were PTK7 changes its cellular localization in response to cultured together with cells expressing and secreting canonical canonical Wnt proteins Wnt2b–GFP or non-canonical Wnt5a–GFP. Subsequently, Recent evidence indicates that PTK7 is a Wnt receptor; however, it colocalization was assessed in living cells (Fig. 3A). As tagging remains unclear how the PTK7 protein is affected by this of Wnt proteins may affect their function, we used here GFP-tagged interaction. Previously, we have shown that PTK7 interacts with Wnt constructs that had previously been shown to be functionally canonical Wnt3a and Wnt8, but not with non-canonical Wnt5a and active in canonical and non-canonical Wnt signaling, respectively Wnt11 (Peradziryi et al., 2011). As ligand binding can cause (Holzer et al., 2012; Wallkamm et al., 2014). Indeed, we found receptor endocytosis, we analyzed whether PTK7 protein evidence that a PTK7–Wnt2b complex translocates to the localization is affected by canonical Wnt ligands. To this end cytoplasm. Secreted Wnt2b–GFP was found to colocalize with PTK7–GFP was stably expressed in MCF7 cells and its localization PTK7–RFP in vesicle-like structures in the cytoplasm (Fig. 3B,C). in presence or absence of recombinant Wnt proteins was analyzed An orthogonal cell view confirms that these PTK7–Wnt2b-positive by confocal microscopy. In the absence of Wnt ligands, PTK7 is vesicles are located in the cytoplasm (Fig. S2A). In contrast, predominantly localized at the cell membrane (Fig. 1A; Fig. S1). In colocalization of PTK7–RFP with Wnt5a–GFP was only rarely contrast, treatment with canonical Wnt3a caused a significant shift observed (Fig. 3B,C; Fig. S2B). Taken together, these data indicate of the PTK7 protein from the plasma membrane to the cytoplasm. that PTK7 interacts with canonical Wnts at the plasma membrane This translocation did not occur in cells treated with non-canonical and subsequently enters the cell as a PTK7–Wnt complex. In Wnt5a, where the PTK7 localization pattern was comparable to that contrast, non-canonical Wnt5a, which does not interact with PTK7, in untreated cells. Next, we asked whether the kinase domain of does not affect PTK7 membrane localization. PTK7, which is required for Dsh recruitment (Shnitsar and Borchers, 2008; Wehner et al., 2011), is necessary for Wnt- PTK7 internalization requires the Fz7 but not the Ror2 mediated PTK7 translocation. Like full-length PTK7, a PTK7 co-receptor deletion mutant lacking the kinase homology domain (ΔkPTK7) PTK7 is a Wnt co-receptor that interacts with other known Wnt shifted from the plasma membrane to the cytoplasm in the presence receptors (Bin-Nun et al., 2014; Martinez et al., 2015; Peradziryi of Wnt3a but not in the presence of Wnt5a (Fig. 1B). Thus, the et al., 2011; Podleschny et al., 2015). As the Fz7 receptor is required Wnt3a-mediated translocation of the PTK7 protein is independent for the interaction of PTK7 with canonical Wnt ligands (Peradziryi of its kinase homology domain. To further verify the Wnt3a- et al., 2011), we analyzed whether the Wnt-mediated translocation dependent shift of the PTK7 receptor from the plasma membrane to of PTK7 is dependent on Fz7. To this end, we used Xenopus the cytoplasm, cell surface biotinylation analyses were performed ectodermal (animal cap) cells expressing PTK7–Myc and combined (Fig. 1C). The fraction of membrane-localized PTK7 and its kinase them either with control or Wnt-expressing animal cap cells deletion mutant was determined in untreated cells versus cells to create larger cell aggregates (Fig. 3D,E). Confirming our Journal of Cell Science

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Fig. 1. Canonical, but not non-canonical Wnt ligands, induce PTK7 translocation independently of its kinase homology domain. (A,B) MCF7 cells stably expressing PTK7–GFP or ΔkPTK7–GFP were treated with recombinant human canonical Wnt3a or non-canonical Wnt5a proteins for 1 h. (A) PTK7–GFP is predominantly localized at the plasma membrane in control cells (top) and cells treated with Wnt5a (bottom). In contrast, in the presence of Wnt3a, PTK7 is mainly localized in the cytoplasm (middle). The graph shows the mean±s.e.m. distribution pattern of PTK7 in three independent experiments. (B) As with full-length PTK7, ΔkPTK7-GFP is mainly localized at the membrane in control cells (top) or cells treated with Wnt5a (bottom), while its localization shifts to the plasma membrane in cells treated with Wnt3a (middle). The graph summarizes the distribution pattern of ΔkPTK7–GFP in three independent experiments. *P<0.01; **P<0.005 (Student’s t-test). m, membrane localization; m/c, membrane and cytoplasmic localization; c, cytoplasmic localization. Scale bars: 10 µm. (C) Cell surface biotinylation assay. PTK7–GFP- or ΔkPTK7–GFP-expressing MCF7 cells were treated with recombinant Wnt3a or Wnt5a for 1 h. Cell surface proteins were biotinylated and precipitated using neutravidin beads. Cell surface and total PTK7 (left panel) or ΔkPTK7 (right panel) levels were detected by using an anti- GFP antibody. Molecular masses (kDa) are indicated. (D) The graph shows the mean±s.e.m. relative membrane localization (AU, arbitrary units) of PTK7 and ΔkPTK7 in three independent experiments. The ratio of cell surface to total protein levels was normalized to the untreated control. mammalian cell culture data, we observe that PTK7 is localized at Wnt2b-mediated internalization of PTK7. As PTK7 can also the plasma membrane in control cells, but is internalized from the interact with the non-canonical Wnt receptor Ror2 (Martinez et al., membrane in the presence of Wnt2b (Fig. 3Da). PTK7 localized in 2015; Podleschny et al., 2015), we also tested whether Ror2 is vesicle-like structures, where it also colocalized with Wnt2b. required. However, Ror2 loss of function did not affect the Wnt2b- Conversely, Wnt5a did not affect the membrane localization of mediated translocation of PTK7 (Fig. 3Dc,E). Compared to the PTK7. In contrast, co-injection of PTK7 with a morpholino results obtained with MCF7 cells (Fig. 3C), fewer vesicles per cell oligonucleotide (MO), which blocks the translation of the Fz7 were detected in Xenopus ectodermal cells (Fig. 3E). Furthermore, protein, significantly inhibited the Wnt2b-mediated internalization in addition to an increase in Wnt and PTK7 double-positive vesicles of PTK7 (Fig. 3Db,E). This suggests that Fz7 is required for the there is also an increase in the PTK7-positive but Wnt-negative Journal of Cell Science

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Fig. 2. PTK7 is internalized in the presence of canonical Wnt proteins. (A) PTK7–GFP-expressing MCF7 cells were imaged before and after Wnt3a or Wnt5a treatment by TIRF microscopy. Penetration depth in the Z-plane was 150 nm. The graph shows the total number (mean±s.e.m.) of PTK7-positive vesicles before (control) and after Wnt3a or Wnt5a treatment. For quantification vesicles were analyzed over a time period of 5 min. The total number of PTK7-positive vesicles (black) includes the number of internalized vesicles (gray). N, numbers of cells; n, numbers of vesicles. *P<0.005 (Student’s t-test). (B) The graph quantifies the average vesicle diameter of PTK7-positive vesicles before (control) and after Wnt3a or Wnt5a treatment in three independent experiments. On average, ten vesicles per cell were analyzed in Wnt3a-treated cells in a given 5 min interval, while fewer vesicles were visible in control or Wnt5a-treated cells. N, numbers of cells; n, numbers of vesicles. *P<0.05 (Student’s t-test). (C) Vesicle tracking of ten vesicles per condition shows the increase in vesicle dynamics after Wnt3a treatment in comparison to control or Wnt5a-treated conditions. Scale bar: 10 µm. (D) The time series shows PTK7 protein accumulation at a specific spot at the membrane and its subsequent internalization. The line scans depict PTK7–GFP fluorescence intensities over a 1 µm range. Scale bar: 1 µm. vesicle population after Wnt2b-treatment, which was not observed longer time period in the presence of Wnt-secreting cells. Thus, in control or Wnt5a-treated cells. An explanation for these Wnt2b and PTK7 double-positive vesicles may have lost the Wnt2b observations is that, in contrast to the co-culture experiment using signal over time. To test this hypothesis, we examined whether we

MCF7 cells, the Xenopus ectodermal cells were incubated for a would obtain similar results when we also prolonged the co-culture Journal of Cell Science

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Fig. 3. PTK7 is internalized together with Wnt2b in a Fz7-dependent process. (A–C) PTK7 and Wnt2b colocalize in the cytoplasm. (A) Scheme of experimental procedure. MCF7 cells were transfected with either PTK7–RFP or Wnt–GFP (canonical Wnt2b–GFP or non-canonical Wnt5a–GFP), respectively, and subsequently co-cultured. (B) Upon co-culture of PTK7–RFP with Wnt2b– GFP-expressing cells, the PTK7-expressing cells showed colocalization of both proteins in intracellular vesicles (upper panels). In contrast, in co-cultures of PTK7–RFP and Wnt5a–GFP no colocalization of PTK7 and Wnt5a was apparent (lower panels). However, a few Wnt5a-positive (but PTK7-negative) puncta were detected in the cytoplasm. The small panels on the right show higher magnifications (2.8×) of the areas indicated by a dashed square. (C) The graph shows the mean±s.e.m. number of PTK7- positive vesicles per living cell. PTK7-positive vesicles were determined by RFP fluorescence. Colocalization with Wnt2b or Wnt5a was assessed through means of the PCCs. Three independent experiments are summarized. n, number of analyzed PTK7-positive vesicles; N, cell number. *P<0.0005 (Student’s t-test). (D) Xenopus animal cap cells were injected with PTK7–Myc (PTK7-MT; red) in combination with MOs, and combined either with animal cap explants expressing Wnt2b–GFP or Wnt5a– GFP, or PTK7 as a control. Subsequently, PTK7 and Wnt proteins were detected by immunostaining. (a) In PTK7 cell aggregates injected with control MO (Co MO), PTK7 was localized at the cell membrane (a, left panel). In contrast, in cell aggregates containing Wnt2b- expressing cells, PTK7 was localized in cytoplasmic vesicles (a, middle panels), where it also frequently colocalized with Wnt2b. Conversely, aggregates containing Wnt5a- expressing cells resembled controls (a, right panels). (b) In contrast, co-injection of Fz7 MO inhibited Wnt2b-mediated endocytosis. As in Wnt5a-containing and control cell aggregates, PTK7 was mainly localized at the plasma membrane. (c) Conversely, Ror2 MO injection showed no effect and aggregates resembled the MO control. Arrowheads highlight Wnt2b and PTK7 colocalization in vesicles. (E) The graph quantifies the mean±s.e.m. number of PTK7- positive and Wnt and PTK7 double-positive vesicles per cell from three independent experiments. The quantification of PTK7 and Wnt colocalization is based on the calculated PCCs. N, number of cells; n, number of analyzed vesicles. Scale bars: 10 µm.

experiment with MCF7 cells. Indeed, we find that this is the case. In canonical Wnt ligands and requires Fz7 to enter the cytoplasm, cells incubated with Wnt ligands for 2 or 4 h, the number of PTK7- but this internalization is independent of the non-canonical Wnt positive but Wnt-negative vesicles per cell is comparable between receptor Ror2. the different treatments (Fig. S3). However, for the 8 h co-culture this population increases in the cells co-cultured with Wnt2b in PTK7 internalization is mediated by caveolin-1α rather than comparison to cells co-cultured with Wnt5a. This suggests that clathrin Wnt2b and PTK7 double-positive vesicles lose their Wnt2b signal Ligand-mediated endocytosis is a common process through which over time, either because Wnt2b is faster degraded or it leaves the transmembrane receptors are transported into the cytoplasm. To vesicles. Taken together, these data suggest that PTK7 interacts with examine whether PTK7 is internalized in the presence of canonical Journal of Cell Science

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Wnts through a clathrin- or caveolin-mediated route, MCF7 cells treatment prevented the Wnt3a-induced PTK7 (Fig. 6A,B) expressing PTK7–GFP were co-stained with antibodies detecting internalization. Consistent with these findings, the Wnt3a- endogenous caveolin-1α or clathrin. In control cells or cells treated dependent endocytosis of the PTK7 construct lacking the kinase with Wnt5a, PTK7–GFP and caveolin-1α colocalized at the homology domain, ΔkPTK7, was also abolished in cells treated with membrane, but cytoplasmic colocalization was rarely observed MβCD (data not shown). Furthermore, loss of function of caveolin- (Fig. 4A,B). Conversely, in the presence of Wnt3a, PTK7–GFP 1α in Xenopus ectodermal cells also prevented the Wnt2b-mediated colocalized with caveolin-1α in vesicle-like structures in the endocytosis of PTK7. Ectodermal aggregates composed of cells co- cytoplasm. In addition, we analyzed whether the kinase deletion injected with PTK7–Myc and control MO in combination with mutant of PTK7, ΔkPTK7–GFP, colocalizes with caveolin-1α in the Wnt2b-expressing cells showed PTK7 internalization. As expected, cytoplasm. Consistent with the findings for full-length PTK7–GFP, PTK7 largely remained at the membrane in aggregates combined ΔkPTK7–GFP colocalized with caveolin-1α in the cytoplasm after with control or Wnt5a-expressing cells (Fig. 6C). This is also Wnt3a but not after Wnt5a treatment (Fig. S4A,B). In addition to reflected by the quantification of Wnt and PTK7 double-positive the observed colocalization of fluorescent PTK7 and caveolin-1α in and PTK7-positive vesicles in these aggregates (Fig. 6D). In human and Xenopus cells, biochemical interaction of these proteins contrast, co-injection of caveolin-1α MO with PTK7–Myc was analyzed by performing co-immunoprecipitation experiments. significantly decreased the number of Wnt and PTK7 double- In lysates of MCF7 cells Myc-tagged PTK7 proteins (full-length positive and PTK7-positive vesicles in Wnt2b-containing PTK7 and ΔkPTK7) co-precipitated HA-tagged caveolin-1α aggregates and PTK7 remained at the membrane (Fig. 6C,D). (Fig. 4C) and vice versa (Fig. S4C), suggesting that caveolin-1α Thus, these data indicate that the Wnt-mediated PTK7 endocytosis interacts with PTK7 independently of its kinase homology domain. is dependent on caveolin-1α. Taken together, these data indicate that PTK7 is endocytosed by a caveolin-mediated pathway. Canonical Wnt treatment results in lysosomal degradation To assess the role of clathrin-mediated endocytosis of PTK7, of PTK7 endogenous clathrin was visualized in MCF7 cells expressing Next, we examined whether caveolin-1α-mediated PTK7 PTK7–GFP by immunostaining. Colocalization of PTK7–GFP with internalization affects PTK7 protein stability. MCF7 cells were clathrin was infrequently detected (Fig. S5). Stimulation of the cells incubated with increasing concentrations of canonical Wnt3a or with Wnt3a, only led to a minor increase in the PTK7–clathrin non-canonical Wnt5a, and endogenous PTK7 protein levels colocalization in comparison to that seen in untreated or Wnt5a- normalized to actin were determined. Endogenous PTK7 protein treated cells (Fig. S5A,B). Similar results were obtained for intensity decreased in the presence of increasing concentrations of ΔkPTK7–GFP in respect to colocalization with clathrin (Fig. S5C,D). Wnt3a but not Wnt5a (Fig. 7A,B). Similar results were obtained These experiments confirm that Wnt3a-induced PTK7 when testing the effect of different canonical (Wnt3a and Wnt8) and internalization is mediated by caveolin-1α rather than by clathrin. non-canonical (Wnt11) Wnt proteins on the PTK7 protein in In contrast to caveolin-1α, which is an integral membrane protein Xenopus ectodermal explants (Fig. S6). To analyze whether the and therefore stably associated with the plasma membrane, clathrin decrease of PTK7 protein in the presence of canonical Wnt is the localizes dynamically to the plasma membrane. Hence, after vesicle result of protein degradation, lysosomal degradation was inhibited release into the cytoplasm clathrin disassembles quickly from the using chloroquine. Chloroquine raises the lysosomal pH and vesicular membrane (Mundy et al., 2012; Parton and del Pozo, thereby inhibits lysosomal enzymes that require an acidic pH for 2013). Consequently, cytoplasmic colocalization of a cargo protein their activity (Steinman et al., 1983). Endogenous PTK7 levels in with caveolin-1α is clearly detectable. However, clathrin MCF7 cells were determined in the presence or absence of Wnt3a, colocalization might not be detected as it may have dissociated and in addition when lysosomal degradation was inhibited using from the vesicles. To further analyze whether PTK7 receptor chloroquine. While the relative PTK7 signal intensity decreased internalization is exclusively mediated by caveolin, its with increasing concentrations of Wnt3a, chloroquine treatment colocalization with PTK7 was studied by TIRF microscopy. This prevented this effect (Fig. 7C,D). Similar effects were observed allows the visualization of the plasma membrane and adjacent when using ammonium chloride to inhibit lysosomal degradation cytoplasmic regions and would therefore detect clathrin if it was still (Fig. S7), while a proteasomal inhibitor (MG 132) did not prevent attached to the vesicles. MCF7 wild-type cells stained with PTK7 degradation (data not shown). Furthermore, immunostaining antibodies detecting endogenous PTK7, caveolin-1α and clathrin, detects a significant increase in colocalization of PTK7 with the respectively, were analyzed by TIRF microscopy. PTK7-positive lysosomal marker Lamp-2 in intracellular vesicles of cells treated dots were analyzed for colocalization with either caveolin-1α or with Wnt3a, compared to that seen in Wnt5a-treated cells or controls clathrin, and quantified through determining the Pearson correlation (Fig. 7E,F). These results indicate that canonical Wnt proteins lead coefficients (PCCs) of single dots. Consistent with the previous to endocytosis and subsequently lysosomal degradation of PTK7 in findings obtained using confocal microscopy, colocalization of a dose-dependent manner. PTK7 with caveolin-1α increased significantly upon treatment with Wnt3a (Fig. 5A,C). In contrast, PTK7-clathrin colocalization Caveolin-1α contributes to the PTK7-mediated inhibition remained largely unchanged in the presence or absence of Wnt3a of canonical Wnt signaling (Fig. 5B,C). These results confirm that PTK7 internalization is Previously, we observed that PTK7 inhibits canonical Wnt mediated by caveolin-1α and seems to be independent of the signaling (Peradziryi et al., 2011); however, the molecular clathrin-mediated endocytosis pathway. mechanism is unclear. Our data suggest that PTK7 interacts with Finally, to examine the role of caveolin for PTK7 internalization, canonical Wnt proteins and removes them via caveolin-mediated MCF7 cells were treated with methyl-β-cyclodextrin (MβCD). endocytosis from the extracellular matrix, thereby likely preventing MβCD removes cholesterol from membranes leading to a disruption their interaction with canonical Wnt co-receptors. Thus, to of lipid rafts and consequently destroys caveolae (Rodal et al., determine whether caveolin function contributes to PTK7-

1999). Inhibition of caveolin-mediated endocytosis by MβCD mediated inhibition of canonical Wnt signaling we performed Journal of Cell Science

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Fig. 4. PTK7 colocalizes with caveolin-1α in the cytoplasm in presence of Wnt3a. (A) MCF7 cells stably expressing PTK7–GFP were treated with recombinant Wnt3a or Wnt5a protein for 1 h, and immunostained for caveolin-1α. PTK7 and caveolin-1α colocalize at the plasma membrane in control and Wnt5a-treated cells (arrows). Conversely, yellow cytoplasmic puncta indicating PTK7 and caveolin-1α double- positive vesicles (arrowheads) appear in the presence of Wnt3a. Scale bar: 10 µm. (B) Graph showing the mean±s.e.m. percentage of cells with cytoplasmic PTK7 and caveolin-1α colocalization in three independent experiments. Cells showing PTK7 and caveolin-1α colocalization were determined based on calculated PCCs. N, numbers of analyzed cells. *P<0.005; **P<0.001 (Student’s t-test). (C) Myc-tagged (MT) PTK7 or Myc-tagged ΔkPTK7 co-precipitate HA-tagged caveolin-1α in lysates of MCF7 cells. The upper panel shows the precipitated caveolin-1α using anti- Myc (IP α-MT) antibodies; the lower panel shows the respective cell lysates. Antibodies used for protein detection and molecular masses (kDa) are indicated on the right.

Xenopus second axis assays. Secondary axes were induced by protein activity or stability (Andre et al., 2012; Gao et al., 2011). overexpressing Wnt8 on the ventral side of Xenopus embryos. As However, vertebrates express 19 Wnt ligands and diverse Wnt expected, co-injection of PTK7 significantly inhibited second axis receptors, allowing the formation of various distinct receptor induction. However, this effect was less severe if the embryos were complexes. Therefore, analyzing the role of Wnt ligands and their co-injected with caveolin-1α MO (Fig. 8), indicating that PTK7 respective receptor complexes in the establishment of vertebrate requires caveolin-1α for successful inhibition of canonical Wnt PCP will likely still hold some surprises. signaling. Thus, these data suggest that caveolin-mediated In this respect, PTK7 is a very interesting Wnt co-receptor in that endocytosis supports the inhibition of canonical Wnt signaling by it regulates PCP signaling in vertebrates, but also interacts with PTK7. canonical Wnt ligands. Here, we find that PTK7 interacts with canonical Wnt ligands at the plasma membrane and is subsequently DISCUSSION endocytosed via a caveolin-mediated process. Interestingly, non- During development and tissue homeostasis Wnt signals control canonical Wnt ligands did not affect PTK7 localization. Consistent various cellular behaviors by activating distinct signaling pathways. with this, canonical-Wnt-dependent PTK7 internalization required The establishment of PCP is an evolutionarily conserved process the Fz7 receptor, but not the non-canonical Ror2 receptor. These in development and organogenesis, and requires the polarized data are consistent with our previous findings that PTK7 interacts localization of core PCP proteins. Wnt ligands have been shown to with canonical Wnt3a and Wnt8, but not non-canonical Wnt5a and serve as instructive signals affecting PCP protein localization and Wnt11. Furthermore, the PTK7–Wnt interaction is not direct, but is polarity axis orientation (Gao, 2012; Yang and Mlodzik, 2015). For mediated by Fz7 (Peradziryi et al., 2011); hence, Fz7 is also required vertebrate PCP, a role of non-canonical Wnt ligands, like Wnt5a and for Wnt-mediated endocytosis of PTK7. The intracellular kinase Wnt11, has been well documented (Gao et al., 2011; Heisenberg homology domain of PTK7 is not required for interaction with Wnt et al., 2000; Kilian et al., 2003; Qian et al., 2007; Tada and Smith, and Fz7, and indeed we confirm here that this domain is also not 2000). Concerning the molecular mechanism, recent data indicate necessary for Wnt-mediated endocytosis of PTK7. As the kinase that Wnt gradients provide directional information to a field of cells domain recruits Dsh and PKCδ to the plasma membrane (Shnitsar by forming Wnt-induced receptor complexes, thereby modulating and Borchers, 2008; Wehner et al., 2011), a function associated with Journal of Cell Science

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Fig. 5. PTK7 endocytosis is mediated by caveolin-1α rather than by clathrin. Endogenous colocalization of PTK7 with caveolin-1α or clathrin was determined by using TIRF microscopy. PTK7, caveolin-1α or clathrin proteins were detected through immunostaining. Penetration depth in the Z-plane was 130 nm. (A) Colocalization of PTK7 and caveolin-1α increased significantly in the presence of Wnt3a compared to that seen in untreated control cells. (B) In contrast, colocalization of PTK7 and clathrin was only detected in a low percentage of fluorescent puncta and remained low after Wnt3a treatment. (C) The graph summarizes the mean±s.e.m. percentage of PTK7 puncta colocalizing with caveolin-1α or clathrin, based on the calculated PCCs for single PTK7-positive puncta. Numbers of analyzed PTK7 puncta are indicated for each column. *P<0.002 (Student’s t-test).

the activation of PCP signaling (Kinoshita et al., 2003; Park et al., efficiently removes canonical Wnt ligands from the extracellular 2005), this suggests that the Wnt-mediated endocytosis of PTK7 is space, thereby preventing their interaction with bona fide canonical independent of its interaction with Dsh. Furthermore, we note that Wnt receptor complexes. Thus, the PTK7–Wnt interaction may PTK7 is subject to lysosomal degradation in the presence of provide a molecular mechanism of mutual regulation of PCP and canonical Wnt ligands, while it is not affected by non-canonical Wnt signaling pathways. Wnt ligands. Thus, canonical Wnt gradients may affect PTK7 Currently, we can only speculate about the endogenous role of the localization and stability, thereby likely modulating its role in PCP PTK7 receptor at the intersection of Wnt signaling pathways. PTK7 signaling. In respect to Wnt/β-catenin signaling, we find that PTK7 is an acknowledged regulator of vertebrate PCP and, as such, is inhibits this signaling pathway suggesting that PTK7 may trap involved in various morphogenetic processes (Hayes et al., 2013; canonical Wnt ligands in a non-canonical Wnt co-receptor complex, Podleschny et al., 2015; Shnitsar and Borchers, 2008; Williams thereby preventing their interaction with receptors favoring Wnt/β- et al., 2014; Xu et al., 2016; Yen et al., 2009). Concerning the catenin signaling. Consistent with this hypothesis, PTK7 inhibits functional relevance of the canonical Wnt-mediated endocytosis of canonical Wnt-induced Xenopus double axis formation (Peradziryi PTK7, this could be a mechanism by which a Wnt gradient affects et al., 2011). Interestingly, this PTK7-mediated inhibition is PTK7 localization. In this respect, one would expect that PTK7 is significantly less severe if caveolin-1α function is knocked down. not evenly distributed at the cell membrane, but shows some – Furthermore, we observed here that fluorescently labeled canonical possibly also dynamic – polarization. A system where the dynamic Wnt2b colocalized with PTK7 in intracellular vesicles, while this localization of PCP proteins has been demonstrated are migrating was only rarely observed for Wnt5a. This suggests that PTK7 neural crest cells. PCP signaling components are, for example, Journal of Cell Science

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Fig. 6. PTK7 endocytosis is prevented by inhibition of caveolin-mediated endocytosis. (A,B) Chemical inhibition using methyl-β-cyclodextrin (MβCD). (A) PTK7–GFP-expressing MCF7 cells were cultured with or without MβCD and incubated with or without Wnt3a for 1 h. PTK7 and caveolin-1α localization was determined by immunostaining. PTK7 localized to the membrane in control cells and cells treated with MβCD. In the presence of Wnt3a, PTK7 colocalized with caveolin-1α in the cytoplasm; however, this was prevented by MβCD treatment. (B) Graph summarizing the mean±s.e.m. PTK7 localization pattern of three independent experiments. N, number of analyzed cells. *P<0.0001; NS, not significant (Student’s t-test). (C,D) Loss of function of caveolin-1α. (C) Xenopus animal cap cells were injected with PTK7–Myc (PTK-MT; red) in combination with different MOs, and combined either with similar treated control cells or animal cap cells exclusively overexpressing Wnt2b–GFP or Wnt5a–GFP. PTK7 (red) and Wnt proteins (green) were detected by immunostaining. PTK7 cells aggregates injected with control MO (Co MO) show PTK7 at the cell membrane (a, left panel). In aggregates containing Wnt2b-expressing cells (a, middle panel), PTK7 is detected in intracellular vesicles where it frequently colocalizes with Wnt2b (arrowheads), while Wnt5a-expressing aggregates resemble controls (a, right panel). In contrast, co-injection of a caveolin-1α MO (Cav1α MO) abolished the Wnt2b-mediated PTK7 endocytosis, and membrane-localized PTK7 was detected in all conditions (b). (D) The graph quantifies the mean±s.e.m. total number of PTK7-positive and Wnt and PTK7 double-positive vesicles based on calculated PCCs in three independent experiments. N, number of cells. *P<0.05 (Student’s t-test). Scale bars: 10 µm. required for contact inhibition of locomotion, which is a at these cell–cell contact zones, but is removed if cell contacts are phenomenon whereby PCP components are transiently localized broken (data not shown). Thus, Wnt ligands may affect PTK7 at cell contacts and mediate the change of cell polarization and the dynamics in migrating neural crest cells. For example, Wnt2b, subsequent movement of cells in the opposite direction (Mayor and which we have shown leads to internalization of PTK7 and

Theveneau, 2014). Interestingly, we also find that PTK7 is localized colocalizes with PTK7 in intracellular vesicles, is expressed in the Journal of Cell Science

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Fig. 7. Canonical Wnt proteins target PTK7 for lysosomal degradation. (A) Wild-type MCF7 cells were incubated for 2 h with increasing concentrations of recombinant Wnt3a (upper panel) or Wnt5a (lower panel) protein as indicated at the top, and endogenous PTK7 expression was analyzed by western blotting. Molecular masses (kDa) are indicated. (B) The graph summarizes three independent experiments showing that the mean±s.e.m. PTK7 protein concentration decreases with increasing Wnt3a concentrations, while it is not affected by Wnt5a treatment. AU, arbitrary units. (C) MCF7 cells were incubated with 50 or 400 ng/ml recombinant Wnt3a in the presence or absence of chloroquine. In the absence of chloroquine, the PTK7 protein decreased with increasing concentrations of Wnt3a. In contrast, after inhibition of lysosomal degradation using chloroquine, PTK7 protein levels remained constant. Molecular masses (kDa) are indicated. (D) The graph shows the mean±s.e.m. PTK7 protein concentration for three independent experiments as in C. The relative signal intensities were determined in relation to the actin expression. (E) MCF7 cells stably expressing PTK7–GFP were treated with recombinant Wnt3a or Wnt5a protein for 1 h and immunostained for the lysosomal marker Lamp-2. PTK7 localizes in lysosomes in the presence of Wnt3a but not under control or Wnt5a-treated conditions. (F) The graph shows the mean±s.e.m. percentage of cells with PTK7 and Lamp-2 colocalization. Colocalization was determined by calculating the PCCs. N, number of analyzed cells. *P<0.0001 (Student’s t-test).

Xenopus branchial arches at the time of neural crest migration (Maj et al., 2016; Rabadán et al., 2016). Thus, neural crest cells (Rankin et al., 2012). Neural crest cells also provide an explanation require controlled basal levels of canonical Wnt signaling to enable for the relevance of the inhibition of canonical Wnt signaling by their migration. PTK7 is expressed in migrating neural crest cells PTK7. Canonical Wnt activity decreases at the onset of neural crest and also has the ability to inhibit canonical Wnt signaling; therefore, migration, and ectopic activation inhibits neural crest migration it could be a molecular tool to achieve these controlled levels. Journal of Cell Science

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Fig. 8. Caveolin-1α loss of function inhibits PTK7 activity. (A) Xenopus embryos were injected with 5 pg Wnt8 RNA to generate secondary axes. Wnt8 activity was significantly inhibited by co-injection of 250 pg PTK7 mRNA. Co-injection of PTK7 and 10 ng caveolin-1α MO reduced the inhibitory effect of PTK7 on canonical Wnt signaling. Scale bar: 1 mm. (B) The graph summarizes the mean±s.e.m. of four independent experiments, normalized to the axis-inducing activity of Wnt8. Numbers of analyzed embryos are indicated. *P<0.001, **P<0.0005 (Student’s t-test).

As PTK7 affects diverse processes ranging from embryonic 6 μg/ml blasticidin. PTK7–GFP or ΔkPTK7–GFP expression in stably morphogenesis to wound repair it likely also contributes to the transfected MCF7 cell lines was induced by the addition of doxycycline to a mutual interaction of Wnt signaling pathways in these systems. final concentration of 1 µg/ml.

MATERIALS AND METHODS Immunofluorescence Generation of stable transfected cell lines Cells were seeded on 10 mm glass coverslips (Menzel-Gläser, Thermo MCF7 (provided by the Department of Hematology, University Medical Scientific), and PTK7–GFP or ΔkPTK7–GFP expression was induced by Center Göttingen, Göttingen, Germany) cell lines were grown in RPMI the addition of doxycycline at a final concentration of 1 µg/ml for at least 1640 medium (Biochrom) supplemented with 10% fetal calf serum (FCS) 15 h. For treatment with Wnt proteins, cells were washed three times with and 5% penicillin-streptomycin (Biochrom) at 37°C and 5% CO2. For the PBS, and 200 ng/ml recombinant human (rh)Wnt3a (R&D Systems, 5036- generation of stable and inducible PTK7–GFP or ΔkPTK7–GFP MCF7 WN) or rhWnt5a (R&D Systems, 645-WN) diluted in RPMI medium was cells, the tetracycline-regulated mammalian expression system T-REx™ added to the cells. For inhibition of caveolin-mediated endocytosis, cells (Invitrogen, Life Technologies) was used. Cells were transfected with the were incubated in 5 mM MβCD (Sigma-Aldrich, C4555) for 4 h before Wnt regulatory plasmid pcDNA™6/TR and selected using blasticidin (6 μg/ml). treatment. Cells were washed with PBS, fixed in 4% paraformaldehyde in Cells that responded strongly to tetracycline were additionally transfected PBS for 20 min at room temperature, permeabilized with 0.2% Triton X-100 with the expression plasmid pcDNA™5/TO carrying the PTK7–GFP or in PBS and blocked in blocking buffer [1% bovine serum albumin (BSA) in ΔkPTK7–GFP gene. Resistant cells were selected with blasticidin and PBS] for 1 h. Cells were incubated with anti-GFP (Roche, 11814460001, hygromycin (1 μg/ml). For cloning of PTK7–GFP into the pcDNA™5/TO, 1:1000; Abcam, ab290, 1:2000), anti-caveolin-1 (Abcam, ab2910, 1:500), first the coding sequence of hPTK7 was amplified using the primers 5′-C- anti-clathrin (Abcam, ab14409, 1:500) or anti-Lamp-2 (BD Bioscience, ACGTGGCTAGCGCCCTCAGCTCCTTTTCCTGA-3′ and 5′-GACGAA- 555803, 1:500) antibodies overnight at 4°C. Cells were washed with TTCGCGGCTTGCTGTCCACGGT-3′, and introduced into pEGFPN1 at blocking buffer and subsequently incubated with Alexa Fluor-conjugated the NheI and EcoRI restriction sites. For cloning of ΔkPTK7–GFP into the secondary antibodies [Alexa Fluor 594-conjugated goat anti-rabbit-IgG pcDNA™5/TO, ΔkPTK7 was amplified using the primers 5′-CACGTGG- (Life Technologies, A-11012, 1:400), Alexa Fluor 488-conjugated goat CTAGCATGGGAGCTGCGCGGGGATCC-3′ and 5′-CGAGAATTCGG- anti-rabbit-IgG (Life Technologies, A-11008, 1:400), Alexa Fluor® 488- TGCATCTTATCACTTGTGC-3′, and introduced into the NheI and EcoRI conjugated goat anti-mouse-IgG (Life Technologies, A-11029,1:400), restriction sites of the pEGFPN1 vector. From the pEGFPN1 vectors, PTK7– Alexa Fluor 594-conjugated goat anti-mouse-IgG (Life Technologies, EGFP or ΔkPTK7–EGFP were amplified using the primers forward 5′-GT- A-11005, 1:400)] for 1 h at room temperature. Cells were mounted in CGATATCATGGGAGCTGCGCGGGGATCC-3′ and reverse 5′-ACGGC- fluorescence mounting medium (Dako, Agilent Technology) supplemented GGCCGCCTTGTACAGCTCGTCCATGC-3′. The PCR products were with DAPI (Carl Roth) to a final concentration of 1 µg/ml. Stained cells were introduced respectively into the EcoRV and NotI restriction sites of the imaged by confocal laser-scanning fluorescence microscopy (LSM 780, pcDNA™5/TO vector. For cultivation of stable transfected MCF7 cell lines, Carl-Zeiss or TCS SP5, Leica Microsystems). For quantification, the PTK7

RPMI medium was additionally supplemented with 1 μg/ml hygromycin and protein localization was classified into three different categories: membrane Journal of Cell Science

1900 RESEARCH ARTICLE Journal of Cell Science (2017) 130, 1890-1903 doi:10.1242/jcs.198580 localization, membrane and cytoplasmic localization, or cytoplasmic precipitated with Protein A–Sepharose (GE Healthcare) for 1 h at 4°C, localization. The category ‘membrane localization’ was used if PTK7 was washed five times for 5 min with NP-40 lysis buffer and boiled for 5 min at clearly localized at the plasma membrane and weakly or not at all in the 95°C in 6× Laemmli loading buffer (350 mM Tris-HCl pH 6.8, 9.3% cytoplasm. An equal distribution of PTK7 at both membrane and cytoplasm Dithiothreitol, 30% (v/v) glycerol, 10% SDS, 0.02% Bromphenol Blue) and was classified as ‘membrane and cytoplasmic localization’. ‘Cytoplasmic loaded on 10% or 12% SDS-PAGE gels. localization’ was determined as mainly cytoplasmic PTK7 localization with weak or no localization at the plasma membrane. Cell surface biotinylation For co-culture experiments cells were seeded in two- or four-well Lab-Tek MCF7 cells stably expressing PTK7–GFP or ΔkPTK7–GFP cultured in Chambers (Nunc™ Lab-Tek™ Chambered Coverglass, Thermo Scientific) six-well plates were treated with 200 ng/ml rhWnt3a or rhWnt5a for 1 h. and transfected with PTK7–RFP. Further cells were seeded on six-well plates Subsequently, cells were cross-linked with 0.25 mg/ml EZ-Link-Sulpho- and transfected with Wnt2b–GFP (Holzer et al., 2012) or Wnt5a–GFP NHS-SS-biotin for 30 min (Thermo Scientific) and subsequently quenched (Walkamm et al., 2014). Transfections were performed using Lipofectamine® with Quenching solution (Thermo Scientific). Cells were washed in TBS, 2000 (Life Technologies). Cells expressing Wnt2b–GFP or Wnt5a–GFP were and scraped and lysed in 800 µl NP-40 lysis buffer. 50 µl of the sample was subsequently added to the cells expressing PTK7–RFP (Podleschny et al., collected as input control. Cell surface proteins were affinity-purified using 2015) and incubated together for 4 h. Live-cell imaging was performed by NeutrAvidin–agarose beads (Thermo Scientific) for 2 h with end-over-end using a spinning disc confocal microscope (AxioObserver Z1, Zeiss) with a mixing. Beads were washed five times for 5 min with NP-40 lysis buffer, Plan-Apochromat 63×, NA 1.40 oil objective. Protein colocalization was boiled at 95°C for 5 min in 6× Laemmli loading buffer and loaded onto 10% quantified with ImageJ software (coloc 2 plugin). or 12% SDS-PAGE gels. Quantification of the relative signal intensities of For analysis of protein colocalization, the PCC was calculated with the cell surface to total protein levels was performed by using ImageJ. ImageJ. PCCs for single vesicles or areas to be analyzed within each cell were determined by drawing a region of interest (ROI) of equal size. PCC Western blotting – values range from 1 to 1, whereby 1 reflects a perfect correlation between MCF7 cells were cultured in six-well plates for at least 15 h. For treatment the pixels in two channels (here, red and green), 0 stands for a random with Wnt proteins, cells were washed three times with PBS before rhWnt correlation and -1 means perfect but negative correlation (Adler and proteins were added at different concentrations (50–400 ng/ml) diluted in ≥ Parmryd, 2010). PCC values of 0.5 are considered as positive. According RPMI medium. For inhibition of caveolin-mediated endocytosis, cells were to this, colocalization of single vesicles or cells was counted as positive if the incubated with 5 mM MβCD for 4 h prior to and during Wnt incubation. For ≥ calculated PCC was 0.5. inhibition of lysosomal degradation cells were incubated with 100 µM chloroquine (or 50 mM NH4Cl) for four hours before and during Wnt TIRF imaging treatment. Cells were washed three times in TBS, lysed in NP-40 lysis buffer For TIRF microscopy, PTK7–GFP-expressing MCF7 cells were seeded on and homogenized using a 30G syringe. Protein extracts of MCF7 cells were 42 mm glass coverslips (H. Saur Laborbedarf). For analysis of endogenous separated by 10% or 12% SDS-PAGE, transferred to a nitrocellulose PTK7, caveolin-1α and clathrin protein expression, wild-type MCF7 cells membrane (Whatman) by electroblotting and blocked in TBST buffer were fixed and blocked as described above. Cells were incubated with anti- [50 mM Tris-HCl pH 7.5, 150 mM NaCl and 0.5% (v/v) Tween 20] caveolin-1 (Abcam, ab2910, 1:500), anti-clathrin (Abcam, ab1440, 1:500) containing 5% nonfat dried milk. The following antibodies were used for or anti-PTK7 (CCK4) (R&D systems, AF4499, 1:200) antibodies overnight detection of proteins: anti-PTK7 (CCK4) (R&D Systems, AF4499, 1:1000), at 4°C. As secondary antibodies, Alexa Fluor 647-conjugated chicken anti- anti-HA.11 (Covance, MMS-101P, 1:1000), anti-GFP (Roche, rabbit-IgG and Alexa Fluor® 488 goat anti-mouse-IgG were used. For TIRF 11814460001, 1:2000), anti-Myc (abcam, ab19234, 1:1000), anti-actin live-cell imaging, PTK7–GFP expression was induced by the addition of (Merck Millipore, MAB1501, 1:2000) antibodies. Horseradish peroxidase- doxycycline at a final concentration of 1 µg/ml for at least 15 h. Cells were conjugated secondary antibodies used were anti-mouse-IgG (Santa Cruz imaged for 15 or 30 min and then either canonical Wnt3a or non-canonical Biotechnology, sc-2005, 1:5000), anti-goat-IgG (Santa Cruz Biotechnology, Wnt5a protein was added and cells were further imaged for 15 or 30 min. sc-2020, 1:10,000) and anti-rabbit-IgG (Cell Signaling, 7074, 1:2000). TIRF imaging was performed by using a Leica DMI6000B microscope with Proteins were detected using Pierce™ ECL Western Blotting Substrate a HCX Plan-Apochromat 100×, NA 1.47 oil objective. The penetration (Thermo Scientific) and exposed to an X-ray film or Odyssey® Fc Imaging depth was 130 nm for the experiments using fixed cells and 150 nm for System (LI-COR Bioscience). live-cell imaging. Xenopus injection and second axis assay Co-immunoprecipitation Xenopus embryo microinjections were performed as described previously For transfection, MCF7 cells were seeded in six-well plates with a surface (Borchers et al., 2001). All animal experiments were performed according to 2 – Δ – area of 10 cm per well. Transfection of PTK7 Myc, kPTK7 Myc approved guidelines. Capped sense RNA for microinjections was prepared α– (Shnitsar and Borchers, 2008) and caveolin-1 HA was carried out by using by using the mMessage mMachine kit (Ambion, Life Technologies). The ® α Lipofectamine 2000 Reagent (Life Technologies). Caveolin-1 was following plasmids and morpholino oligonucleotides (MOs) were used for Xenopus α amplified by PCR from caveolin-1 cDNA (RZPD, catalogue sense mRNA synthesis: PTK7 (Shnitsar and Borchers, 2008), PTK7–Myc ′ number IRBMp990B0725D) with the forward primer, 5 -TTGAATTCA- (Shnitsar and Borchers, 2008), Wnt2b–GFP (Holzer et al., 2012), Wnt5a– ′ ′ GCATGTCTGGTGGCAAATACATAG-3 , which contains a 5 EcoRI GFP (Wallkamm et al., 2014) and lacZ (Smith and Harland, 1991), cave- ′ restriction site and the reverse primer, 5 -TTCTCGAGCACTTCTTTGC- olin-1α MO (5′-CATCTATGTATTTGCCACCAGACAT-3′, Gene Tools, ′ ′ GTAAGGAA-3 , which contains a 3 XhoI restriction site. The PCR LLC) and standard control MO (5′-CCTCTTACCTCAGTTACAATTTA- product was cut with EcoRI and XhoI and inserted into the respective sites TA-3′, Gene Tools, LLC). of the pCS2+/HA vector. At 48 h after transfection, cells were washed in Axis duplication was induced by the injection of 5 pg Wnt8 mRNA into Tris-buffered saline (TBS; 50 mM Tris-HCl pH 7.5 and 150 mM NaCl), four-cell-stage embryos, marginally in one ventral blastomere. 250 pg of scraped and lysed in NP-40 lysis buffer [50 mM Tris-HCl pH 7.5, 150 mM PTK7 RNA, 10 ng of control MO or caveolin-1α MO were co-injected. NaCl, 0.5% (v/v) NP-40 containing Complete EDTA-free protease inhibitor Embryos were quantified for second axis generation at early tadpole stages. cocktail tablet (Roche)] and supplemented with SDS to a final concentration of 0.1%. A total volume of 800 µl lysate was pre-cleared for 1 h with Protein-A–Sepharose CL-4B (GE Healthcare) at 4°C with end-over-end Xenopus ectodermal explants assay mixing. After pre-clearing, 50 µl of the lysate were collected as input One-cell-stage Xenopus embryos were injected with 500 pg PTK7-Myc control. For the antigen–antibody reaction, supernatants were incubated mRNA alone or in combination with 10 or 20 ng standard control MO, with anti-HA.11 (Covance, MMS-101P, 1:150) or anti-Myc 9E10 (Sigma 20 ng caveolin-1α MO, 10 ng Fz7 MO (Abu-Elmagd et al., 2006) or 10 ng

Aldrich, M4439, 1:250) antibodies for 2 h at 4°C. Complexes were Ror2 MO (5′-GTCAGGCGAGGTAAGGGGCAACACT-3′). Additionally, Journal of Cell Science

1901 RESEARCH ARTICLE Journal of Cell Science (2017) 130, 1890-1903 doi:10.1242/jcs.198580 one-cell embryos were injected with 100 pg Wnt2b-GFP or 100 pg Wnt5a- Borchers, A., David, R. and Wedlich, D. (2001). Xenopus cadherin-11 restrains GFP mRNA. Ectodermal explants (animal caps) were cut at stage 8 as cranial neural crest migration and influences neural crest specification. described previously (Wallingford and Harland, 2001). Ten PTK7–Myc- Development 128, 3049-3060. Dunn, N. R. and Tolwinski, N. S. (2016). Ptk7 and Mcc, unfancied components in expressing animal caps were mixed with ten Wnt-expressing animal caps in non-canonical Wnt signaling and cancer. Cancers 8, E68. 2+ 24-well plates and afterwards dissociated in Ca -free 0.8× MBS buffer Gao, B. (2012). Wnt regulation of planar cell polarity (PCP). Curr. Top. Dev. Biol. [10 mM Hepes (pH 7.0), 88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO3, 101, 263-295. 0.82 mM MgSO4 and 0.66 mM KNO3] by gentle pipetting. For controls, 20 Gao, B., Song, H., Bishop, K., Elliot, G., Garrett, L., English, M. A., Andre, P., PTK7-expressing caps were used. At 1 h after dissociation, the Ca2+-free Robinson, J., Sood, R., Minami, Y. et al. (2011). Wnt signaling gradients buffer was removed and the cells were allowed to reaggregate in 0.8× MBS establish planar cell polarity by inducing Vangl2 phosphorylation through Ror2. Dev. Cell 20, 163-176. [10 mM Hepes (pH 7.0), 88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO3, Hayes, M., Naito, M., Daulat, A., Angers, S. and Ciruna, B. (2013). Ptk7 promotes 0.82 mM MgSO4, 0.41 mM CaCl2, 0.66 mM KNO3]. The cells were non-canonical Wnt/PCP-mediated morphogenesis and inhibits Wnt/beta-catenin- incubated until the control Xenopus embryos reached stage 14–15, fixed in dependent cell fate decisions during vertebrate development. Development 140, 1807-1818. MEMFA (4% formaldehyde, 0.1 M MOPS, 2 mM EGTA, 1 mM MgSO4) for 20 min and blocked for 1 h in PTw containing 10% FCS. PTK7–Myc Heisenberg, C.-P., Tada, M., Rauch, G.-J., Saúde, L., Concha, M. L., Geisler, R., Stemple, D. L., Smith, J. C. and Wilson, S. W. (2000). Silberblick/Wnt11 was stained using a Cy3-tagged anti-Myc antibody (Sigma-Aldrich, C6594, mediates convergent extension movements during zebrafish gastrulation. Nature 1:100), Wnt proteins were visualized by using anti-GFP (Abcam, ab290, 405, 76-81. 1:1000) primary antibodies and secondary Alexa Fluor® 488-conjugated Holzer, T., Liffers, K., Rahm, K., Trageser, B., Özbek, S. and Gradl, D. (2012). goat anti-rabbit-IgG (Life Technologies, A-11008, 1:400) antibodies. The Live imaging of active fluorophore labelled Wnt proteins. FEBS Lett. 586, reaggregated animal caps were analyzed by spinning disk confocal 1638-1644. microscopy (AxioObserver Z1, Zeiss). The total number of PTK7- Kilian, B., Mansukoski, H., Barbosa, F. C., Ulrich, F., Tada, M. and Heisenberg, C.-P. (2003). The role of Ppt/Wnt5 in regulating cell shape and movement during positive vesicles in PTK7-expressing cells was determined in cells zebrafish gastrulation. Mech. Dev. 120, 467-476. adjacent (in close proximity) to Wnt-expressing cells. Wnt and PTK7 Kinoshita, N., Iioka, H., Miyakoshi, A. and Ueno, N. (2003). PKC delta is essential colocalization of specific vesicles was determined by analyzing the PCC. for Dishevelled function in a noncanonical Wnt pathway that regulates Xenopus convergent extension movements. Genes Dev. 17, 1663-1676. Acknowledgements Kroiher, M., Miller, M. A. and Steele, R. E. (2001). Deceiving appearances: “ ” “ ” We thank Dietmar Gradl (Dept. of Cell and Developmental Biology, Zoological signaling by dead and fractured receptor protein-tyrosine kinases. BioEssays Institute, Karlsruhe Institute of Technology, Karlsruhe, Germany) for supplying 23, 69-76. Linnemannstöns, K., Ripp, C., Honemann-Capito, M., Brechtel-Curth, K., plasmids. Furthermore, we thank Ingrid Bohl-Maser and Christiane Rohrbach for Hedderich, M. and Wodarz, A. (2014). The PTK7-related transmembrane technical assistance and Melanie Bernhardt for excellent care of the Xenopus proteins off-track and off-track 2 are co-receptors for Drosophila Wnt2 required for colony. Spinning disk experiments were performed using the Core Facility Cellular male fertility. PLoS Genet. 10, e1004443. Imaging, Philipps University Marburg and TIRFM images were recorded in the Lu, X., Borchers, A. G. M., Jolicoeur, C., Rayburn, H., Baker, J. C. and Tessier- Δ Bioimaging Facility Marburg. The results on PTK7- and kPTK7-mediated caveolin Lavigne, M. (2004). PTK7/CCK-4 is a novel regulator of planar cell polarity in and clathrin endocytosis in MCF7 cells (colocalization and precipitation, TIRF vertebrates. Nature 430, 93-98. β imaging of fixed cells, biotinylation assay, M CD and chloroquine treatment) as well MacDonald, B. T. and He, X. (2012). Frizzled and LRP5/6 receptors for Wnt/beta- as the Xenopus second axis experiments were previously presented as part of a catenin signaling. Cold Spring Harbor Perspect. Biol. 4, a007880. PhD thesis by Hanna Berger, Georg-August University Göttingen, Germany, 2016. MacDonald, B. T., Tamai, K. and He, X. (2009). Wnt/beta-catenin signaling: components, mechanisms, and diseases. Dev. Cell 17, 9-26. Competing interests Maj, E., Künneke, L., Loresch, E., Grund, A., Melchert, J., Pieler, T., Aspelmeier, The authors declare no competing or financial interests. T. and Borchers, A. (2016). Controlled levels of canonical Wnt signaling are required for neural crest migration. Dev. Biol. 417, 77-90. Author contributions Martinez, S., Scerbo, P., Giordano, M., Daulat, A. M., Lhoumeau, A. C., Thome, Conceptualization: R.J., A.B.; Validation: H.B., M.B., H.P.; Formal analysis: H.B.; V., Kodjabachian, L. and Borg, J. P. (2015). The PTK7 and ROR2 protein Investigation: H.B., M.B., H.P., M.P., R.J.; Resources: R.J., A.B.; Writing - original receptors interact in the vertebrate WNT/planar cell polarity (PCP) pathway. draft: H.B., R.J., A.B.; Writing – review & editing: H.B., M.B., H.P., M.P., R.J., A.B.; J. Biol. Chem. 290, 30562-30572. Visualization: H.B., M.B., R.J., A.B.; Supervision: R.J., A.B.; Project administration: Mayor, R. and Theveneau, E. (2014). The role of the non-canonical Wnt-planar cell A.B.; Funding acquisition: R.J., A.B. polarity pathway in neural crest migration. Biochem. J. 457, 19-26. Mikels, A. J. and Nusse, R. (2006). Purified Wnt5a protein activates or inhibits beta- Funding catenin-TCF signaling depending on receptor context. PLoS Biol. 4, 570-582. The authors thank the Deutsche Forschungsgemeinschaft (DFG) for funds to A.B. Miller, M. A. and Steele, R. E. (2000). Lemon encodes an unusual receptor protein- tyrosine kinase expressed during gametogenesis in Hydra. Dev. 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