Abstract
The epithelial Zonula adherens (ZA) is a main adhesion compartment that enables organogenesis by allowing epithelial cells to assemble into sheets. How ZA assembly is regulated during epithelial cell morphogenesis is not fully understood. We show that during ZA morphogenesis, the function of the small GTPase Rap1 and the F-actin binding protein AF6/Cno are both linked to that of the P21-activated kinase Pak4/Mbt. We find that Rap1 and Mbt regulate each other’s localization at the ZA and cooperate in promoting ECadherin stabilization. During this process Cno regulates the recruitment of Baz at the ZA, a process that is also regulated by Arm phosphorylation by Mbt. Altogether, we propose that Rap1, Cno and Mbt regulate ZA morphogenesis by coordinating ECadherin stabilization and Baz recruitment and retention. In addition, our work uncovers a new link between two main oncogenes, Rap1 and Pak4/Mbt, in a model developing epithelial cell.
Introduction
The epithelial ZA consists of a lateral circumferential belt of Adherens Junction (AJ) material that allows for epithelial cells to assemble into sheets. Loss of epithelial adhesion is a hallmark of cancer and there is therefore a strong interest in better understanding how ZA morphogenesis, remodeling and maintenance are regulated. The adhesion molecule ECadherin/Shotgun (ECad) and its effector βcatenin/Armadillo (Arm) are main AJ components of the ZA in animal epithelial cells. Work in Drosophila and vertebrate cells points to multiple pathways that regulate AJ material morphogenesis during epithelial morphogenesis, including membrane delivery, endocytosis, local accumulation and stabilization at the plasma membrane (Bryant and Stow, 2004; Tepass, 2012). However, we still lack an integrated view of how these pathways and the corresponding molecular players come together to regulate ZA morphogenesis and remodeling during development.
The fly retina has long been used as a model system to study the genetic and molecular basis of ZA morphogenesis and remodeling during organogenesis. During pupal development, photoreceptors build a new ZA to accommodate the nascent apical light-gathering structure, called the rhabdomere (Ready, 2002). During this process the Par complex, which consists of Cdc42-Par6-aPKC and Par3/Bazooka (Baz), regulates the specification of the photoreceptor cortex and plasma membrane into a sub-apical domain (stalk membrane) and ZA (Hong et al., 2003; Nam and Choi, 2003; Walther et al., 2016; Walther and Pichaud, 2010). During ZA morphogenesis, phosphorylation of Baz by aPKC at the conserved S980 leads to the apical exclusion of P-S980-Baz, a step of molecular sorting that also depends on Crumbs (Crb) capturing Par6-aPKC and Stardust (Sdt) at the stalk membrane (Krahn et al., 2010; Morais-de-Sa et al., 2010; Walther and Pichaud, 2010). Confined to the apical-lateral border of the cell, P-S980-Baz is thought to promote ZA assembly by binding to Arm and Echinoid (Ed), two main AJ components (Wei et al., 2005).
Next to Baz, the Cdc42 effector type-2 p21-activated kinase Mushroom bodies tiny (Mbt/Pak4) has also been shown to regulate ZA morphogenesis in several epithelial cell types by promoting Baz retention at the ZA and regulating the accumulation of the ECad-Arm complex via phosphorylating βCat/Arm (Jin et al., 2015; Law and Sargent, 2014; Menzel et al., 2008 Schneeberger, 2003 #1892; Walther et al., 2016). In vertebrate cells, Pak4 has also been linked to the Par complex via Par6b phosphorylation, indicating a potential cross talk between Par6b-aPKCγ/ζ and Pak4, downstream of Cdc42 (Jin et al., 2015; Wallace et al., 2010). While in fly photoreceptors both Mbt and Baz are main regulators of AJ accumulation at the developing ZA, they seem to operate as part of parallel convergent pathways. This is demonstrated by the fact that while Arm accumulation is reduced in mbt null mutant photoreceptors, AJ material is no longer present at the plasma membrane in cells mutant for both mbt and baz (Walther et al., 2016). How exactly Baz contributes to regulating AJ material accumulation at the ZA is not fully understood. Similarly, phosphorylation of Cat/Arm by Pak4/Mbt (Law and Sargent, 2014; Menzel et al., 2008) cannot fully account for mbt function during ZA morphogenesis, as re-introducing a phosphomimetic form of Arm does not rescue the loss of mbt function (Walther et al., 2016). Therefore other factors must regulate AJ morphogenesis during ZA maturation, either in concert with Mbt and Baz, or as part of the Mbt and Baz pathways.
An interesting candidate in contributing to AJ accumulation at the epithelial ZA is Rap1. This member of the Ras subfamily of small GTPases has been shown to localize at the AJ in various fly epithelia, and to be an essential AJ regulator (Boettner et al., 2003; Boettner and Van Aelst, 2007; Choi et al., 2013; Knox and Brown, 2002; O’Keefe et al., 2009; Spahn et al., 2012; Wang et al., 2013). In the early embryo, Rap1 and its effector F-actin binding protein AF6/Cno (Boettner et al., 2003; Mandai et al., 2013; Sawyer et al., 2009), regulate the apical localization of both Baz and Arm, with Baz reciprocally influencing Cno localization. Later in embryonic development, Baz is required at the ZA to capture preassembled AJ material, thus promoting ZA morphogenesis (McGill et al., 2009). In addition, work in human MCF7 cells has shown a role for Rap1 during AJ maturation via promoting ECad recruitment at the sites of cell-cell contact, a function that has been shown to be mediated, at least in part, by Cdc42 (Hogan et al., 2004). Here we sought to examine the relationship between Rap1, its GEF Dizzy/PDZ-GEF and the protein network that drives epithelial apical cortex and plasma membrane specification using the pupal photoreceptor as a model system.
Results
Dizzy and Rap1 regulate pupal photoreceptor ZA morphogenesis
In the fly retina, Rap1 has been previously shown to regulate AJ remodeling between newly specified photoreceptors, and between retinal accessory cells (cone and pigment cells) (O’Keefe et al., 2009). To test whether dizzy and Rap1 are required during ZA morphogenesis in the pupal photoreceptor, we made use of the rap1-Rap1::GFP and dizzy-Dizzy::GFP transgenes, which allow for expression of these proteins under their endogenous promoter. We found that Rap1::GFP accumulates predominantly at the developing pupal photoreceptor ZA (Figure 1A), and can also be detected at lower levels at the apical photoreceptor membrane, which includes the stalk membrane. Dizzy::GFP (Figure 1B’) shows a similar pattern, with perhaps less accumulation at the apical membrane when compared to Rap1::GFP. Accumulation at the ZA and low levels of expression at the apical membrane is also observed for Arm (Walther and Pichaud, 2010); and (Figure 1A”, 1B). Therefore in the developing pupal photoreceptor, the expression pattern of Dizzy, Rap1 and Arm are very similar.
Rap1 is required to preserve the integrity of the retina (Supplementary Figure 1). Generating mutant clones using the strong allele Rap1CD3, or expressing high levels of a previously validated Rap1IR construct (O’Keefe, 2009 #1401), leads to severe defects in recruiting the full complement of retinal accessory cells including the cone cells (Supplementary Figure 1A). Missing cone and pigment cells lead to retinal cell delamination, with many photoreceptors found below the floor of the retina (Supplementary Figure 1B-D), preventing us from assessing polarity and ZA morphogenesis. In order to bypass this strong phenotype we limited the expression of Rap1IR to the pupal phase. Decreasing the expression of Rap1 at pupal stages did not affect photoreceptor apical-basal polarity in the majority of ommatidia examined (Figure 1C-E). However, we could measure shorter ZA (as measured along the apical-basal axis of the cell), and thus an overall decrease in the quantity of Arm at the ZA of the Rap1 deficient photoreceptors (Figure 1C’, D’, E’ and 1F). A survey of ZA-associated proteins revealed that in addition to Arm, the length of the Mbt (Figure 1D” and 1F’) and Baz (Figure 1E” and 1F”) domains were also significantly reduced in Rap1IR photoreceptors. Furthermore, Cno could no longer be detected at the ZA (Figure 1C”) and Mbt levels were significantly decreased when compared to wild type (Figure 1D” and 1G’). In some cases, ZA domains were present that did not contain Mbt, resulting in a significant alteration of the ratio between Mbt and Arm. In wild type, ZA levels of Mbt/Arm are correlated and follow a normal distribution. In Rap1IR ZA this correlation was disrupted, with significantly more junctions presenting either high or low Mbt/Arm ratios that fall outside of a normal Gaussian distribution (Figure 1H). In these shortened ZA, levels of Arm and Baz were comparable to wild type (Figure 1G, 1G”) and the ratios of Baz/Arm in Rap1IR cells remained similar to wild type (Figure 1H’). Apical levels of F-actin (Figure 1C”’), aPKC (Figure 1D”’), and Crb (Figure 1E”’), were not affected in Rap1IR photoreceptors when compared to wild type. These data indicate that Rap1 is required for the accumulation of AJ material at the developing ZA. This function appears most critical when considering the ZA levels of Cno and Mbt,
Next, to examine the function of dizzy during ZA morphogenesis, we made use of the strong dizzyΔ12 allele. We found that reducing dizzy expression leads to a phenotype similar to that seen in Rap1IR photoreceptors, including a shortening of the ZA along the apical-basal axis (Figure 1I-J). Consistent with Dizzy acting as a Rap1-GEF in photoreceptors, removing a copy of the dizzy locus enhances the mild rough-eye phenotype obtained when reducing the expression of Rap1 using RNAi (Supplementary Figure 2A-D).
Rap1 promotes AJ stabilization during ZA remodeling
We have previously shown that in pupal photoreceptors, loss of mbt function leads to an increase in the mobile fraction of ECad at the ZA when compared to wild type over 250 secondes (Walther et al., 2016). Our analysis of Rap1IR indicates that Mbt accumulation is reduced in the corresponding ZA (Figure 1D” and 1G’), which should therefore be accompanied by an increased in ECad mobility. To assess whether this is the case, we made use of FRAP and compared the recovery after photo-bleaching of a ubi-ECad::GFP transgene in wild type and Rap1IR photoreceptors. In wild type cells, over approximately 250 sec, we estimated that 25% of ECad::GFP is mobile, which is consistent with previous estimations from our lab (Walther et al., 2016) (not shown).
However, while ECad::GFP shows a stronger recovery over this relatively short time scale in Rap1IR when compared to wild type, the GFP signal failed to plateau (not shown), preventing us from extrapolating the mobile fraction. We therefore performed FRAP over a longer time scale (1000 sec). Over this long time scale, we found approximately 35% of ECad::GFP is mobile in wild type ZA, while ~70% is mobile in Rap1IR photoreceptors (Figure 2A-C). These data indicate that Rap1 promotes ECad stabilization at the ZA, and are compatible with Mbt mediating part of Rap1 function during this process.
Cno couples Arm and Baz at the ZA and is required for the apical accumulation of aPKC and Crb
Next to regulating Mbt accumulation at the ZA, one likely mechanism whereby Rap1 might promote ECad stabilization is through the F-actin linker Cno (Kooistra et al., 2007). In the pupal photoreceptor, Cno localizes at the ZA and this localization is also strongly decreased in Rap1IR photoreceptors (Figure 1C”). Decreasing cno expression using the strong cnoR2 allele leads to delamination of the mutant photoreceptors through the floor of the retina (Figure 3A-B), a phenotype resembling that obtained when strongly reducing Rap1 expression. As for Rap1 loss of function, delamination of the cnoR2 mutant retinal cells is likely due to strong defects in assembling the full complement of interommatidial accessory cells, and polarity of the delaminated photoreceptors is strongly compromised (Figure 3B). The delamination phenotype complicates the analysis of cno function. In order to circumvent this issue we made use of cno RNAi (cnoIR). Examining retinas mosaic for cnoIR revealed that this factor is required for the accumulation of Arm (Figure 3C’, 3E’), Baz (Figure 3C”) and Mbt (Figure 3E”) at the developing photoreceptor ZA. Examining cnoIR mutant ZA, we noted instances where Arm was present at the ZA but Baz or Mbt were absent (Figure 3D, 3F). The change in the relative accumulation of Arm and Mbt measured in cnoIR resembles that quantified in Rap1IR (Figure 1H), suggesting that Rap1 and Cno function during ZA morphogenesis are linked. However, in the case of cnoIR, we also detect uncoupling between Arm and Baz, a phenotype not detected in Rap1IR, indicating that part of Cno function is independent of Rap1. In addition, levels of Crb and aPKC were decreased in cnoIR mutant cells (Figure 3C”’ and 3E”’), indicating a Cno regulates the accumulation of these factors during apical membrane morphogenesis.
Linking Rap1 function to that of baz and mbt
Our results suggest that during ZA morphogenesis, Rap1 could function through Cno, and Mbt. In addition, Cno accumulation at the ZA depends on mbt (Figure 4A”), indicating multiple cross talks exist between Rap1, Cno and Mbt. To examine the relationship between Rap1, Cno and Mbt, we asked whether expressing Mbt or Cno could ameliorate the Rap1IR ZA phenotype. Expressing mbt in Rap1IR cells did not ameliorate the length of the ZA and did not restore levels of Cno (Figure 4B”, 4D). Expressing cno in Rap1IR cells did not ameliorate the length of the ZA (Figure 4C-D). These results indicate that Rap1 function on ZA morphogenesis is pleiotropic.
Next, to examine the relationship between Rap1 and mbt in more detail, we asked whether expressing the rap1-Rap1::GFP transgene could ameliorate the decrease in AJ material accumulation measured in mbtP1 null mutant photoreceptors. mbtP1 mutant cells are characterized by a decreased accumulation of Arm, Baz (Walther et al., 2016) (Supplementary Figure 3A-B), and Cno (Figure 4A”) at their ZA. When expressing rap1-Rap1::GFP in mbtP1 mutant cells (Figure 4E-G), we did not measure any significant recovery in the length of the Arm (Figure 4F’, G’ and 4H) or Baz domains (Figure 4E’, 4H’) when compared to mbtP1 mutant cells, and Cno levels were not restored (Figure 4G”). However, we noted that Rap1::GFP expression was more widespread than in wild type photoreceptors, as the preferential ZA accumulation (Figure 1A) was no longer readily detected. Instead Rap1::GFP was localized all over the apical membrane (Figure 4E, 4F, 4G and 4I). These results indicate that Mbt regulates the distribution of Rap1 between the apical membrane and the ZA.
Finally, we made use of genetics to probe the relationship between Rap1 and baz. Firstly, we found that Rap1 and baz genetically interact during eye development, as decreasing the expression of baz using RNAi (bazIR), enhances the Rap1IR rough eye phenotype (Supplementary Figure 2A-B, 2E-F). Secondly, to assay whether Rap1 function during ZA morphogenesis relates to that of Baz we generated photoreceptors deficient for both baz (using the bazxi106 allele) and Rap1 (using the NP-Gal42631-Rap1IR strain) (O’Keefe et al., 2009). As we have show before (Walther et al., 2016), AJ material such as Arm is detected at the plasma membrane in bazxi106 and mbtP1 single mutant cells (Figure 5A and Supplementary Figure 3). However, no AJ material is detected in bazxi106, mbtP1 double mutant cells (Figure 5B) indicating that baz and mbt converge in promoting AJ material accumulation at the plasma membrane. We found that expressing Rap1IR in bazxi106 photoreceptors led to fewer cortical domains positive for Arm shared by flanking photoreceptors when compared to bazxi106 and Rap1IR single mutant cells (Figure 5C, 5E). This was accompanied by a loss of Mbt accumulation (Figure 5C”’), which is consistent with our observation that Rap1 is required for the accumulation of Mbt at the ZA (Figure 1D”; quantified in 1F’ and 1G’). These data further indicate that during ZA morphogenesis, the function of Rap1 and mbt are interlinked.
To complement these experiments, we next asked whether decreasing Rap1 expression could modify the mbt phenotype. Combining the null allele mbtP1 to Rap1IR led to a very strong additive effect as nearly all photoreceptors delaminated from the retina, a phenotype due to strong defects in recruiting cone and pigment cells around the photoreceptor clusters (Figure 5D). Nevertheless, a majority of the delaminated, photoreceptors still presented Arm domain linking flanking photoreceptors (Figure 5D”” and 5E). Altogether, these genetic experiments argue in favor of Rap1 functioning together with mbt during Baz-dependent ZA morphogenesis.
Discussion
In the pupal photoreceptor, ZA morphogenesis is orchestrated by a conserved protein network that includes the Par complex, Crb and its binding partners Sdt and PATJ, together with the lateral kinase Par1 (Berger et al., 2007; Hong et al., 2003; Izaddoost et al., 2002; Nam and Choi, 2003; Pellikka et al., 2002; Richard et al., 2006; Walther et al., 2016; Walther and Pichaud, 2010). While loss of ZA, for example in arm3 mutant photoreceptors, leads to complete failure in pupal photoreceptor apical-basal polarization (Walther et al., 2016), the connection between the ZA and the protein network that drives its morphogenesis is not fully understood. We and others have previously shown that Mbt regulates pupal photoreceptor development by promoting ZA morphogenesis (Menzel et al., 2007; Walther et al., 2016). During this process Mbt contributes in preventing Baz from spreading to the lateral membrane, a regulation we have found depends on the phosphorylation of Arm by Mbt at S561 and S688 (Walther et al., 2016).
Our results show that Mbt function is linked to that of Rap1 and Cno. We find that Cno couples Arm and Baz accumulation at the ZA, as we detect ZA domains that do not contain Baz in cnoIR photoreceptors. This phenotype resembles that seen when substituting Arm by an Mbt-phospho-dead version of Arm (Walther et al., 2016). Such uncoupling between Arm and Baz is not detected in Rap1IR photoreceptors. While this indicates that part of cno function is independent of Rap1, it suggests that Mbt and Cno function during photoreceptor morphogenesis are linked. Our work shows that Cno is nearly absent at the ZA of mbt mutant photoreceptors. We therefore propose that part of Mbt’s function in promoting ZA accumulation of Baz is mediated by Cno. Whether Cno accumulation at the photoreceptor ZA is linked to Arm phosphorylation at S561 and S688 is an interesting possibility that remains to be investigated. mbt mutant photoreceptors are frequently found below the floor of the retina, and unlike for cno and Rap1, this phenotype is not due to defects in retinal accessory cells, but instead is autonomous to the photoreceptor (Walther et al., 2016). These mbt photoreceptors present strong defects in apical-basal polarity when considering aPKC and Crb for example (Walther et al., 2016). We show here that cnoIR photoreceptors can fail to accumulate aPKC and Crb properly at their apical membrane. We interpret these results as further evidence of a functional link between Mbt and Cno during polarized photoreceptor morphogenesis.
Our results also show that in mbt mutant photoreceptors, Rap1 localization is no longer restricted to the ZA but instead spreads apically. Conversely, Rap1 and cno promote the accumulation of Mbt at the ZA. Therefore Rap1, Cno and Mbt localization and accumulation at the ZA are interlinked. To probe Rap1 function during photoreceptor ZA morphogenesis, we assessed the effect of decreasing Rap1 expression on ECad stability. Consistent with the notion that the function of mbt and Rap1 are linked, we find that they are both required to stabilize ECad::GFP at the photoreceptor ZA. However, ECad mobile fraction is much higher in Rap1IR cells than in mbt null cells, evaluated over approximately 1000 sec at approximately 70% for Rap1IR and 45% over 250 sec for mbtP1 (Walther et al., 2016). Together with our finding that Mbt accumulation at the ZA is decreased in Rap1IR cells, our FRAP data are therefore compatible with Mbt mediating part of Rap1’s function in promoting ECad stability. The much larger mobile fraction we estimate in Rap1IR when compared to mbt null mutant photoreceptors indicates that Rap1 must also regulate ECad stability independently of Mbt. The longer time scale for ECad to recover in Rap1IR cells when compared to mbt mutant cells is compatible with Rap1 functioning in part through promoting ECad delivery. Previous work in MCF7 cells has shown that Rap1 function in promoting ECad delivery depends on Cdc42 (Hogan et al., 2004). Our work raises the possibility that Pak4 is one of the downstream effector of Cdc42 during this process.
Rap1 and cno have been shown to regulate apical-basal polarity in the cellularizing embryo, a system that allows for examination of the net contribution of the Par complex and AJ material toward epithelial cell polarization (Choi et al., 2013). In this model system, Rap1 and cno regulate the apical localization of both Baz and Arm, with Baz reciprocally influencing Cno localization. Later in embryonic development, Baz is required at the ZA to capture preassembled AJ material, which includes ECad, thus promoting ZA morphogenesis (McGill et al., 2009). Our work indicates that similar regulations might be at play during epithelial polarity remodeling. However, unlike in the early embryo, AJ material (Arm) is absolutely required for Baz (and Par6-aPKC) accumulation at the cell cortex in the developing pupal photoreceptor (Walther et al., 2016). In this cell, we therefore favor a model whereby Mbt, Cno and Rap1 influence ZA morphogenesis primarily through regulating ECad/Arm, through Arm phosphorylation and coupling of ECad/Arm to the actomyosin cytoskeleton and Baz via Cno. Our work shows that in turn these regulations influence apical membrane morphogenesis including aPKC, Part6 and Crb accumulation.
Material and Methods
Fly strains:
The following fly strains were used:
rap1-Rap1::GFP and NP-Gal42631, UAS-Rap1IR (O’Keefe et al., 2009)
Rap1IR (BL #29434); bazIR (BL #39072); cnoIR (BL #33367)
dizzyΔ12, FRT40A (Huelsmann et al., 2006)
dizzy-Dizzy::GFP (Boettner and Van Aelst, 2007)
ubi-Cad::GFP (Oda and Tsukita, 2001)
mbtP1 (Schneeberger and Raabe, 2003)
mbtP1, FRT19A and mbtP1, bazxi106, FRT9.2 (Walther et al., 2016)
w,bazxi106, FRT9.2 (Nusslein-Volhard et al., 1987).
FRT82B, cnoR2 (Sawyer et al., 2009)
Analysis of gene function
Clonal analysis of mutant alleles in the retina was performed using the standard FLP-FRT technique (Xu and Rubin, 1993) with appropriate FRT, ubi-GFP chromosomes used to generate negatively marked mutant tissue in combination with eyFLP (Newsome et al., 2000). Retina expressing RNAi in clones were generated using the coinFLP system (Bosch et al., 2015). Clones of retinal tissue expressing RNAi against Rap1 were generated both with and without UAS-dicer, while clones of retinal tissue expressing RNAi against cno were generated without UAS-dicer only. In order to mitigate the strong Rap1 loss of function phenotype, Rap1IR animal were raised at 20 degrees and shifted to appropriate temperature (25 or 29 degrees) at puparium formation.
Antibodies and immunological methods
Whole mount retinas at 40% after puparium formation (APF) were prepared as previously described (Walther and Pichaud, 2006). The following antibodies were used: rabbit anti-PKC, 1/600 (SAB4502380, Sigma), mouse anti-Arm, 1/200 (N27-A1, Developmental Studies Hybridoma Bank), rat anti-Baz, 1/1000 (Gift from A.Wodarz, University of Cologne), rabbit anti-Cno, 1/200 (Gift from L. Van Aelst, (Boettner et al., 2003)), rabbit anti-Baz, 1/2000, rat anti-Crb, 1/200, Guinea Pig anti-Mbt 1/200, Guinea Pig anti-PATJ 1/400 (Walther et al., 2016), with the appropriate combination of mouse, guinea pig, rabbit and rat secondary antibodies conjugated to Dy405, Alexa488, Cy3 or Cy5 as appropriate at 1/200 each (Jackson ImmunoResearch) or TRITCconjugated Phalloidin (P1951, Sigma) at 2μg/mL. Retinas were mounted in VectaShield™ with or without DAPI as appropriate and imaging was performed using a Leica SP5 confocal. Images were edited using ImageJ and Adobe Photoshop 7.0.
Data analysis
For length and pixel intensity measurements, a threshold was applied to define the ZA domain, and a line was drawn along the apical-basal axis of the cell, running in the middle of the ZA to measure the length of the Arm, Baz, Mbt domains. Mean pixel intensity was measured using the wand (tracing) tool in Fiji (Schindelin et al., 2012). In all cases, at least four independent mosaic retinas were used for each genotype. To compare the Mbt/Arm or Baz/Arm ratios in individual ZA, mean pixel intensity individual measurements for Mbt or Baz and Arm were normalized using the average mean intensity calculated for each epitope for a given set of experiments (i.e Mbt/Arm and Arm/Baz). These normalized data were used to calculate the Mbt/Arm and Baz/Arm ratio for a collection of individual ZA. To measure the ratio of apical Rap1::GFP/ZA Rap1::GFP, GFP intensity was measured using an ROI covering the ZA which was then applied to the apical domain. Statistical analysis was done using Prism 7.0. Data sets were tested for normality (D’Agostino and Pearson normality test) and p-values were calculated using the student’s t-test or the Mann-Whitney test as appropriate.
Fluorescence recovery after photobleaching (FRAP)
FRAP analysis was performed as previously described (Walther et al., 2016). At 40% APF the pupal cuticle was removed to expose the retina and the animal was mounted in Voltalef oil. Live imaging was performed on a Leica SP5 confocal using a 63x 1.4 NA oil immersion objective at the following settings: pixel resolution 512 x 512, speed 400 Hz, 10% 488 nm laser power at 20% argon laser intensity and 5x zoom. FRAP analysis of ubi-ECad::GFP was performed by marking the basal tip of the AJ with a 5 pixel-diameter circle ROI followed by photo-bleaching with a single pulse using 90 % 488 nm laser power at 20 % argon laser intensity. AJ recovery was recorded every 1.293 seconds with the previously mentioned settings for approximately 1000 sec. FRAP data were drift corrected in Fiji (Schindelin et al., 2012) using the StackReg plugin. Three different z axis profiles were analysed: (1) from the photo-bleached area; (2) from an equivalent area of a neighbouring non-photo-bleached AJ; and (3) from an equivalent area of background. The data were normalized using easyFRAP. ECad::GFP data were fitted to a two-phase association curve in GraphPad Prism. The p values were calculated with an unpaired two-tailed Student’s t test with Welch’s correction.
Scanning Electron Microscopy
Flies were fixed in 2% paraformaldehyde, 2% glutaraldehyde and 0.1 M cacodylate for 2 hours and then dehydrated in ethanol, as previously described (Richardson and Pichaud, 2010). The samples were then critical-point dried and mounted on aluminum stubs before gold coating. Imaging was carried out on a JEOL Variable Pressure scanning electron microscope (SEM).
Acknowledgements
The authors wish to thank all members of the Pichaud lab for helpful discussion, in particular Francisca Nunes Almeida for help with the FRAP assay and for critical reading of the manuscript. We are grateful to Linda Van Aelst and Andreas Wodarz for generously sharing reagents. The N2 A71 monoclonal antibody, developed by Eric Weischaus, was obtained from the Developmental Studies Hybridoma Bank, created by the NICHD of the NIH and maintained at The University of Iowa, Department of Biology, Iowa City, IA 52242. Stocks obtained from the Bloomington Drosophila Stock Center (NIH P40OD018537) were used in this study. This work, including support to RFW, MB and NP was funded by an MRC grant to FP (award code MC_UU_12018/3)