Drosophila ATP6AP2/VhaPRR functions both as a novel planar cell polarity core protein and a regulator of endosomal trafficking
2013; Springer Nature; Volume: 32; Issue: 2 Linguagem: Inglês
10.1038/emboj.2012.323
ISSN1460-2075
AutoresTobias Hermle, María Clara Guida, Samuel J. Beck, Susanne Helmstädter, Matias Simons,
Tópico(s)Genetics and Neurodevelopmental Disorders
ResumoArticle4 January 2013free access Source Data Drosophila ATP6AP2/VhaPRR functions both as a novel planar cell polarity core protein and a regulator of endosomal trafficking Tobias Hermle Corresponding Author Tobias Hermle Center for Systems Biology (ZBSA), University of Freiburg, Freiburg, Germany Renal Division, University Hospital Freiburg, Freiburg, Germany Search for more papers by this author Maria Clara Guida Maria Clara Guida Center for Systems Biology (ZBSA), University of Freiburg, Freiburg, Germany Renal Division, University Hospital Freiburg, Freiburg, Germany Graduate Program GRK1104, University of Freiburg, Freiburg, Germany Search for more papers by this author Samuel Beck Samuel Beck Center for Systems Biology (ZBSA), University of Freiburg, Freiburg, Germany Renal Division, University Hospital Freiburg, Freiburg, Germany Search for more papers by this author Susanne Helmstädter Susanne Helmstädter Center for Systems Biology (ZBSA), University of Freiburg, Freiburg, Germany Renal Division, University Hospital Freiburg, Freiburg, Germany Search for more papers by this author Matias Simons Corresponding Author Matias Simons Center for Systems Biology (ZBSA), University of Freiburg, Freiburg, Germany Renal Division, University Hospital Freiburg, Freiburg, Germany BIOSS Centre for Biological Signalling Studies, University of Freiburg, Freiburg, Germany Search for more papers by this author Tobias Hermle Corresponding Author Tobias Hermle Center for Systems Biology (ZBSA), University of Freiburg, Freiburg, Germany Renal Division, University Hospital Freiburg, Freiburg, Germany Search for more papers by this author Maria Clara Guida Maria Clara Guida Center for Systems Biology (ZBSA), University of Freiburg, Freiburg, Germany Renal Division, University Hospital Freiburg, Freiburg, Germany Graduate Program GRK1104, University of Freiburg, Freiburg, Germany Search for more papers by this author Samuel Beck Samuel Beck Center for Systems Biology (ZBSA), University of Freiburg, Freiburg, Germany Renal Division, University Hospital Freiburg, Freiburg, Germany Search for more papers by this author Susanne Helmstädter Susanne Helmstädter Center for Systems Biology (ZBSA), University of Freiburg, Freiburg, Germany Renal Division, University Hospital Freiburg, Freiburg, Germany Search for more papers by this author Matias Simons Corresponding Author Matias Simons Center for Systems Biology (ZBSA), University of Freiburg, Freiburg, Germany Renal Division, University Hospital Freiburg, Freiburg, Germany BIOSS Centre for Biological Signalling Studies, University of Freiburg, Freiburg, Germany Search for more papers by this author Author Information Tobias Hermle 1,2,‡, Maria Clara Guida1,2,3,‡, Samuel Beck1,2, Susanne Helmstädter1,2 and Matias Simons 1,2,4 1Center for Systems Biology (ZBSA), University of Freiburg, Freiburg, Germany 2Renal Division, University Hospital Freiburg, Freiburg, Germany 3Graduate Program GRK1104, University of Freiburg, Freiburg, Germany 4BIOSS Centre for Biological Signalling Studies, University of Freiburg, Freiburg, Germany ‡These two authors contributed equally to this work *Corresponding authors. Center for Systems Biology (ZBSA)/Renal Division, University Hospital Freiburg, Habsburgerstr. 49, 79104 Freiburg, Germany. Tel.:+49 761 20397206; Fax:+49 761 20397188; E-mail: [email protected] Division, University Hospital Freiburg, Habsburgerstr. 55, 79104 Freiburg, Germany. Tel.:+49 761 20397210; Fax:+49 761 20397188; E-mail: [email protected] The EMBO Journal (2013)32:245-259https://doi.org/10.1038/emboj.2012.323 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Planar cell polarity (PCP) controls the orientation of cells within tissues and the polarized outgrowth of cellular appendages. So far, six PCP core proteins including the transmembrane proteins Frizzled (Fz), Strabismus (Stbm) and Flamingo (Fmi) have been identified. These proteins form asymmetric PCP domains at apical junctions of epithelial cells. Here, we demonstrate that VhaPRR, an accessory subunit of the proton pump V-ATPase, directly interacts with the protocadherin Fmi through its extracellular domain. It also shows a striking co-localization with PCP proteins during all pupal wing stages in Drosophila. This localization depends on intact PCP domains. Reversely, VhaPRR is required for stable PCP domains, identifying it as a novel PCP core protein. VhaPRR performs an additional role in vesicular acidification as well as endolysosomal sorting and degradation. Membrane proteins, such as E-Cadherin and the Notch receptor, accumulate at the surface and in intracellular vesicles of cells mutant for VhaPRR. This trafficking defect is shared by other V-ATPase subunits. By contrast, the V-ATPase does not seem to have a direct role in PCP regulation. Together, our results suggest two roles for VhaPRR, one for PCP and another in endosomal trafficking. This dual function establishes VhaPRR as a key factor in epithelial morphogenesis. Introduction The planar cell polarity (PCP) pathway is a highly conserved pathway that polarizes cells in the plane of a tissue and equips cells with a defined orientation. In Drosophila, PCP is evident in the organization of cuticular structures, such as wing hair or body bristles, and in the orientation of photoreceptor clusters of the eye. In vertebrates, the coordinated beating of cilia and the directional cell migration during gastrulation or epidermal wound healing are examples of important processes that rely on PCP (Simons and Mlodzik, 2008; Bayly and Axelrod, 2011; Goodrich and Strutt, 2011). As a consequence, defective PCP signalling contributes to many diseases including tissue fusion disorders (e.g., neural tube defects) and ciliopathies (e.g., polycystic kidney disease; Simons and Walz, 2006). On the molecular level, there are two conserved PCP protein cassettes: the Fat/Dachsous group and the classical PCP core group consisting of Frizzled (Fz), Dishevelled (Dsh), Flamingo/Starry night (Fmi/Stan), Strabismus/Van Gogh (Stbm/Vang), Prickle and Diego. Fz and Dsh also function in the Wingless (Wg) or canonical Wnt pathway (Veeman et al, 2003). An excellent system to study epithelial PCP is the pupal wing epidermis of Drosophila. Genetic approaches in pupal wings have uncovered several key features of PCP, including the asymmetric localization of the PCP complex. It was shown that Fz and Dsh localize to the distal plasma membrane, whereas Stbm and Pk localize to the proximal membrane (Axelrod, 2001; Strutt, 2001; Bastock et al, 2003). The protocadherin Fmi shows homophilic-binding behaviour and localizes to both membranes (Usui et al, 1999). It has also become clear that these PCP domains are already formed in larval stages and are temporarily lost during a phase of junctional remodelling in pupal wing morphogenesis (Aigouy et al, 2010). Recent evidence suggests that the formation of the complex requires polarized transport of PCP proteins to the proximal–distal (P–D) boundaries along microtubules (Shimada et al, 2006). The maintenance of the complex involves rapid turnover of non-complexed components by endocytosis and recycling (Shimada et al, 2006; Strutt et al, 2011). These dynamic membrane trafficking events seem to involve the recruitment of components of the endocytic machinery by Fmi (Classen et al, 2005; Mottola et al, 2010). However, the underlying mechanisms remain poorly understood. We and others recently demonstrated that an accessory subunit of the vacuolar (V)-ATPase, VhaPRR (also called PRR or ATP6AP2 in mammals), plays a role in both canonical Wnt and PCP signalling (Buechling et al, 2010; Cruciat et al, 2010; Hermle et al, 2010). The V-ATPase is a large protein complex consisting of a peripheral V1 domain with eight subunits for ATP hydrolysis, and a V0 domain with six subunits for proton translocation (Forgac, 2007). Many of these subunits are expressed in different isoforms and splice variants, further increasing the complexity of the V-ATPase. The two accessory subunits (ATP6AP1/Ac45 and ATP6AP2/PRR) are not found in unicellular organisms, suggesting that they are not essential for proton pump activity. The V-ATPase acidifies intracellular organelles and the extracellular space depending on its subcellular localization. One prominent function of the V-ATPase is to ensure a low intraluminal pH required for protein degradation by lysosomal hydrolases. Additional functions of the V-ATPase in signalling, membrane trafficking and the regulation of post-translational modifications of proteins have also been proposed (Forgac, 2007; Sihn et al, 2010). During an effort to purify the V-ATPase complex from adrenal chromaffin cells, VhaPRR was discovered as an 8.9-kDa fragment containing the transmembrane domain and the cytosolic tail (Ludwig et al, 1998). Later, the full-length protein (37 kDa) was cloned as a receptor for (pro)renin (PRR; Nguyen et al, 2002). The Drosophila name for ATP6AP2/PRR, VhaPRR, reflects the proposed dual function of the mammalian protein. Previous studies demonstrated that ATP6AP2/PRR undergoes proteolytic cleavage generating the 8.9-kDa-transmembrane stump and a 28-kDa-sized N-terminal soluble fragment (sPRR) that can be detected in urine and plasma samples (Cousin et al, 2009). The significance of this cleavage event remains unknown. Moreover, tissue-specific conditional knockout approaches have revealed important functions of ATP6AP2/PRR in the survival of murine cardiomyocytes and podocytes (Kinouchi et al, 2010; Oshima et al, 2011; Riediger et al, 2011). The only described human ATP6AP2/PRR mutation so far is a hypomorphic mutation that results in mental retardation and epilepsy in the affected individuals (Ramser et al, 2005). In the context of Wnt signalling, ATP6AP2/PRR was shown to function as an adaptor between the V-ATPase and Fz in acidic endosomal compartments. The Fz-V-ATPase clusters also contained activated forms of the Wnt co-receptor LRP6, thus, promoting Wnt signalling (Cruciat et al, 2010). Similarly, knockdown of VhaPRR in the Drosophila wing led to defects in both Wg and PCP signalling. VhaPRR was shown to interact genetically and physically with Fz, and the absence of VhaPRR caused mislocalization of Fz in pupal wing cells (Buechling et al, 2010; Hermle et al, 2010). However, lack of VhaPRR also generated other phenotypes, including vein alterations and bristle duplications, suggestive of additional unknown functions. Here, we use genetics and biochemistry in a well-characterized PCP system to demonstrate that VhaPRR fulfills all criteria of a PCP core protein. In addition, we show that VhaPRR acts as a regulator of endosomal sorting and protein degradation. Our findings suggest that VhaPRR is an important regulator of epithelial morphogenesis. Results VhaPRR is required for PCP core protein localization To characterize the role of VhaPRR in PCP in more detail, we generated a mutant allele by imprecise P-element excision. The excision deleted 860 bp of the VhaPRR locus including the first two of the three exons (Supplementary Figure S1A), containing the epitope sequence of our antibody (see below). The specificity of the allele (VhaPRRΔ1) was determined by introducing one copy of a genomic construct harbouring the entire 2.6 kb-genomic locus. The rescue construct restored viability producing adult flies without obvious phenotypes. Consistent with previous RNAi experiments (Buechling et al, 2010; Hermle et al, 2010), clonal elimination of VhaPRR in the pupal wing led to a significant delay in prehair formation and, occasionally, multiple wing hairs emerging from one cell (Figure 1A and B). In the adult wing and notum, VhaPRR clones showed hair and bristle polarity defects, respectively (Supplementary Figure S1D and E). Generally, PCP phenotypes were stronger in pupal than in adult stages. In the adult leg, lack of VhaPRR caused a mispatterning of tarsal segments similar to phenotypes previously observed for PCP mutants (Supplementary Figure S1F and G; Gubb et al, 1999; Lee and Adler, 2002). In addition to the PCP phenotypes, the clones also displayed signs of impaired Notch signalling including ectopic notum bristles and vein thickening and loss (Supplementary Figure S1B–E; Dietzl et al, 2007). Figure 1.Loss of VhaPRR causes PCP defects and PCP core protein mislocalization. All clones are marked by loss of β-galactosidase (β-gal; only shown in (F′, G′) in red). Clone outlines are marked with a dotted white line. In all figures, mutant homozygous areas are marked with ‘−/−’, and heterozygous areas with ‘−/+’. Cells in the latter areas are the control cells. Scale bars represent 10 μm in all figures, unless otherwise stated. The proximal side of the wing is to the left, the distal to the right. Anterior is up, posterior is down. (A, B) Prehairs inside and outside, the VhaPRR mutant clone are stained with Phalloidin (red). In addition to the wing hair mispolarization, more than one hair per cell is occasionally observed (arrowheads). Hairs are shorter, thus showing a significant delay in prehair formation. Phenotypes are usually weaker at later stages ((B) is slightly later as can be seen by the longer hairs; for adult wings see Supplementary Figure S1). (C, D) Fz (C) and Stbm (D; both green) are mislocalized inside VhaPRR mutant clones. Arrowheads mark normal asymmetric localization in control tissue. (E) Fmi (green) is less asymmetric and also shows a diffusely enhanced cytoplasmic staining. (F, F′, F′′) Fz and Stbm show partial overlap in residual PCP domains and intracellular vesicles. (G–I) Fmi antibody uptake assay in live prepupal wings. At this developmental stage, PCP domains point towards the wing margin and are less organized than at 28 h APF. (G) Fmi localization inside VhaPRR clones is comparable to wild-type tissue, when the chase after Fmi antibody binding is performed at 4°C, which attenuates endocytosis. Note that effects on Fmi localization caused by removal of VhaPRR become apparent at later stages (see above). (H, H′) A 45-min chase at 29°C causes a reduction of Fmi at apical junctions inside VhaPRR mutant clones, reflecting increased internalization of Fmi in the absence of VhaPRR compared with the neighbouring wild-type tissue. (I) Quantification of antibody uptake experiment. Average staining intensity of mutant and the surrounding wild-type tissue was measured as a ratio in shown in the y axis. When endocytic activity is abrogated at 4°C, the ratio is slightly below 1.0. Error bars represent standard error of the mean, and statistical significance was determined using unpaired Student's t-test (**P<0.01). Download figure Download PowerPoint Immunostaining of PCP core proteins revealed the following changes inside the mutant clone: both Fz and Stbm were reduced at apical junctions. In addition, both proteins showed a partial overlap in vesicular compartments (Figure 1C, D and F). Fmi also lost its ability to maintain an asymmetric localization and showed a slight cytoplasmic increase inside the clone (Figure 1E). All effects were slightly stronger than those achieved with RNAi knockdown (Hermle et al, 2010), suggesting a more complete elimination of expression with the mutant (also see below in section on acidification). Because these findings indicated that lack of VhaPRR reduces the integrity of the PCP complex, we next attempted to test the stability of the PCP domains using a Fmi antibody internalization assay (Strutt et al, 2011). This experiment is performed in the prepupal wing (around 5 h APF), because at this developmental stage there is no cuticle yet, which allows the application of an antibody. PCP domains do exist at this stage, but they are less coherent and point towards the wing margin (Figure 1G and H; Classen et al, 2005; Aigouy et al, 2010). For the uptake assay, live prepupal wings were dissected, and incubated at 4°C with an antibody against Fmi, followed by chasing at 29°C for 45 min before fixation (Strutt et al, 2011). We detected a stronger reduction of Fmi at the apical junctions of VhaPRR mutant clones compared with the neighbouring wild-type cells, reflecting an increased Fmi internalization inside the clones (Figure 1H). The difference between the mutant and wild-type tissue was much less pronounced when the chase was carried out at 4°C, a temperature that strongly attenuates endocytosis (Figure 1G and I). Collectively, these results suggest that VhaPRR functions in PCP by stabilizing asymmetric PCP domains. VhaPRR localizes to PCP domains To study the localization of VhaPRR in the pupal wing cells, we raised an antibody against VhaPRR. The antibody was directed against the extracellular domain potentially recognizing both uncleaved VhaPRR and sPRR. Antibody specificity was confirmed by western blot analysis of VhaPRR RNAi-treated S2 cells as well as by staining of mutant clones (Figures 2D and 4D). Figure 2.VhaPRR localizes to PCP domains. (A–E) Immunostainings of pupal wings from different developmental stages are shown. (A, A′, A′′) VhaPRR (green) co-localizes with Fmi (red) in prepupal wings (5.5 h APF). At this stage, PCP domains point towards the wing margin. (B) At 18 h APF, VhaPRR localization is more irregular. Junctional staining is still visible, but also intracellular staining, reflecting the dynamic cell rearrangements during this phase (Aigouy et al, 2010). Co-localization with Fz (B′; red) is shown in (B″). (C) VhaPRR shows maximal asymmetric localization beyond 28 h APF. Co-localization with Fz (C′) is shown in (C″). The x–z projection in the inset of (C″) shows co-localization of VhaPRR and Fz at apical junctions. (D) VhaPRR staining without permeabilization shows that the junctional pool is at the surface. The fine vesicular staining usually observed by staining with detergent is strongly decreased with this technique. Interestingly, at sites where the pupal wing tissue has been injured with the forceps during dissection (arrowhead), an additional intracellular pool becomes apparent. In the wound egde cells another intracellular antigen, Engrailed, can also be detected (D′). Engrailed is not detected in cells away from the wound, demonstrating the efficiency of both the surface staining and the wounding. (E, E′) VhaPRR is lost from VhaPRRΔ1 mutant clones at 28 h. Clones are marked by loss of β-gal (in blue). Note that also the cytoplasmic staining of VhaPRR is slightly reduced inside the clone. Arrowheads in (D′) show that VhaPRR can localize to proximal (yellow) and distal (white) clone boundaries (scale bar 5 μm). Download figure Download PowerPoint In immunostainings of pupal wings, we found that VhaPRR co-localized with the PCP domains at all stages of pupal development: in prepupal stages, VhaPRR was polarized towards the wing margin (Figure 2A); during the junctional remodelling phase (Aigouy et al, 2010), the protein partly relocalized to intracellular compartments (Figure 2B); and before prehair formation, the enrichment at P–D membranes reached its maximum (Figure 2C): VhaPRR concentrated with the other PCP core proteins at P–D boundaries and spared anterior–posterior (A–P) boundaries. By performing the staining without permeabilization, we confirmed that this pattern reflects the cell surface pool of VhaPRR (Figure 2D). However, in this experiment we also noted that at sites where the pupal wing epithelium had been injured with the forceps, the antibody was able to bind to an additional intracellular pool of VhaPRR (Figure 3D′). At VhaPRR clone boundaries, VhaPRR appears to localize to both proximal and distal sides of wild-type cells facing the clone (Figure 2D). Figure 3.PCP core proteins control VhaPRR stability. (A, A′) Flp-out clones expressing Fmi RNAi show loss of VhaPRR, also at the clone boundaries (white arrow in (A)). Clone area is marked by GFP and labelled with ‘RNAi’. Neighbouring wild-type tissue is marked with ‘wt’. (B, C) fz mutant clones (B′; marked by loss of β-gal) and Stbm RNAi flp-out (C′; marked by GFP) show reduction of VhaPRR. Note that VhaPRR is still present at clone boundaries (arrowheads in (B, C)). (D) Flp-out clones overexpressing Fmi (marked by GFP and ‘GOF’ for gain-of-function) show strong stabilization of endogenous VhaPRR at the plasma membrane. Fmi, VhaPRR (not shown) and the endosomal marker Hrs co-localize in subapical vesicles (inset in (D′) is a more basal confocal plane compared with (D, D′)). (E, F) By contrast, VhaPRR is strongly reduced upon Fz overexpression (E, E′; in GFP-marked flp-out clones) and less asymmetric upon Stbm overexpression (F, F′). Note that VhaPRR responds to the non-autonomous effects of Fz overexpression, displaying enrichment at clone boundaries (arrowhead in (E)) and reoriented PCP domains surrounding the clone. (G, G′, G′′) Overexpression of myc–Fz with ptc-GAL4 reduces Fmi and VhaPRR at junctions. Both proteins redistribute to small intracellular vesicles. At the posterior ptc expression domain boundary, Fmi and VhaPRR are enriched (arrowhead in (G″)). (H) The co-overexpression of Fmi (in red; H′) and myc–Fz (in blue; H″) causes stabilization of VhaPRR (in green; H). All three proteins co-localize at broadened apical junctions (inset in H″). Download figure Download PowerPoint VhaPRR localization and stability is controlled by Fmi Next, we asked whether the association of VhaPRR with the polarized PCP domains depends on PCP proteins. For this, we induced fz mutant clones and expressed Fmi and Stbm RNAi in flp-out clones, respectively. In all cases, VhaPRR levels were strongly reduced inside the clones (Figure 3A–C). At clone boundaries, VhaPRR recapitulated the typical localization of PCP transmembrane core proteins (Strutt, 2001; Strutt and Strutt, 2008): whereas in fz and Stbm RNAi clones VhaPRR was still present at clone boundaries, it was completely lost at boundaries of Fmi RNAi clones (Figure 3A–C). By contrast, we detected a striking stabilization of VhaPRR in Fmi overexpression clones or other GAL4-controlled expression domains (Figure 3D). VhaPRR accumulated together with Fmi at apical junctions and Hrs-positive subapical vesicles, suggesting that the complex shuttles between the plasma membrane and endosomes (Figure 3D′). In situ hybridization showed no increase of VhaPRR expression, indicating that VhaPRR gain was not caused by increased gene expression (Supplementary Figure S2). The opposite result—a severe destabilization of VhaPRR—was found inside Fz and Stbm overexpression clones (Figure 3E and F). In addition, VhaPRR was enriched at the boundaries of Fz overexpression clones and at non-autonomously reoriented PCP domains surrounding the clones (Figure 3E and E′). The overexpression of Fz with patched(ptc)-GAL4 displaced Fmi from apical junctions into the cytoplasm (Figure 3G), suggesting that VhaPRR loss inside the Fz overexpression clone could be secondary to the reduction of junctional Fmi and/or the masking of VhaPRR-binding sites on Fmi. This is supported by the co-expression of Fz and Fmi (Figure 3H). Here, VhaPRR was stabilized to a similar degree as by the single expression of Fmi, suggesting that Fmi effects are dominant over Fz effects (Figure 3H). Together, these results propose that VhaPRR requires intact PCP domains for its junctional localization. Moreover, recruitment of VhaPRR to the domains appears to depend on Fmi. The extracellular part of VhaPRR is secreted and binds to Fmi We further discovered that the simultaneous overexpression of Fmi and VhaPRR RNAi in the ptc or engrailed domain of the pupal wing displayed an accumulation of VhaPRR. This means that, despite the knockdown, VhaPRR was increased within the Fmi expression area (Figure 4A). Furthermore, beyond 33–34 h APF VhaPRR partially reappeared at the apical junctions within the clones (Figure 4B). The most plausible explanation for these observations is that the reported extracellular cleavage product, sPRR, can be secreted and travel extracellularly. Figure 4.The extracellular part of VhaPRR, sPRR, is cleaved and secreted. (A, A′, A′′) Co-overexpression of Fmi and VhaPRR RNAi (with en-GAL4) causes stabilization of VhaPRR (green) and co-localization with Fmi (red) in spite of RNAi expression in the posterior compartment. The reduced posterior compartment is a result of VhaPRR silencing. (B) VhaPRR reappears within the clones and at clone boundaries beyond 34 h APF. Note that also anterior–posterior (A–P) clone boundaries (arrowhead in (B′)) contain VhaPRR, suggesting that new PCP domains are formed here due to different levels of Fz signalling (scale bar 5 μm). (C) Schematic diagram of the domain structure of VhaPRR, demonstrating the extracellular domain (sPRR) and the transmembrane stump M8.9. The cleavage site (RxxR) is highlighted with a red triangle. (D) S2 cells were transfected with pAc-VhaPRR-mCherry or empty pAc vector, and after 48 h cells were subjected to serum starvation for 24 h for conditioned medium collection. Immunoblots of cell lysates and of the conditioned medium are shown. The extracellular part of VhaPRR (sPRR) in the conditioned medium is enhanced upon VhaPRR-mCherry overexpression. The specificity for full-length VhaPRR (upper panel) and sPRR (lower panel) bands was demonstrated by the knockdown of VhaPRR (right). VhaPRR-mCherry generates two bands with higher molecular weight than endogenous VhaPRR. Actin was used as a loading control of the lysates. (E) Western blot showing the time course of sPRR secretion by S2 cells. Conditioned medium was collected after 0, 4, 8 and 24 h. Cell lysates (two lower panels) show corresponding VhaPRR levels and actin as a loading control.Source data for this figure is available on the online supplementary information page. Source Data for Figure 4 [embj2012323-SourceData-Fig4.pdf] Download figure Download PowerPoint To test whether Fmi could function as a receptor for sPRR, we generated medium conditioned with sPRR. This was achieved either by overexpressing HA-tagged sPRR or by overexpressing full-length VhaPRR in S2 cells (Figure 4C–E). When sPRR–HA conditioned medium was applied onto S2 cells transfected with a Fmi construct, sPRR–HA predominantly bound to cells positive for Fmi and not to non-transfected cells or to cells overexpressing a control transmembrane protein (Figure 5A and B). We also explored whether ectopic sPRR had any effects on PCP signalling in vivo by overexpressing sPRR–HA with ptc-GAL4. sPRR–HA co-localized with Fmi and Hrs in large subapical vesicles outside of the expression domain (Supplementary Figure S3B), suggesting that the complex of Fmi and sPRR–HA is endocytosed. Figure 5.VhaPRR and Fmi physically interact. (A, B) Conditioned medium containing sPRR–HA was applied to S2 cells transfected with Fmi (A) or a control protein Nhe2–YFP (B). (A–A″) sPRR–HA (red), detected by immunostaining with an anti-HA antibody, predominantly bound to Fmi-positive cells (green) and not to untransfected cells (asterisks) or Nhe2 expressing cells (B′, B′′). The non-transfected cells in (A) show a weak signal with anti-Fmi due to endogenous Fmi. (C–C″) Overexpression of Fmi–YFP with ptc-GAL4 causes stabilization of endogenous VhaPRR. (D, E) This effect is mimicked by using a Fmi construct that lacks the intracellular part (ΔC-Fmi; D–D″) and by Fmi that lacks most of the extracellular part (ΔN-Fmi; E–E″). The small so-called HRM-domain and the extracellular transmembrane loops are the only overlapping parts of Fmi between these constructs.(F) V5-tagged PRR (PRR–V5; from X. laevis) was co-expressed with Celsr-1- or Celsr-2–EGFP (from mouse) and a control transmembrane protein (PKD2–V5) in HEK293T cells. After immunoprecipitation with anti-GFP, PRR–V5 bound to immunoprecipitated Celsr-1 and Celsr-2, but not to the control protein mEGFP. The asterisk marks the heavy chain bands of the GFP antibody. (G) In a similar fashion, Celsr-1 and Celsr-2 were present in immunoprecipitates formed by PRR–V5, but not by the control transmembrane protein PKD2–V5. (H) A schematic diagram shows the interaction (arrows) between the extracellular domain of VhaPRR or sPRR with the HRM and/or the extracellular loops of the heptahelical protocadherin Fmi (or Celsr-1/2). Note that the sizes of the boxes and shapes do not represent the actual sizes of the respective domains.Source data for this figure is available on the online supplementary information page. Source Data for Figure 5 [embj2012323-SourceData-Fig5.pdf] Download figure Download PowerPoint Nevertheless, ectopic sPRR was never found at the PCP domains and also did not cause any adult PCP phenotypes (Supplementary Figure S3B and not shown). Similarly, the overexpression of full-length VhaPRR was unable to localize to any cell surface region including the PCP domains. Instead, it was diffusely distributed in the cytoplasm (Supplementary Figure S3C). Together, the results indicate that both endogenous and exogenous sPRR can be secreted in a paracrine manner in the pupal wing epithelium. However, only endogenous sPRR seems to be able to interact with PCP domains. The cleavage site is not required for PCP signalling and survival To study the significance of VhaPRR cleavage, we introduced two arginine-to-alanine conversions into the previously characterized consensus motif (RxxR) for cleavage by the pr
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