Expanded directly binds conserved regions of Fat to restrain growth via the Hippo pathway

Fulford et al. show the cell adhesion molecule Fat regulates Expanded by direct binding, stabilizing, and localizing Expanded. Highly conserved regions of Fat bind Expanded and are needed to control tissue growth in vivo. Fat–Dachsous binding can occur via direct cytoplasmic interactions as well as previously described extracellular interactions.


Introduction
The precise and coordinated control of metazoan growth is essential for correctly sized and proportioned adult organisms, and the highly conserved Hippo pathway is key to its regulation. The Hippo pathway modulates growth by inhibiting the transcriptional coactivator Yorkie (Yki). Yki activity is controlled by the core Hippo kinase cassette consisting of the kinases Hippo (Hpo) (Harvey et al., 2003;Jia et al., 2003;Pantalacci et al., 2003;Udan et al., 2003;Wu et al., 2003) and Warts (Wts) (Justice et al., 1995;Xu et al., 1995), which phosphorylates Yki and inhibits its nuclear localisation (Huang et al., 2005). When free from inhibition by these kinases, Yki concentrates in the nucleus where it interacts with transcription factors such as Scalloped (Sd) to promote transcription of cell cycle and anti-apoptotic genes, for example, cyclinE and diap1, ultimately driving tissue growth (Wu et al., 2008;Zhang et al., 2008). Once Yki is activated, excessive growth is prevented through a negative feedback loop, whereby Yki drives the transcription of its own inhibitors, such as expanded (ex) (Fulford et al., 2018;Zheng and Pan, 2019).
Upstream of the kinase cassette are numerous inputs into the pathway, such as cell polarity, adherens junctions and the cytoskeleton (Fulford et al., 2018;Zheng and Pan, 2019). One important nexus of signalling is through the 4.1, Ezrin, Radixin and Moesin (FERM) protein Ex. Ex forms a complex with Merlin (Mer) and Kibra (Kib) at the apical junctions (termed the KEM complex), where Ex activates the Hippo kinase cassette by scaffolding core pathway members and by recruiting the scaffold protein Schip1, which promotes Hpo phosphorylation by Tao-1 (Baumgartner et al., 2010;Boggiano et al., 2011;Chung et al., 2016;Genevet et al., 2010;Genevet & Tapon, 2011;Hamaratoglu et al., 2006;McCartney et al., 2000;Poon et al., 2011;Sun et al., 2015;Yu et al., 2010). Ex also directly interacts with WW-domains of Yki at the apical junctions through three PPxY motifs in its C-terminus (Badouel et al., 2009;Oh et al., 2009). This limits the translocation of Yki to the nucleus, as well as bringing it into proximity of the kinase cassette, a process inhibited by Activated cdc42 kinase (Ack) phosphorylation of Ex (Hu et al., 2016). Together these mechanisms promote robust inhibition of Yki function.
Ex is thought to sit at the interface between the major epithelial polarity axes -apico-basal and planar cell polarity (PCP) since it is regulated by two transmembrane proteins, Fat (Ft) and Crumbs (Crb), which organise tissues by regulating both polarity and growth (Fulford et al., 2018;Genevet and Tapon, 2011). The apico-basal polarity protein Crb promotes Ex apical localisation through a direct interaction between the FERM-binding motif of the Crb intracellular domain (ICD) and the N-terminal FERM domain of Ex. Crb is the only transmembrane protein that has been shown to directly bind Ex (Chen et al., 2010;Grzeschik et al., 2010;Ling et al., 2010;Robinson et al., 2010). Mutations in crb cause mislocalisation of Ex from the apical membrane to the cytoplasm, associated with an increase in Yki activity (Chen et al., 2010;Grzeschik et al., 2010;Ling et al., 2010;Robinson et al., 2010). In addition to promoting Ex localisation, Crb also regulates Ex levels by promoting its phosphorylation-dependent turnover. Overexpression of Crb stimulates Ex phosphorylation by Casein Kinase 1 (CK1) family kinases. This phosphorylation promotes the interaction of Ex with the F-box E3 ligase Supernumerary Limbs (Slmb) and results in Ex ubiquitination and proteasomal degradation Ribeiro et al., 2014).
Ft is a giant atypical cadherin that localises to the apical junctions where it regulates Hippo signalling and PCP, in part through heterophilic interaction with the atypical cadherin Dachsous (Ds) (Blair and McNeill, 2018;Fulford and McNeill, 2019;Irvine and Harvey, 2015). Ft also regulates Ex localisation and levels (Bennett & Harvey, 2006;Cho et al., 2006;Silva et al., 2006), however if Fat regulates Ex directly is not known. The Ft ICD is crucial in implementing its biological function. Several structure-function studies have identified key regions within the ICD that mediate its signalling, including Hippo functional domains, and highly conserved regions A to F (Bossuyt et al., 2014;Matakatsu & Blair, 2012;Pan et al., 2013;Zhao et al., 2013) Ft inhibits growth in part by limiting levels and activity of the atypical myosin Dachs (Mao et al., 2006), which regulates growth by destabilising and sterically inhibiting Wts, thereby activating Yki function Rauskolb et al., 2011;Vrabioiu & Struhl, 2015). Ft suppresses growth in concert with the CK1 kinase Discs overgrown (Dco) and the F-box E3 ubiquitin ligase Fbxl7. Dco phosphorylates the Ft ICD contributing to its activation Feng & Irvine, 2009;Pan et al., 2013;Sopko et al., 2009), while Fbxl7 interacts with Ft and limits Dachs accumulation (Bosch et al., 2014;Rodrigues-Campos & Thompson, 2014).
The palmitoyltransferase Approximated (App) and the SH3 containing protein Dlish (also known as Vamana) antagonise Ft activity and promote Dachs activity. Single mutations of dachs, app or dlish have only mild undergrowth phenotypes alone, but can suppress the overgrowth induced by loss of ft (Mao et al., 2006;Matakatsu and Blair, 2008;Misra and Irvine, 2016;Zhang et al., 2016). App, Dlish and Dachs form a complex at the apical membrane, stabilising Dachs localisation and enhancing its activity (Matakatsu et al., 2017;Matakatsu and Blair, 2008;Misra and Irvine, 2016;Zhang et al., 2016). App can palmitoylate Dlish and regulate Dachs independently of its enzymatic activity (Matakatsu et al., 2017). App also palmitoylates Ft, antagonising the action of Dco (Matakatsu et al., 2017). The complex relationship between these proteins is thought to precisely tune Hippo pathway activity (Matakatsu et al., 2017).
Ft can regulate Ex localisation and levels through Dlish (Wang et al., 2019). Dlish can regulate Hippo signalling independently of Dachs by regulating Ex turnover (Wang et al., 2019). Dlish directly binds to the C-terminus of Ex, promoting the interaction between Ex and Slmb, thereby stimulating Ex degradation via the proteasome. This process is antagonised by Wts phosphorylation at Ex S1116, which stabilises Ex by protecting it from Slmb-mediated turnover (Zhang et al., 2015). C-terminal regulation of Ex by Slmb appears to be independent of the Crb-mediated regulation of the Ex N-terminus . The E3 ubiquitin ligase Plenty of SH3s (POSH) has also been implicated in regulating Ex levels by binding to the Ex C-terminus and promoting its degradation (Ma et al., 2018), though this appears to be independent of Dlish (Wang et al., 2019).
As detailed above, Ex localisation, stability and activity are finely tuned by a complex molecular machinery. However, whether Ft regulation of Ex is direct, the role of Ds in these processes and the relationship between Ft and Crb to control Ex has remained unclear. Here we report that Ft promotes apical localisation of Ex by tethering it to the apical membrane, mediated by a direct interaction between the Ex FERM domain and the conserved E region of the Ft ICD. These processes occur independently of Crb. Using CRISPR, we determine that deletion of the conserved E region of Ft leads to wing overgrowth, reduced apical Ex localisation and increased levels of Dachs and Dlish. We show binding between the intracellular domains of Ft and Ds. Remarkably, we find that Ft can regulate Ex independently of Ds binding, as loss of ds upregulates Dachs/Dlish but does not downregulate Ex. This intricate regulation of Ex highlights its importance as an integrator of distinct polarity cues in the control of tissue growth. examination we observed that a subset of Ex was still present at the apical membrane in the absence of Crb. Residual apical Ex was seen in crb null tissue using an Ex-specific antibody or a GFP-tagged knock-in Ex allele (Ex::GFP), and observed in clones of two different crb null alleles . We confirmed both loss of Crb protein  and Ex antibody specificity [suppFIG1E]. These data indicate that there is a portion of Ex that localises to the apical membrane independently of Crb.
In addition to Crb, Ft regulates Ex localisation and levels [FIG1C] (Bennett & Harvey, 2006;Cho et al., 2006;Silva et al., 2006;Wang et al., 2019). We confirmed these data and further showed that Crb, Ft and Ex tightly colocalise [suppFIG1F-H], including within apical punctae where Ft has previously shown to localise (Ma et al., 2003a;Brittle et al., 2012;Hale et al., 2015) [suppFIG1I]. We also find loss of ft does not dramatically affect Crb levels [suppFIG1J]. Interestingly, the remaining apical Ex within crb mutant clones colocalises with Ft [FIG1A -B]. We tested the ability of Ft to contribute to Ex apical localisation by comparing crb null clones and crb null clones expressing full-length, HA-tagged Ft (Ft::HA). Notably, increasing levels of Ft within a crb clone significantly rescues apical Ex [FIG1D-F]. This increase in apical Ex is not accompanied by alterations in basal Ex [suppFIG1K]. suggesting an overall increase in Ex protein, consistent with previous studies (Bennett and Harvey, 2006;Silva et al., 2006;Wang et al., 2019). This indicates that Ft can promote Ex apical localisation and stability independently of Crb.
We next investigated whether Ft regulates Ex localisation independently of Crb by examining double mutant null clones in the wing disc. As ft and crb are on different chromosomes, FLP-FRT mediated recombination occurs independently, generating patches of tissue mutant for only ft or crb, as well as ft;crb double mutant tissue. In the single mutant tissue, as expected, loss of crb or ft results in a significant reduction in apical Ex . Notably loss of crb additionally results in an increase in cytosolic Ex not seen in ft mutant clones, consistent with previous studies [FIG1H] (Chen et al., 2010;Ling et al., 2010;Robinson et al., 2010). Interestingly, ft mutation results in a greater loss of apical Ex than crb mutation [FIG1I]. In ft;crb clones there is a dramatic and near total loss of Ex when compared to either single mutant [FIG1G-I]. Thus, loss-of-function experiments also indicate that Ft and Crb regulate Ex independently.

Ex directly binds to Ft
Both gain and loss of function studies indicate that Ft regulates Ex independently of Crb but does not address how Ft controls Ex localisation. To test if Ft physically associates with Ex, we co-expressed a form of Ft (Ft ΔECD ), which rescues growth defects caused by null mutations of ft (Matakatsu & Blair, 2006)  To determine if the interaction between Ft and Ex is direct, we performed a pulldown assay between bacterially expressed and purified GST-Ft ICD and in vitro translated N-terminal (Ex NT ) and C-terminal Ex (Ex CT ) along with GFP as a negative control. Compared to GST alone, GST-Ft ICD significantly binds to Ex NT but not Ex CT [FIG2B]. We also tested Ex 1 -468 and found this smaller fragment of Ex also directly binds Ft ICD [suppFIG2A]. These data reveal Ft binds directly to Ex. These data also indicate the Fat-Ex interaction occurs via the N-terminal Ex FERM domain.
Next, we investigated if the Ft-Ex interaction occurs in vivo by performing an in-situ proximity ligation assay (PLA), an immuno-PCR-based technique producing a positive fluorescent signal when two antibody epitopes are no further than approximately 40 nm apart and presumably interacting directly (Alam, 2018). In addition, PLA provides data on the localisation of protein interactions. To perform this technique, we used the FERM-domain containing Ex 1-468 truncation, driven by the ubiquitin 63E promoter (ubi-Ex 1 -468::GFP) , which colocalises with Ft [suppFIG2B] and a C-terminal FLAG-tagged Ft knock-in allele we generated for this study. Consistent with our biochemical data, we observed PLA signal colocalising with Ex signal at the apical membrane in in XZ [FIG2D] and XY [suppFIG2C] sections of imaginal wing discs, supporting a direct interaction in vivo between Ft and Ex.
Dlish has recently been shown to directly bind the C-terminus of Ex and regulate its turnover downstream of Ft (Wang et al., 2019). Dlish and Ft have been shown to interact via co-IP from cultured cells (Misra and Irvine, 2016;Zhang et al., 2016) but it is unclear if this interaction is direct. We therefore tested whether Dlish could also bind FtICD through an in vitro binding assay similarly to Ex but could see no evidence of direct binding [suppFIG2D].
Taken together, these data support the hypothesis that the Ex FERM domain binds directly to Ft in vivo at the apical membrane and suggest a model where Ex biochemically links Ft and Dlish.

Ft contains two Ex interaction domains
Our data indicates that the Ft ICD binds directly to the Ex FERM [FIG2B, suppFIG2A].
To determine which region of Ft interacts with the FERM domain, we performed co-IPs using truncated and/or internally deleted Ft ΔECD constructs in HEK293 cells (see FIG3E and suppFIG3C). Significantly, Ft c Δ244 (removing 244 residues from the C-terminus of Ft ΔECD ) can effectively co-IP Ex FERM , whereas removing 255 residues from the C-terminus of Ft ΔECD (Ft c Δ255) completely abolished the interaction with Ex FERM [FIG3A]. These data indicate the amino acids between residues 255-244 from the Ft C-terminus are needed to interact with Ex FERM , defining Expanded Binding Region 1 (EBR1). Interestingly, this region is within the Hippo activating domain of the Ft ICD as defined in several structurefunction analyses of Ft (Bossuyt et al., 2014;Matakatsu & Blair, 2012;Pan et al., 2013;Zhao et al., 2013) Further deletion and co-IP analyses revealed the existence of a second Ex binding site in the C-terminus of Ft [suppFIG3A]. This binding site includes the E and F domains (defined in (Pan et al., 2013)), conserved across multiple species including human FAT4. To confirm this interaction, we generated a construct containing the last 124 residues of Ft tagged with a myristoylation sequence targeting it to cell membranes (Ft myr-c124 ). Ft myr-c124 can interact with Ex FERM [FIG3B] indicating that the C-terminal 124 residues of Ft can bind Ex. To narrow down the C-terminal binding domain further, we created an internal deletion within Ft myr -c124, which removes a Ft ICD fragment containing the conserved E region (Ft myr -c124;ΔEBR2). This deletion abolished the interaction between Ft and Ex [FIG3B] indicating an Ex-binding region lies between the C-terminal residues 64-25, which we named EBR2 [FIG3E,FIG4A]. Interestingly, neither EBR1 nor EBR2 contain known FERM binding motifs (Gunn-Moore et al., 2006;Ling et al., 2010).
We confirmed a direct interaction between Ft conserved E region and Ex by generating a biotin-tagged peptide − called EBR2 WT − and performed a streptavidin-pulldown with recombinant Ex NT [FIG3C]. In contrast, a biotin-tagged EBR MUT peptide, with 6 residues mutated to alanine was unable to interact with Ex NT , highlighting the importance of the conserved E region in the binding to Ex [FIG3C].
To explore whether additional conserved regions affect the Ft-Ex interaction we generated Ft ΔECD constructs from EBR1 through to the conserved D region (Ft cΔ492-256;cΔ153 ), removing the C region (Ft cΔ492-256;ΔC;cΔ153 ), D region (Ft cΔ492-256;ΔD-CT ) or both (Ft cΔ492-256;ΔC;ΔD-CT ). Interestingly, we found that loss of either of these conserved regions reduced Ft-Ex interaction, and removal of both completely abolished it, although these constructs contain EBR1 [suppFIG3B]. These data show that the conserved C and D regions contribute to the Ft-Ex interaction. Deleting the entire region from EBR1 to EBR2 (Ft ΔEBR1-EBR2 ) abrogates Ex binding [FIG3D].

Ex binding regions of the Ft ICD are required in vivo for regulation of tissue growth
Having mapped the regions of the Ft ICD that interact with Ex, we next investigated their biological significance in vivo. We used CRISPR to delete EBR1 (Ft EBR1 ) and both EBR1 and EBR2 (Ft EBR1/2 ), from the endogenous ft locus, and added a 3xFLAG tag to the C-terminus. In addition, we used CRISPR to remove the conserved E region (largely overlapping EBR2), and replaced it with a 3xFLAG tag (Ft ΔE ) [FIG4A]. Immunoblot and clonal analysis indicated deletion of these regions in Ft EBR1 , Ft ΔE and Ft EBR1/2 did not affect levels of Ft protein, nor perturb Ft localisation [suppFIG4A -D]. To account for CRISPRinduced second site hits, we performed trans-heterozygous analysis of two independent lines for each mutation, which produced viable and fertile animals for all genotypes [suppFIG4E-I] despite significant pupal lethality in Ft ΔE and Ft EBR1/2 [suppFIG4J]. Due to the pupal lethality seen in Ft ΔE and Ft EBR1/2 , we analysed wing growth phenotypes as trans-heterozygous to the ft fd null allele. Interestingly, in this sensitised background, all three genotypes produced overgrown wings compared to the ft::FLAG control, with Ft EBR1/2 producing overgrowth in excess of either Ft ERB1 or Ft ΔE [FIG4B-F].
Further analysis of the EBR trans-heterozygous flies (independent lines of the same mutation) confirmed Ft EBR1 causes wing overgrowth, consistent with EBR1 residing in the HpoC functional domain that affects Hippo signalling [suppFIG4E-F,K]. However, Ft ΔE flies were not overgrown [suppFIG4G,K], and Ft EBR1/2 wings were mostly undergrown (class 1) [suppFIG4H,K], with a distinct subpopulation of Ft EBR1/2 flies (class 2) that were significantly larger than the controls [suppFIG4I,K]. The reason for this phenotypic separation remains unclear but could be due to developmental defects causing the significant pupal lethality observed (suppFIG4J). Nevertheless, the population of Ft EBR1/2 flies that were overgrown (class 2) all had rounded wings with cross-vein defects [suppFIG4I], which was also observed in Ft ΔE [suppFIG4G], indicating the E region affects wing shape. We calculated the ratio of length to width to measure the wing roundness, a typical phenotype of Ft/Ds pathway mutants (Mao et al., 2006;Matakatsu & Blair, 2006). Both Ft EBR1 and Ft ΔE had significantly rounder wings than controls [suppFIG4L]. As with wing size, Ft EBR1/2 separated into distinct populations, with the class 2 subpopulation generating wings that were significantly rounder than the control [suppFIG4L].
To assess whether the overgrowth was the result of excessive Yki activity, we analysed adult wing phenotypes in a ft, yki haplo-insufficient background. Importantly, overgrowth of EBR mutant flies was suppressed when one copy of Yki was removed, consistent with the hypothesis that overgrowth due to loss of Ex binding to Ft is Hippo pathway dependent [FIG4B-I].

Conserved E region/EBR2 is required to regulate Ex, Dachs and Dlish in vivo
To mechanistically understand the effects of loss of Ex binding regions on the Hippo pathway, we generated mitotic clones of our new alleles in wing discs and stained for Ex, as well as for Dlish and Dachs, critical mediators of Fat-Hippo signalling (Mao et al., 2006;Misra and Irvine, 2016;Zhang et al., 2016). Ft restricts Dlish and Dachs apical localisation, and Dlish stimulates Ex degradation (Wang et al., 2019). Loss of Ft leads to reduction of Ex [FIGsupp1F] and increased expression of Dachs and Dlish as previously reported (Bennett and Harvey, 2006;Mao et al., 2006;Misra and Irvine, 2016;Silva et al., 2006;Zhang et al., 2016). Clones of cells homozygous for ft EBR1 caused no change in levels or distribution of Ex, Dachs or Dlish [FIG5A, D,G], indicating this region is not critical for regulation of these proteins. Importantly, clones of ft ΔE or ft EBR1/2 showed a reduction in apical Ex , and a dramatic increase in both Dlish and Dachs [FIG5E-F,H-I].
Together, these data indicate that conserved region E is essential for restricting Dlish and Dachs and stabilising Ex in vivo. Interestingly, ft EBR1/2 clones appear to cause a greater loss of apical Ex than ft ΔE , which may indicate a contribution of EBR1 [Fig4A].
As Crb and Dlish both regulate Ex, we wondered whether loss of Crb alters Dachs/Dlish, and therefore regulate Ex through these proteins. Neither Dachs nor Dlish was altered in crb clones [suppFIG5A -B], indicating that Ft and Crb independently regulate Ex. As Dlish binds to the Ex C-terminus, these data are also consistent with previous studies showing the Slmb-mediated regulation of the Ex C-terminus is independent of Crb . (Bosch et al., 2014;Rodrigues-Campos & Thompson, 2014). As increased apical Dachs is associated with a concurrent increase in apical Dlish, we investigated whether loss of Fbxl7 could also regulate Ex. Knockdown of Fbxl7 did not alter levels of endogenous Ex observed through staining, or levels of the ubi-Ex 1 -468::GFP construct that does not respond to changes in Yki transcription [suppFIG5C -D]. These data suggest Fbxl7 does not regulate Ex.

Increased Dachs and Dlish do not reduce Ex levels in Ds mutants
Ds is the only known ligand of Ft, and spatial gradients of Ds expression are thought to regulate Ft activity (Strutt & Strutt, 2021). Null mutations of ft or ds result in increased levels of apical Dachs and Dlish and Hippo pathway dependent overgrowth (Brittle et al., 2012;Misra and Irvine, 2016;Zhang et al., 2016). We confirmed Ds repression of Dachs Given the current model where increased levels of Dlish-Ex complex stimulates Ex degradation (Wang et al., 2019), loss of Ds should lead to reduced Ex. We therefore investigated whether Ds regulates Ex. Remarkably, ds loss in wing discs did not decrease Ex levels and often resulted in a subtle increase in Ex, particularly in the imaginal disc hinge region, where Ds expression is highest . This is in stark contrast to ft mutant clones, which dramatically decrease Ex [suppFIG1F]. Loss of ds has a limited effect on Ft staining in the wing pouch, with subtly diffuse but still apical Ft [suppFIG5I] (Strutt and Strutt, 2002;Mao et al., 2009). These surprising data indicate that, despite Ds regulating Dachs and Dlish, there is no reciprocal reduction in Ex, and further indicate that levels of Dachs/Dlish can be uncoupled from levels of Ex.
Our finding that Ft and Ds both regulate Dachs and Dlish levels, yet loss of Ds does not cause a reduction in Ex, prompted us to investigate whether Ds and Ex could bind. However, we found no evidence of a direct interaction between Ds and Ex NT or Ex CT , consistent with Ft regulation of Ex being independent of Ds [FIGsupp5J]. As Ds and Dlish interact through co-IP (Misra and Irvine, 2016;Zhang et al., 2016), we tested if they could directly bind, but found no interaction [FIGsupp5K] suggesting these proteins likely need an intermediary to interact.
Although representations of Ft and Ds emphasize asymmetric distribution of these molecules, co-staining reveals that there is substantial overlap in Ft and Ds staining at cell membranes (Ma et al., 2003b). In addition, Ds puncta visualised by immunofluorescence are stabilised by the presence of Ft (Hale et al., 2015), and genetic data suggest that Ft and Ds may interact in cis (within cells) as well as in trans (Sharma & McNeill, 2013).
We therefore tested if Ft and Ds could interact independently of their previously documented extracellular domain interactions by performing co-IP of Ft ΔECD with Ds ICD in S2R+ cells. Ft ΔECD removes most of the extracellular domain, including all cadherin repeats, EGF-like domains and Laminin-G domains, and retains the transmembrane domain, and the full ICD (Matakatsu & Blair, 2006). Ds ICD removes the entire extracellular and transmembrane domains, and only retains the intracellular domain. Remarkably, Ds-ICD can co-immunoprecipitate with Ft ΔECD [Fig5L], indicating that Ft and Ds can form a complex mediated by their cytoplasmic domains.
The co-IP of Ft and Ds mediated by their intracellular domains could be direct, or via intermediary proteins. We tested if this interaction was direct through a binding assay between GST-Ft ICD and in vitro translated Ds ICD and observed binding between the ICDs of these proteins [Fig5M]. These data indicate cis interactions between Ft and Ds within the cytoplasm can be mediated via their ICDs. Thus, Ft and Ds can interact both across cell borders via their extracellular cadherin repeats, and within cells via their intracellular domains. These intracellular interactions imply that complexes that are independently formed on Ft and Ds can be brought together, and this cross-regulation has the potential for regulating Hippo or PCP activity.

Discussion
Ex is a critical nexus of Hippo signalling and is highly regulated, with its levels and localisation being controlled by two transmembrane proteins, Crb and Ft (Bennett and Harvey, 2006;Ling et al., 2010;Ribeiro et al., 2014;Silva et al., 2006;Wang et al., 2019). Cell-cell interactions are a key aspect of Hippo pathway upstream regulation that is thought to underpin their role in maintaining tissue homeostasis (Fulford et al., 2018;Misra and Irvine, 2018;Zheng and Pan, 2019). Previously, Crb was the only transmembrane protein known to directly bind and regulate Ex function (Ling et al., 2010), and has been established as an apical hub of Hippo signalling (Genevet & Tapon, 2011;Su et al., 2017;Sun et al., 2015 Combined, these data indicate that this conserved region regulates Hippo signalling, and that Ft interaction with Ex provides a Crb-independent hub for Hippo signalling at the apical membrane. While several Hippo pathway proteins have been identified as interacting with Ft (App, Dco, Dlish, Ds, FbxL7, Fj and Lft), or are regulated by Ft, so far none have been identified as a direct intracellular binding partner (Bosch et al., 2014;Brittle et al., 2010;Feng and Irvine, 2009;Ishikawa et al., 2008;Mao et al., 2009;Matakatsu et al., 2017;Sopko et al., 2009;Matakatsu and Blair, 2004;Misra and Irvine, 2016;Simon et al., 2010;Zhang et al., 2016). We show here that Ex binds directly to Ft. Ex also binds directly to Dlish, which promotes Slmb-dependent proteasomal degradation of Ex (Wang et al., 2019). Ex-Dlish binding occurs through the Ex C-terminus, whereas Ft binds to the N-terminal Ex FERM domain [FIG2B]. The region of the Ft ICD which interacts with Dlish  overlaps with the conserved E region that binds Ex. Therefore, Dlish may interact with Ft via Ex. In the simplest model, a Ft-Ex-Dlish complex may conformationally inhibit Dlish, sequestering it away from Ex and preventing Ex degradation [FIG6]. Alternatively, the Ft-Ex-Dlish complex could inhibit the ability of App to palmitoylate Ft and/or Dlish (Matakatsu et al., 2017;Zhang et al., 2016). In this case, the Ex-Ft interaction would promote Ft signalling and limit the apical localisation of Dlish and Dachs, therefore inhibiting Dlish-dependent Ex degradation. Moreover, App antagonises activating phosphorylation of Ft by the CK1 kinase Dco (Matakatsu et al., 2017).
Unexpectedly, although loss of Ds results in increased Dachs/Dlish, Ex levels do not decrease [FIG5J-K, suppFIG5F-G]. We hypothesize that increased Dachs/Dlish levels are unable to stimulate Ex degradation in ds mutant cells because intact Ft can still bind Ex, protecting it from Dlish-dependent degradation [FIG6]. This is consistent with the ability of Ft to suppress growth independently of Ds (Matakatsu & Blair, 2006). Together our data shows Ft both directly contributes to apical localisation of Ex and promotes Ex stability, ensuring consistent levels of Ex protein, and Hippo pathway homeostasis.
We also discovered that in addition to their known extracellular trans interaction (Brittle et al., 2010;Matakatsu and Blair, 2004;Simon et al., 2010), the Ft and Ds ICDs interact directly via their cytoplasmic domains . Previous studies have shown that although loss of Ds promotes growth, the Ds ICD itself can promote growth. This may occur through interaction with Dachs and Dlish to promote their activity (Bosveld et al., 2012;Misra and Irvine, 2016;Zhang et al., 2016) and through the phosphorylation and inhibition of Wts by the Minibrain kinase (Degoutin et al., 2013).

The subtle increase in Ex within ds clones [FIG5J-K] despite the strong increase in Dachs
and Dlish suggests another potential mechanism by which the Ds ICD can promote growth − by antagonising Ft to promote Ex degradation. This could be influenced by the interaction of the Ft-Ds ICDs and suggests binding, in trans and cis, may modulate the interaction of Ft-Ex and Hippo signalling more broadly [FIG6].
Ft-Ex binding is dependent on regions that are highly conserved in Ft orthologues, including in mammals [FIG4A]. Interestingly, in mammals, Fat4 and Crb3 both regulate a functional orthologue of Ex, Amot. In mammalian cells, Amot binds to the Crb3 complex to regulate YAP/TAZ activity and, in the heart, Fat4 binds to Amotl1 to mediate YAP1 nuclear exclusion (Ragni et al., 2017;Varelas et al., 2010). It will be interesting to see whether the conserved regions of Fat4 contribute to its interaction with Amotl1 or other FERM domain orthologues of Ex and regulate YAP signalling.

Drosophila genetics and genotypes
Ex::GFP and ft 5 -5 were generated by CRISPR/Cas9-mediated gene editing. Ex::GFP contains a C-terminal GFP tag. The ex genomic locus was cut near the stop codon by Cas9 guided by a gRNA (sequence: ATTAGCTTGTCGAGTCTAGC) and repaired from a co-injected plasmid template containing homologous sequence from the ex locus (2.4 kb upstream and 2.0 kb downstream of the stop codon), in which the eGFP coding sequence had been inserted immediately before the ex stop codon. ft 5 -5 is a remake of the ft fd allele. The entire Ft locus was sequenced in wildtype yw and mutant ft fd flies, which identified a single nucleotide mutation in Tyr982 (TAT>TAA) generating a premature stop Fulford et al. Page 10 J Cell Biol. Author manuscript; available in PMC 2023 May 01.

Europe PMC Funders Author Manuscripts
Europe PMC Funders Author Manuscripts codon in the first exon. This mutation was re-generated using Cas9 guided by gRNA (sequence: GGGATGCGGGCGTGAATAGT) and repaired from a co-injected plasmid template containing homologous sequence (1.3 kb upstream and 1.3kb downstream of the ft fd mutation site) incorporating site directed mutagenesis to generate the T>A mutation.
Progeny were genotyped and validated by sequencing.
ft::FLAG, ft EBR1 ::FLAG and ft EBR1/2 ::FLAG, all with C-terminal 3x FLAG tags and ft ΔE ::FLAG with the conserved E region replaced by a 3x FLAG tag were generated by CRISPR/Cas9-mediated gene editing performed by GenetiVision. Sequences removed or replaced are indicated in [FIG4A]. Two independently generated lines for each genotype were analysed: ft::FLAG (2) and (5)
Cloning into cell expression vectors was performed us using standard PCR/restriction enzyme-based cloning, Gateway technology (Thermo Fisher Scientific), Q5 Site-Directed Mutagenesis (New England BioLabs) or the pCDNA3.1/V5-His TOPO TA Expression Kit (Thermo Fisher Scientific) and confirmed by sequencing.

Binding assay between GST, Biotin-tagged or FLAG-tagged and in vitro translated protein:
In vitro Dlish, GFP, Ex 1 -468, Ex NT , Ex CT and Ds ICD were generated using TnT T7 Quick Coupled Transcription/Translation System (Promega) as per the manufacturer's protocol. N-terminally Biotin-tagged Ft peptide was generated by GenScript. 100 pmol purified GST-protein, 10 ug Biotin-peptide or 10 uL FLAG-tagged TnT T7 product was incubated with 10 µL TnT T7 product in a total of 300 µL binding buffer: 0.02% NP40 (IGEPAL CA-630), 10% glycerol, PBS supplemented with 0.5 mM DTT 0.1 mM PMSF and HALT protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific) for 2 h at 4°C. GST-protein binding reactions were then incubated with Glutathione Sepharose (Sigma Aldrich) and Biotin-peptide binding reactions were incubated with Pierce Streptavidin Agarose (Thermo Fisher Scientific) for 2 h at 4°C before purification by centrifugation. Beads were washed with PBS and were eluted by incubation with 2x Laemmli, 5% 2-Mercaptoethanol at 95°C for 8 min. FLAG-binding reactions were incubated with anti-FLAG M2 affinity agarose gel (Sigma Aldrich) overnight at 4°C before purification by centrifugation. Beads were washed with PBS and eluted by incubation with 150 ng/µl FLAG peptide (Sigma Aldrich) for 30-60 min. 2x SDS sample buffer was added to supernatant and incubated for 5 min at 95°C. Binding was analysed by immunoblotting.

Pupal lethality analysis:
The percentage of pupal lethality was calculated by counting the total number of pupal cases and the number of non-eclosed pupal cases from the same vials and presented as a ratio. Statistical analysis by one-way ANOVA with the Dunnett's post-hoc test was performed in GraphPad Prism.
width, which were presented as a ratio to measure shape. Statistical analysis by one-way ANOVA with the Tukey's or Dunnett's post-hoc test was performed in GraphPad Prism.
Immunofluorescence Quantification and Processing: For quantification of apical fluorescence inside vs outside a clone, regions of interest were manually defined using the fluorescent clonal marker. Apical or basal mean pixel intensity was measured using NIS-Elements (Nikon) or Image J. Data points represent the averaged signals from at least two transverse sections per wing disc normalised to the average signal from the wild-type tissue. Statistical analysis by unpaired t-test or one-way ANOVA with the Tukey's post-hoc test was performed in GraphPad Prism. Where indicated, images were denoised using NIS-Elements (Nikon).

Supplementary Material
Refer to Web version on PubMed Central for supplementary material. UAS-Ft::HA (E-E"'). Clones are marked by GFP (green in D' and D"' and E' and E"') and are stained with Ex (grey) and Ft (visualised by HA staining, red in E' and E"').
(F) Quantification of the ratio between apical Ex inside versus outside the MARCM clone normalised to the wildtype tissue. Data points represent an average of a single disc with the mean and standard deviation indicated. **P= 0.0018 using an unpaired T-test. (G-H"') Loss of Crb and Ft have an additive effect causing dramatic loss of apical Ex. XY (G-G"') and transverse (H-H"') confocal micrographs of third instar wing imaginal discs containing ft 5 -5 (marked by absence of GFP − grey in G' and H', green in G"' and H"' and by green asterisks) and crb 11A22 mutant clones (marked by absence of RFP − grey in G" and H", red in G"' and H"' and by red asterisks), with Ex staining (grey in G, G"', H and H"'). ft 5 -5 is a remake of ft fd and is a null allele. Double mutant clones are marked by absence of GFP and RFP and by yellow asterisks.
(I) Quantification of the ratio between apical Ex inside indicated clone verses outside the clone. All quantification was performed on the genotype used to create double clones. Data points represent an average of a single disc with the mean and standard deviation indicated. *P= 0.0177, **P= 0.0054 and ****P< 0.0001 using one-way ANOVA with a Tukey's post-hoc test.
All XY images are orientated as dorsal up and all transverse images are apical up. Clonal boundaries are marked by yellow dotted lines. Scale bars are 10 µm.  Ex FL or Ex FERM in the presence of Ft ΔECD , compared to FLAG-bead controls. Ft presents as multiple bands due to proteolytic processing (Feng & Irvine, 2009;Sopko et al., 2009). (B) Ft ICD directly binds Ex NT . In vitro transcribed and translated GFP as a control, Ex NT and Ex CT were incubated with bacterially expressed and purified GST alone or GST::Ft ICD and subjected to GST-purification.  peptide containing 6 alanine substitutions) and subjected to streptavidin-purification. EBR2 sequence defined by co-IP and the conserved-E region (highlighted in red) are also indicated. (D) Identification of the Expanded interacting region of the Ft-ICD. HEK293 cell expression and IP of indicated FLAG-tagged Ft ΔECD constructs in the presence of Ex FERM compared to FLAG-bead controls. The expression and presence of proteins was analysed by immunoblotting with the indicated antibodies. Ft presents as multiple bands due to proteolytic processing (Feng & Irvine, 2009;Sopko et al., 2009). (E) Graphical scheme highlighting the Ft constructs used in figure 3. In addition, the transmembrane domain (TM), EBR1, EBR2 and established conserved and function domains of the Ft-ICD are depicted. In binding column: '++' denotes constructs that interact strongly to Ex FERM and '-' denotes no interaction with Ex FERM . Graphical illustration of findings (not to scale). In wildtype conditions, Ft regulates Ex independently of Crb and there is a homeostasis of Ex levels. Ft promotes apical localisation of Ex. Degradation of Ex is stimulated by Dlish, which is balanced by Ft-mediated inhibition of Dlish. Loss of Ds increases the amount of apical Dlish available to interact with and degrade Ex, however this is counteracted by Ft actively inhibiting this process. Upon loss of Ft, or the conserved E region responsible for Ft-Ex interaction, Dlish is derepressed, increasing Ex-degradation. Ft and Ds cis interaction may act to antagonise Ft-mediated inhibition of Dlish, which may also promote mutual antagonism between Ft and Ds to support PCP.