_:b33392303 . _:b33392350 . _:b33392296 . . _:b33392305 "Overexpression of Fbxl7 reduced expression of an ex-lacZ reporter (Boedigheimer and Laughon, >>1993<<; Hamaratoglu et al., 2006) in a cell-autonomous manner (Figure 1O\u2013O\u2032)." . _:b33392297 . . _:b33392305 . _:b33392282 "dCul1DN is a C-terminal truncation (1-451aa) which corresponds to 1-452aa of dominant negative human hCul1DN (Wu et al., >>2000<<) and was cloned into pMT-HA as for full length dCul1." . _:b33392298 . _:b33392351 . _:b558790714 . . _:b33392299 . _:b33392308 . _:b33392309 . _:b33392338 . _:b33392260 "However, the overall levels of D protein are not obviously changed (Mao et al., >>2006<<). It has been proposed that Ft restricts growth by negatively regulating the levels of D at the apical membrane and that it regulates the D-dependent PCP functions by maintaining D asymmetry (Rogulja et al., 2008)." . _:b33392287 "In two different genetic screens, one for mutations that caused cells to outgrow their neighbors (described in Tapon et al., >>2001<<) and another for mutations that enabled cells to promote the elimination of their slower-growing neighbors by cell competition (Hafezi et al., 2012), we identified mutant alleles of the Drosophila Fbxl7 gene (CG4221), which encodes a" . _:b33392310 . _:b558790743 . _:b33392250 "Additionally, mutations in ft disrupt the localization of Expanded (Ex), a FERM-domain protein that functions upstream of Hippo (Hpo) (Bennett and Harvey, 2006; Silva et al., 2006; Willecke et al., >>2006<<), though other studies suggest Ft and Ex act in parallel (Feng and Irvine, 2007)." . _:b33392311 . _:b558790779 . _:b33392304 . _:b33392305 . _:b33392362 "Cindr and its mammalian orthologues Cin85 and CD2AP are thought to regulate interactions between membrane proteins and actin cytoskeleton (Haglund et al., 2002; Petrelli et al., >>2002<<; Soubeyran et al., 2002; Johnson et al., 2011, 2012)." . _:b33392234 "In addition to regulating growth, Ft also regulates planar cell polarity (PCP), oriented cell division and proximodistal patterning of appendages (reviewed in Thomas and Strutt, >>2012<<; Sharma and McNeill, 2013) and its regulated activity therefore impacts the size and shape of organs." . _:b33392306 . . _:b33392337 . _:b33392307 . _:b33392316 . _:b33392353 "Indeed, it has previously been suggested that Ft regulates growth by restricting the levels of apical D, and regulates PCP by influencing the planar asymmetry of apical D (Rogulja et al., >>2008<<; Pan et al., 2013)." . _:b33392317 . _:b558790760 . . _:b33392318 . . _:b33392319 . _:b33392263 "In an attempt to identify signaling pathways downstream of Ft, several recent studies have made systematic deletions in the intracellular domain (ICD) of Ft (Matakatsu and Blair, 2012; Bossuyt et al., >>2013<<; Pan et al., 2013; Zhao et al., 2013)." . _:b33392267 . _:b33392270 "materials and methods" . _:b33392312 . . . _:b33392313 . . _:b33392314 . _:b33392346 _:b33392348 . _:b33392346 _:b33392349 . _:b33392346 _:b33392350 . _:b33392315 . _:b33392346 _:b33392351 . _:b33392245 "While cadherins are known to have important functions in cell\u2013cell adhesion, a key aspect of Ft function is its role as a signaling molecule (Matakatsu and Blair, >>2006<<). Ft regulates the Hippo pathway in two ways." . _:b33392289 "We also found that a Mi{MIC} minos insertion in the first intron of Fbxl7 (Venken et al., >>2011<<) results in a strong loss-of-function phenotype similar to our other mutant alleles (Figure 1\u2014figure supplement 1A, Figure 1\u2014figure supplement 2A\u2013B)." . _:b33392252 "Additionally, PCP defects in ft mutants are partially rescued by loss of D (Mao et al., >>2006<<). D localizes to the apical membrane where, in cells of the wing disc, it localizes preferentially to the distal edge of the cell (Mao et al., 2006; Mao et al., 2011; Ambegaonkar et al., 2012; Bosveld et al., 2012; Brittle et al., 2012)." . _:b33392324 . _:b33392346 _:b33392347 . _:b33392299 . _:b33392332 "et al., 2013; Hippo-N, Hippo-C, Matakatsu and Blair, 2012; H2, Zhao et al., 2013), region required for Dco binding (green) (Sopko et al., 2009), mutV region (orange) (Pan et al., 2013), Su(DN) region (purple) (Matakatsu and Blair, >>2012<<), and two point mutations, ftsum (Bossuyt et al., 2013) and ft61 (this study). Size and position of regions are drawn to scale relative to the ICD." . _:b33392325 . _:b33392354 . _:b33392326 . _:b33392355 . _:b33392346 _:b33392364 . _:b33392346 _:b33392365 . _:b558790786 . _:b33392327 . _:b33392230 . . _:b33392346 _:b33392366 . _:b33392346 _:b33392367 . _:b33392346 _:b33392360 . _:b33392347 "One or more regions between amino acids 4834 and 4899 in full-length Ft appear responsible for Ft\u2019s ability to regulate Hippo signaling (labeled HM in Figure 7M) (Matakatsu and Blair, >>2012<<; Bossuyt et al., 2013; Zhao et al., 2013)." . _:b33392346 _:b33392361 . _:b33392346 _:b33392362 . _:b33392320 . _:b33392346 _:b33392363 . _:b33392346 _:b33392356 . . _:b33392346 _:b33392357 . _:b33392346 _:b33392358 . _:b33392321 . _:b33392346 _:b33392359 . _:b33392241 "Ft\u2013Ds interactions are modulated by the kinase Four-Jointed (Fj), which resides in the Golgi and phosphorylates the extracellular domains of both Ft and Ds (Ishikawa et al., 2008; Brittle et al., >>2010<<; Simon et al., 2010)." . _:b33392346 _:b33392352 . _:b33392346 _:b33392353 . _:b33392346 _:b33392354 . _:b33392322 . _:b33392274 . _:b33392346 _:b33392355 . . _:b558790784 . _:b33392323 . _:b558790785 . _:b558790786 . _:b33392343 "However, in wild-type cells, endogenous D is preferentially observed on the proximal/distal edges and is found at lower levels at the other edges (Brittle et al., >>2012<<; Figure 6E,G)." . _:b558790787 . . _:b33392332 . . . . _:b33392333 . _:b33392284 . _:b33392334 . _:b33392335 . _:b558790738 . . . _:b558790739 . _:b33392328 . . _:b33392329 . . _:b33392248 . _:b558790705 . _:b33392330 . _:b33392266 . _:b33392334 "These sites were identified as candidates for phosphorylation by Dco (Pan et al., >>2013<<). However, since Fbxl7 localizes normally in dco mutant clones, the inability of Fbxl7 to bind to FtmutV might be caused by a change in its conformation that does not depend on phosphorylation by Dco. Indeed ftGrv/ft8; ftmutV flies also" . _:b33392331 . _:b33392314 "These changes in Fbxl7 localization could simply be a consequence of the more diffuse localization of Ft that is observed in ds clones (Strutt and Strutt, 2002; Ma et al., 2003; Mao et al., >>2009<<). Fj is required for normal localization of Ds and Ft (Strutt and Strutt, 2002; Ma et al., 2003). In agreement with this, we see subtle effects on Fbxl7 localization in fj clones, which appears similar to that seen in ds clones (Figure" . . _:b33392340 . _:b33392285 "Quantifications were performed as in Brittle et al. (>>2012<<) using ImageJ. Wing discs were immunostained for Dachs and F-actin and imaged under identical settings at 20x to determine P-D orientation, and at 63x to image the dorsal portion of the wing pouch where Dachs asymmetry is highest." . _:b33392341 . _:b33392342 . _:b33392366 "In addition, some F-box proteins have SCF-independent roles (Nelson et al., >>2013<<). Fbxl proteins are thought to recruit substrates to the SCF complex through the interaction with their LRR domains, and substrates have been identified for several Fbxls such as Skp2 (Fbxl1), which degrades p27 (Carrano et al., 1999;" . _:b33392343 . . _:b33392280 "Other antibodies used: rat anti-Dachs (1:500, Brittle et al., >>2012<<), rat anti-Fat (1:1600, Feng and Irvine, 2009), rat anti-Dachsous (1:5000, Yang et al., 2002) rat anti-Ecad (1:" . . _:b33392336 . . . _:b33392337 . _:b33392244 . _:b33392292 . . _:b33392338 . . _:b33392339 . . _:b33392348 . _:b558790710 . _:b33392349 . _:b33392284 "Other plasmids used are pMT-Dco-V5 (Ko et al., 2002), pUAS-Dachs-V5 (Mao et al., >>2006<<)." . _:b33392350 . _:b33392348 "One or more regions between amino acids 4834 and 4899 in full-length Ft appear responsible for Ft\u2019s ability to regulate Hippo signaling (labeled HM in Figure 7M) (Matakatsu and Blair, 2012; Bossuyt et al., >>2013<<; Zhao et al., 2013)." . _:b33392231 "The Hippo signaling pathway has emerged as a key regulator of organ size (reviewed by Pan, >>2010<<; Halder and Johnson, 2011; Tapon and Harvey, 2012)." . _:b33392351 . . _:b33392344 . _:b33392267 "several proteins have been shown to bind to the Ft ICD including the transcriptional repressor Atrophin/Grunge which regulates PCP (Fanto et al., 2003), the novel protein Lowfat that regulates Ft protein levels (Mao et al., >>2009<<), and the casein kinase I protein Discs overgrown (Dco) that phosphorylates the Ft ICD (Feng and Irvine, 2009; Sopko et al., 2009)." . _:b33392345 . _:b33392296 "The combination of overgrowth and reduced spacing of the cross veins is especially reminiscent of mutations in the Ft branch of the Hippo signaling pathway (Bryant et al., 1988; Mahoney et al., >>1991<<; Clark et al., 1995; Villano and Katz, 1995; Mao et al., 2006; Matakatsu and Blair, 2008; Mao et al., 2009)." . _:b33392346 . . _:b558790781 . _:b33392347 . . . _:b33392356 . _:b33392249 "Additionally, mutations in ft disrupt the localization of Expanded (Ex), a FERM-domain protein that functions upstream of Hippo (Hpo) (Bennett and Harvey, 2006; Silva et al., >>2006<<; Willecke et al., 2006), though other studies suggest Ft and Ex act in parallel (Feng and Irvine, 2007)." . _:b558790747 . _:b33392244 "Both Ds and Fj are expressed in gradients in Drosophila imaginal discs where they function in patterning the disc along a major axis (e.g., equatorial to polar or proximodistal) (Yang et al., 2002; Ma et al., >>2003<<)." . _:b33392357 . _:b33392261 . _:b558790732 . _:b33392358 . _:b33392260 . _:b33392354 "Indeed, it has previously been suggested that Ft regulates growth by restricting the levels of apical D, and regulates PCP by influencing the planar asymmetry of apical D (Rogulja et al., 2008; Pan et al., >>2013<<)." . _:b33392359 . _:b33392273 "Additional stocks used were: FRT82B dcole88 (Jursnich et al., 1990), P[acman]-Fat+; P[acman]-Fat\u0394D, P[acman]-Fat\u0394F (Pan et al., 2013), Diap1 3.5-GFP (Zhang et al., >>2008<<), FRT42D fjN7, FRT40A ftGrv, FRT40A ds38k, UAS-Fat (Simon, 2004), ykiB5 (Huang et al., 2005), Tub-EGFP." . _:b33392352 . _:b33392353 . _:b33392337 "The overgrowth and lethality of a ft null background (ftGrv/ft8) can be rescued by ubiquitous expression of Ft (Matakatsu and Blair, 2012; Bossuyt et al., >>2013<<; Pan et al., 2013; Zhao et al., 2013; Figure 5\u2014figure supplement 3D,I)." . _:b33392354 . . _:b33392355 . _:b558790736 . _:b33392322 "A region between the conserved blocks \u2018B\u2019 and \u2018C\u2019 seems necessary for the major growth-suppressive function of Ft (Matakatsu and Blair, 2012; Bossuyt et al., >>2013<<; Zhao et al., 2013)." . _:b33392258 . _:b33392364 . _:b558790783 . _:b33392365 . _:b558790731 . _:b558790708 . _:b558790712 . _:b33392366 . . _:b33392367 . _:b558790723 . _:b33392360 . . . _:b33392361 . _:b33392236 . . _:b33392264 "In an attempt to identify signaling pathways downstream of Ft, several recent studies have made systematic deletions in the intracellular domain (ICD) of Ft (Matakatsu and Blair, 2012; Bossuyt et al., 2013; Pan et al., >>2013<<; Zhao et al., 2013)." . _:b33392362 . . _:b33392363 . _:b33392350 "Several mutations within this region compromise this function of Ft and cause massive tissue overgrowth (Bossuyt et al., >>2013<<). Intriguingly, an allele of ft, ft61, which harbors such a mutation, showed neither an effect on the recruitment of Fbxl7 to the apical membrane nor on the binding of Ft to Fbxl7. Thus, signaling via this region of the ICD appears to be" . . _:b33392316 "Fj is required for normal localization of Ds and Ft (Strutt and Strutt, 2002; Ma et al., >>2003<<). In agreement with this, we see subtle effects on Fbxl7 localization in fj clones, which appears similar to that seen in ds clones (Figure 4\u2014figure supplement 1A).10.7554/eLife.03383.009Figure 4.Relationship between Fbxl7 and the Fat" . _:b33392315 . _:b33392312 . _:b33392342 "D is localized at higher levels at the distal edge of the cell (Mao et al., 2006; Brittle et al., 2012) where it is likely stabilized by physical interaction with the cadherin Ds (Bosveld et al., >>2012<<). Therefore, a likely explanation is that the formation of multimeric Ft\u2013Ds complexes between cells results in the concomitant accumulation of Fbxl7 at the FatICD and D at the DsICD (Figure 6C).10.7554/eLife.03383.011Figure 6.Fbxl7" . . _:b558790709 "5"^^ . _:b33392248 "Additionally, mutations in ft disrupt the localization of Expanded (Ex), a FERM-domain protein that functions upstream of Hippo (Hpo) (Bennett and Harvey, >>2006<<; Silva et al., 2006; Willecke et al., 2006), though other studies suggest Ft and Ex act in parallel (Feng and Irvine, 2007)." . _:b558790708 "6"^^ . . . . _:b558790711 "5"^^ . . _:b33392239 . _:b558790710 "5"^^ . _:b558790706 "6"^^ . _:b558790707 "6"^^ . _:b558790768 . _:b33392320 "However, six blocks of sequence (labeled A\u2013F in Figure 5A based on the nomenclature of Pan et al. (>>2013<<)) are conserved with the ICD of mammalian Fat4." . _:b33392324 "ft61 displays phenotypic abnormalities that are very similar to those described for ftsum, which also changes a single amino acid two residues N-terminal to ft61 (Bossuyt et al., >>2013<<). Additionally, in a ft null background, deletion of one of the conserved blocks (block D in Figure 5A) in a ft genomic rescue transgene was shown to cause overgrowth (Pan et al., 2013) albeit to a much lesser extent than for ft61 and" . _:b558790704 "8"^^ . . _:b558790761 . _:b558790724 . _:b558790705 "7"^^ . _:b33392344 . _:b33392253 "D localizes to the apical membrane where, in cells of the wing disc, it localizes preferentially to the distal edge of the cell (Mao et al., >>2006<<; Mao et al., 2011; Ambegaonkar et al., 2012; Bosveld et al., 2012; Brittle et al., 2012)." . _:b33392323 "A region between the conserved blocks \u2018B\u2019 and \u2018C\u2019 seems necessary for the major growth-suppressive function of Ft (Matakatsu and Blair, 2012; Bossuyt et al., 2013; Zhao et al., >>2013<<). In our screen, we identified an allele of ft, ft61 (Figure 5A), which displays strong overgrowth (Figure 5C,K) and is caused by a single amino acid change (T to I) within this region. ft61 displays phenotypic abnormalities that are very" . _:b558790717 "4"^^ . . _:b33392302 . _:b558790716 "5"^^ . . _:b558790719 "4"^^ . _:b33392289 . _:b558790748 . _:b33392255 . _:b558790706 . _:b558790718 "4"^^ . _:b33392327 "domain of Fat showing the transmembrane domain (TM), regions conserved with mammalian Fat4 (blue, A\u2013F) (defined by Pan et al., 2013), regions associated with the major growth suppressive function of Fat (red) (HM, Bossuyt et al., >>2013<<; Hippo-N, Hippo-C, Matakatsu and Blair, 2012; H2, Zhao et al., 2013), region required for Dco binding (green) (Sopko et al., 2009), mutV region (orange) (Pan et al., 2013), Su(DN) region (purple) (Matakatsu and Blair, 2012), and two point" . _:b558790713 "5"^^ . . _:b558790712 "5"^^ . _:b33392297 "The combination of overgrowth and reduced spacing of the cross veins is especially reminiscent of mutations in the Ft branch of the Hippo signaling pathway (Bryant et al., 1988; Mahoney et al., 1991; Clark et al., >>1995<<; Villano and Katz, 1995; Mao et al., 2006; Matakatsu and Blair, 2008; Mao et al., 2009)." . _:b33392358 . _:b558790715 "5"^^ . _:b33392364 . _:b558790704 . . _:b33392288 . _:b558790714 "5"^^ . . _:b558790725 "3"^^ . . _:b558790724 "3"^^ . _:b33392340 "D is localized at higher levels at the distal edge of the cell (Mao et al., >>2006<<; Brittle et al., 2012) where it is likely stabilized by physical interaction with the cadherin Ds (Bosveld et al., 2012)." . _:b33392240 "Ft\u2013Ds interactions are modulated by the kinase Four-Jointed (Fj), which resides in the Golgi and phosphorylates the extracellular domains of both Ft and Ds (Ishikawa et al., >>2008<<; Brittle et al., 2010; Simon et al., 2010)." . _:b558790727 "3"^^ . _:b33392336 "The overgrowth and lethality of a ft null background (ftGrv/ft8) can be rescued by ubiquitous expression of Ft (Matakatsu and Blair, >>2012<<; Bossuyt et al., 2013; Pan et al., 2013; Zhao et al., 2013; Figure 5\u2014figure supplement 3D,I)." . _:b558790726 "3"^^ . _:b33392338 "The overgrowth and lethality of a ft null background (ftGrv/ft8) can be rescued by ubiquitous expression of Ft (Matakatsu and Blair, 2012; Bossuyt et al., 2013; Pan et al., >>2013<<; Zhao et al., 2013; Figure 5\u2014figure supplement 3D,I)." . _:b33392288 "for mutations that caused cells to outgrow their neighbors (described in Tapon et al., 2001) and another for mutations that enabled cells to promote the elimination of their slower-growing neighbors by cell competition (Hafezi et al., >>2012<<), we identified mutant alleles of the Drosophila Fbxl7 gene (CG4221), which encodes a protein with an F-box and 11 leucine-rich repeats (LRRs) (Figure 1A, Figure 1\u2014figure supplement 1A)." . _:b558790721 "4"^^ . _:b33392269 . . . _:b558790720 "4"^^ . _:b558790723 "4"^^ . _:b558790722 "4"^^ . _:b33392255 "D localizes to the apical membrane where, in cells of the wing disc, it localizes preferentially to the distal edge of the cell (Mao et al., 2006; Mao et al., 2011; Ambegaonkar et al., >>2012<<; Bosveld et al., 2012; Brittle et al., 2012)." . _:b33392351 "region of the Ft ICD (Region D in Figure 7M) that extends between amino acids 4975 and 4993 of full-length Ft, is removed by the ft\u0394D deletion and also has a growth-suppressive function albeit weaker than that of HM (Pan et al., >>2013<<). This second growth-suppressive pathway requires the function of Fbxl7, as the protein generated by the ft\u0394D allele cannot bind to Fbxl7 nor can it localize Fbxl7 to the apical membrane." . _:b558790733 "3"^^ . _:b33392256 "D localizes to the apical membrane where, in cells of the wing disc, it localizes preferentially to the distal edge of the cell (Mao et al., 2006; Mao et al., 2011; Ambegaonkar et al., 2012; Bosveld et al., >>2012<<; Brittle et al., 2012)." . _:b558790732 "3"^^ . . . _:b558790735 "3"^^ . . . _:b558790780 . _:b558790769 . _:b558790734 "3"^^ . _:b558790729 "3"^^ . _:b558790754 . _:b33392340 . . . _:b33392240 . _:b558790728 "3"^^ . _:b33392271 . _:b558790731 "3"^^ . _:b558790709 . _:b558790730 "3"^^ . _:b33392296 . _:b33392292 "Consensus sequence definitions according to Bella et al. (>>2008<<) and Kobe and Deisenhofer (1994)." . _:b558790708 . _:b33392257 "D localizes to the apical membrane where, in cells of the wing disc, it localizes preferentially to the distal edge of the cell (Mao et al., 2006; Mao et al., 2011; Ambegaonkar et al., 2012; Bosveld et al., 2012; Brittle et al., >>2012<<). In ft mutants, increased levels of D are observed apically and D is redistributed around the entire perimeter of the cell (Mao et al., 2006; Brittle et al., 2012)." . _:b558790741 "3"^^ . _:b558790711 . _:b33392232 "The Hippo signaling pathway has emerged as a key regulator of organ size (reviewed by Pan, 2010; Halder and Johnson, >>2011<<; Tapon and Harvey, 2012)." . _:b558790757 . . _:b558790740 "3"^^ . _:b33392246 "First, Ft influences the protein levels of Warts (Wts), a kinase that regulates the activity and subcellular location of the pro-growth transcriptional co-activator Yorkie (Yki) (Cho et al., >>2006<<; Rauskolb et al., 2011)." . _:b558790763 . _:b558790710 . _:b558790743 "2"^^ . . . . _:b558790705 . _:b558790742 "2"^^ . _:b558790704 . . _:b558790737 "3"^^ . _:b33392306 "A similar phenomenon was observed with the bantam sensor (Figure 1\u2014figure supplement 2O\u2013O\u2032) (Brennecke et al., >>2003<<), which is expressed at higher levels when Yki activity is reduced." . _:b33392355 "The asymmetric localization of D depends on at least two different regions of Ft (Pan et al., >>2013<<). One is the region that binds to Fbxl7 (Region D) and the other is composed of the last three amino acids at the C-terminus of the protein (Region F in Figure 7M), which is not necessary for Fbxl7 localization to the apical membrane." . _:b558790707 . _:b558790736 "3"^^ . _:b558790706 . _:b558790739 "3"^^ . _:b558790717 . _:b558790756 . _:b558790738 "3"^^ . _:b33392275 "were: FRT82B dcole88 (Jursnich et al., 1990), P[acman]-Fat+; P[acman]-Fat\u0394D, P[acman]-Fat\u0394F (Pan et al., 2013), Diap1 3.5-GFP (Zhang et al., 2008), FRT42D fjN7, FRT40A ftGrv, FRT40A ds38k, UAS-Fat (Simon, 2004), ykiB5 (Huang et al., >>2005<<), Tub-EGFP.ban (\u2018bantam sensor\u2019, Brennecke et al., 2003), FRT40A, FRT82B (Xu and Rubin, 1993), UAS-GFP-cindr-PC (Johnson et al., 2008), Dachs-GFP (Bosveld et al., 2012)." . _:b558790716 . _:b33392304 "In Fbxl7 mutant clones in the eye imaginal disc, expression of a diap1-GFP reporter gene (Zhang et al., >>2008<<) was increased, especially posterior to the morphogenetic furrow (Figure 1N\u2013N\u2033) consistent with increased Yki activity." . _:b558790749 "2"^^ . _:b33392321 "A region between the conserved blocks \u2018B\u2019 and \u2018C\u2019 seems necessary for the major growth-suppressive function of Ft (Matakatsu and Blair, >>2012<<; Bossuyt et al., 2013; Zhao et al., 2013)." . _:b558790737 . _:b33392367 . _:b558790719 . _:b558790711 . _:b558790748 "2"^^ . _:b33392262 "In an attempt to identify signaling pathways downstream of Ft, several recent studies have made systematic deletions in the intracellular domain (ICD) of Ft (Matakatsu and Blair, >>2012<<; Bossuyt et al., 2013; Pan et al., 2013; Zhao et al., 2013)." . . _:b558790718 . . _:b558790759 . _:b558790751 "2"^^ . _:b558790713 . _:b558790750 "2"^^ . _:b33392254 . _:b33392360 "Another known regulator of apical Ft levels is lowfat (lft) (Mao et al., >>2009<<). Fbxl7 and Lft appear to regulate Ft in different ways." . . _:b558790712 . _:b558790745 "2"^^ . _:b558790767 . _:b558790715 . _:b558790744 "2"^^ . _:b558790714 . _:b558790727 . _:b558790747 "2"^^ . _:b33392294 . _:b558790725 . . _:b558790746 "2"^^ . _:b558790724 . _:b558790757 "2"^^ . _:b558790727 . . _:b558790756 "2"^^ . _:b33392330 . _:b558790726 . _:b558790759 "2"^^ . _:b558790773 . _:b558790721 . _:b33392242 "Ft\u2013Ds interactions are modulated by the kinase Four-Jointed (Fj), which resides in the Golgi and phosphorylates the extracellular domains of both Ft and Ds (Ishikawa et al., 2008; Brittle et al., 2010; Simon et al., >>2010<<). Both Ds and Fj are expressed in gradients in Drosophila imaginal discs where they function in patterning the disc along a major axis (e.g., equatorial to polar or proximodistal) (Yang et al., 2002; Ma et al., 2003)." . _:b33392339 "The overgrowth and lethality of a ft null background (ftGrv/ft8) can be rescued by ubiquitous expression of Ft (Matakatsu and Blair, 2012; Bossuyt et al., 2013; Pan et al., 2013; Zhao et al., >>2013<<; Figure 5\u2014figure supplement 3D,I)." . . _:b558790758 "2"^^ . . _:b558790720 . _:b558790753 "2"^^ . _:b558790723 . _:b558790752 "2"^^ . _:b33392300 . _:b558790722 . _:b558790755 "2"^^ . _:b33392299 "and reduced spacing of the cross veins is especially reminiscent of mutations in the Ft branch of the Hippo signaling pathway (Bryant et al., 1988; Mahoney et al., 1991; Clark et al., 1995; Villano and Katz, 1995; Mao et al., >>2006<<; Matakatsu and Blair, 2008; Mao et al., 2009)." . _:b33392235 "In addition to regulating growth, Ft also regulates planar cell polarity (PCP), oriented cell division and proximodistal patterning of appendages (reviewed in Thomas and Strutt, 2012; Sharma and McNeill, >>2013<<) and its regulated activity therefore impacts the size and shape of organs." . _:b33392278 "ban (\u2018bantam sensor\u2019, Brennecke et al., 2003), FRT40A, FRT82B (Xu and Rubin, 1993), UAS-GFP-cindr-PC (Johnson et al., >>2008<<), Dachs-GFP (Bosveld et al., 2012)." . _:b558790733 . _:b558790754 "2"^^ . _:b558790732 . _:b558790765 "2"^^ . . _:b558790735 . _:b558790764 "2"^^ . _:b558790734 . _:b558790767 "2"^^ . _:b558790729 . _:b558790766 "2"^^ . _:b33392313 "These changes in Fbxl7 localization could simply be a consequence of the more diffuse localization of Ft that is observed in ds clones (Strutt and Strutt, 2002; Ma et al., >>2003<<; Mao et al., 2009)." . _:b558790728 . _:b558790761 "2"^^ . . . _:b558790731 . _:b558790760 "2"^^ . _:b558790730 . _:b558790765 . _:b33392365 "Cindr and its mammalian orthologues Cin85 and CD2AP are thought to regulate interactions between membrane proteins and actin cytoskeleton (Haglund et al., 2002; Petrelli et al., 2002; Soubeyran et al., 2002; Johnson et al., 2011, >>2012<<). D is an atypical myosin with a predicted actin binding domain in its conserved head domain." . _:b558790763 "2"^^ . . _:b558790741 . . _:b33392310 "In cells of the wing imaginal disc, Ft is preferentially expressed on the proximal side of cells and Ds and D on the distal surface (Ambegaonkar et al., 2012; Brittle et al., >>2012<<). We generated small clones that expressed FLAG-Fbxl7, which enabled us to examine the borders between FLAG-Fbxl7-expressing cells and wild-type cells." . _:b558790762 "2"^^ . _:b558790740 . _:b558790773 "2"^^ . . _:b558790743 . _:b33392293 "Consensus sequence definitions according to Bella et al. (2008) and Kobe and Deisenhofer (>>1994<<)." . _:b558790772 "2"^^ . _:b558790742 . . _:b558790775 "2"^^ . . _:b558790737 . _:b558790774 "2"^^ . _:b33392294 "The same alterations in wing area and spacing between the cross veins were also observed when Fbxl7 function was reduced by RNAi (Figure 1L, Figure 1\u2014figure supplement 2A\u2013B) (Dui et al., >>2012<<). The combination of overgrowth and reduced spacing of the cross veins is especially reminiscent of mutations in the Ft branch of the Hippo signaling pathway (Bryant et al., 1988; Mahoney et al., 1991; Clark et al., 1995; Villano and" . _:b558790716 . _:b33392243 "Both Ds and Fj are expressed in gradients in Drosophila imaginal discs where they function in patterning the disc along a major axis (e.g., equatorial to polar or proximodistal) (Yang et al., >>2002<<; Ma et al., 2003)." . _:b558790736 . . . _:b558790769 "2"^^ . _:b558790739 . _:b33392309 . _:b558790768 "2"^^ . _:b33392352 . _:b558790738 . _:b558790771 "2"^^ . _:b33392363 "Cindr and its mammalian orthologues Cin85 and CD2AP are thought to regulate interactions between membrane proteins and actin cytoskeleton (Haglund et al., 2002; Petrelli et al., 2002; Soubeyran et al., >>2002<<; Johnson et al., 2011, 2012)." . _:b33392234 . _:b558790749 . _:b558790770 "2"^^ . _:b33392367 "Fbxl7 has one described substrate in mice, Aurora A (Coon et al., >>2012<<). However we do not believe Aurora A is a relevant substrate in Drosophila, as we do not observe Ft signaling defects when Aurora A is knocked down or overexpressed (not shown)." . _:b33392265 "an attempt to identify signaling pathways downstream of Ft, several recent studies have made systematic deletions in the intracellular domain (ICD) of Ft (Matakatsu and Blair, 2012; Bossuyt et al., 2013; Pan et al., 2013; Zhao et al., >>2013<<). These deletion studies implicate multiple non-overlapping regions in the ICD that differentially affect growth, PCP and organ shape, suggesting that Ft signals via multiple effector pathways." . _:b558790748 . _:b558790781 "2"^^ . _:b33392231 . _:b558790751 . _:b33392303 "Thus Fbxl7 mutant cells do indeed behave as supercompetitors similar to loss-of-function mutations in ft or in core components of the Hippo pathway such as hpo or wts (Tyler et al., >>2007<<)." . _:b558790780 "2"^^ . . _:b33392237 "It has a large extracellular domain composed of 34 cadherin domains as well as 4 EGF-like domains and 2 laminin G domains (Mahoney et al., >>1991<<) that binds to another large cadherin, Dachsous (Ds) (Clark et al., 1995), on adjacent cells (Matakatsu and Blair, 2004)." . _:b33392283 . _:b558790750 . _:b558790783 "2"^^ . _:b33392313 . _:b558790717 . . . _:b558790745 . _:b558790782 "2"^^ . _:b33392290 "LRRs identified using SMART software (Letunic et al., >>2012<<). Black box indicates the Drosophila Fbxl7C616Y amino acid mutation that affects a conserved cysteine." . _:b558790744 . _:b558790777 "2"^^ . . _:b558790747 . _:b558790776 "2"^^ . _:b558790746 . . _:b558790779 "2"^^ . _:b33392317 . _:b558790709 . _:b33392262 . _:b558790757 . _:b558790778 "2"^^ . _:b33392316 . _:b558790756 . _:b33392353 . . _:b558790759 . _:b33392256 . _:b33392333 "2012; H2, Zhao et al., 2013), region required for Dco binding (green) (Sopko et al., 2009), mutV region (orange) (Pan et al., 2013), Su(DN) region (purple) (Matakatsu and Blair, 2012), and two point mutations, ftsum (Bossuyt et al., >>2013<<) and ft61 (this study). Size and position of regions are drawn to scale relative to the ICD." . _:b558790758 . . _:b33392239 "domain composed of 34 cadherin domains as well as 4 EGF-like domains and 2 laminin G domains (Mahoney et al., 1991) that binds to another large cadherin, Dachsous (Ds) (Clark et al., 1995), on adjacent cells (Matakatsu and Blair, >>2004<<). Ft\u2013Ds interactions are modulated by the kinase Four-Jointed (Fj), which resides in the Golgi and phosphorylates the extracellular domains of both Ft and Ds (Ishikawa et al., 2008; Brittle et al., 2010; Simon et al., 2010)." . _:b558790753 . _:b33392361 "trafficking, similar phenotypic abnormalities are observed with gain-of-function and loss-of-function mutations, since the normal execution of the process requires the protein to shuttle efficiently between two states (Park et al., >>1993<<). Thus dynamic aspects of the localization of Ft, Ds and D clearly merit more attention." . _:b558790722 . _:b558790752 . _:b558790785 "2"^^ . . _:b33392282 . _:b558790755 . _:b558790784 "2"^^ . _:b558790754 . _:b558790745 . _:b558790787 "2"^^ . _:b558790765 . . _:b558790786 "2"^^ . _:b558790733 . _:b558790720 . _:b558790764 . . _:b558790767 . _:b33392265 . _:b558790766 . _:b558790746 . _:b558790761 . _:b33392307 . . _:b558790760 . _:b33392230 _:b33392252 . _:b33392230 _:b33392253 . _:b33392318 "Ds binding to Ft induces the phosphorylation of the ICD of Ft, which requires the protein kinase, Dco (Feng and Irvine, 2009; Sopko et al., >>2009<<). Since some F-box proteins bind to phosphorylated proteins (Skaar et al., 2013), we tested whether the apical localization of Fbxl7 was dependent upon Dco function." . _:b33392302 "Since we identified one of the Fbxl7 alleles in a screen for mutations that made cells capable of eliminating their neighbors (Hafezi et al., >>2012<<), we examined imaginal discs for evidence of cell death. We observed elevated levels of activated caspase-3, a marker of apoptosis, especially in wild-type cells adjacent to Fbxl7 mutant clones (Figure 1M\u2013M\u2033)." . _:b33392230 _:b33392254 . _:b558790763 . _:b33392230 _:b33392255 . _:b33392238 . _:b33392230 _:b33392248 . _:b33392230 _:b33392249 . _:b33392230 _:b33392250 . _:b558790777 . _:b558790762 . _:b33392230 _:b33392251 . _:b33392230 _:b33392244 . _:b33392230 _:b33392245 . _:b33392358 "Previous observations of the localization of Ft, Ds, and D to vesicles are suggestive of trafficking events being involved in Ft signaling (Ma et al., 2003; Matakatsu and Blair, 2004; Mao et al., >>2006<<). We have demonstrated that, in addition to the apical membrane, Fbxl7 localizes to vesicles." . _:b33392230 _:b33392246 . _:b558790773 . _:b33392230 _:b33392247 . _:b33392230 _:b33392240 . _:b33392230 _:b33392241 . _:b33392230 _:b33392242 . _:b558790772 . . _:b33392266 "Additionally, several proteins have been shown to bind to the Ft ICD including the transcriptional repressor Atrophin/Grunge which regulates PCP (Fanto et al., >>2003<<), the novel protein Lowfat that regulates Ft protein levels (Mao et al., 2009), and the casein kinase I protein Discs overgrown (Dco) that phosphorylates the Ft ICD (Feng and Irvine, 2009; Sopko et al., 2009)." . _:b33392230 _:b33392243 . _:b558790764 . _:b33392230 _:b33392236 . _:b33392230 _:b33392237 . _:b33392230 _:b33392238 . _:b558790775 . _:b33392230 _:b33392239 . _:b558790775 . _:b33392230 _:b33392232 . _:b558790719 . _:b33392230 _:b33392233 . . _:b558790774 . _:b33392230 _:b33392234 . _:b33392230 _:b33392235 . _:b558790769 . . _:b33392230 _:b33392231 . _:b33392259 "In ft mutants, increased levels of D are observed apically and D is redistributed around the entire perimeter of the cell (Mao et al., 2006; Brittle et al., >>2012<<). However, the overall levels of D protein are not obviously changed (Mao et al., 2006). It has been proposed that Ft restricts growth by negatively regulating the levels of D at the apical membrane and that it regulates the D-dependent" . . _:b558790768 . . _:b558790771 . _:b33392238 "It has a large extracellular domain composed of 34 cadherin domains as well as 4 EGF-like domains and 2 laminin G domains (Mahoney et al., 1991) that binds to another large cadherin, Dachsous (Ds) (Clark et al., >>1995<<), on adjacent cells (Matakatsu and Blair, 2004)." . _:b33392283 "Other plasmids used are pMT-Dco-V5 (Ko et al., >>2002<<), pUAS-Dachs-V5 (Mao et al., 2006)." . . . _:b558790770 . _:b558790758 . _:b33392279 . _:b558790781 . . _:b33392311 "In the dorsal part of the wing pouch, where polarization of D is most evident (Brittle et al., >>2012<<), FLAG-Fbxl7 localizes preferentially to the proximal side of cells (Figure 2J,K\u2013K\u2034)." . _:b558790780 . . _:b558790730 . _:b33392357 "Previous observations of the localization of Ft, Ds, and D to vesicles are suggestive of trafficking events being involved in Ft signaling (Ma et al., 2003; Matakatsu and Blair, >>2004<<; Mao et al., 2006). We have demonstrated that, in addition to the apical membrane, Fbxl7 localizes to vesicles." . _:b558790783 . _:b33392306 . . _:b558790782 . _:b558790777 . _:b33392268 . _:b558790776 . _:b558790751 . . _:b558790779 . . _:b558790778 . _:b33392278 . . . _:b558790776 . . _:b33392328 "domain (TM), regions conserved with mammalian Fat4 (blue, A\u2013F) (defined by Pan et al., 2013), regions associated with the major growth suppressive function of Fat (red) (HM, Bossuyt et al., 2013; Hippo-N, Hippo-C, Matakatsu and Blair, >>2012<<; H2, Zhao et al., 2013), region required for Dco binding (green) (Sopko et al., 2009), mutV region (orange) (Pan et al., 2013), Su(DN) region (purple) (Matakatsu and Blair, 2012), and two point mutations, ftsum (Bossuyt et al., 2013) and" . _:b558790728 . _:b33392258 "In ft mutants, increased levels of D are observed apically and D is redistributed around the entire perimeter of the cell (Mao et al., >>2006<<; Brittle et al., 2012)." . . _:b558790772 . _:b558790785 . _:b558790778 . _:b558790784 . _:b33392252 . _:b558790787 . . _:b33392253 . _:b558790786 . . _:b33392331 "growth suppressive function of Fat (red) (HM, Bossuyt et al., 2013; Hippo-N, Hippo-C, Matakatsu and Blair, 2012; H2, Zhao et al., 2013), region required for Dco binding (green) (Sopko et al., 2009), mutV region (orange) (Pan et al., >>2013<<), Su(DN) region (purple) (Matakatsu and Blair, 2012), and two point mutations, ftsum (Bossuyt et al., 2013) and ft61 (this study)." . . _:b33392308 "to an N-terminal portion of Fbxl7 detects uniform Fbxl7 expression throughout the wing imaginal disc (Figure 2A), with a slight enrichment at the dorsal-ventral boundary in the pouch as is also observed for Ft protein (Mao et al., >>2009<<). At the cellular level, punctate staining is observed outlining the apical profiles of cells, which is absent in homozygous mutant clones of the Fbxl7Q201X allele (Figure 2B\u2013B\u2032) indicating that the truncated protein generated by this" . _:b558790766 . _:b33392336 . _:b558790726 . _:b558790713 . _:b33392356 "Previous observations of the localization of Ft, Ds, and D to vesicles are suggestive of trafficking events being involved in Ft signaling (Ma et al., >>2003<<; Matakatsu and Blair, 2004; Mao et al., 2006). We have demonstrated that, in addition to the apical membrane, Fbxl7 localizes to vesicles." . _:b33392285 . _:b33392303 . . . _:b33392319 . _:b33392280 . . _:b33392318 . _:b33392276 "ban (\u2018bantam sensor\u2019, Brennecke et al., >>2003<<), FRT40A, FRT82B (Xu and Rubin, 1993), UAS-GFP-cindr-PC (Johnson et al., 2008), Dachs-GFP (Bosveld et al., 2012)." . . _:b33392245 . . _:b33392301 "is especially reminiscent of mutations in the Ft branch of the Hippo signaling pathway (Bryant et al., 1988; Mahoney et al., 1991; Clark et al., 1995; Villano and Katz, 1995; Mao et al., 2006; Matakatsu and Blair, 2008; Mao et al., >>2009<<)." . _:b33392273 . _:b33392345 . _:b33392291 "Structural model shows the predicted LRR \u03B2-sheet (arrow) and \u03B1-helix (helix) modeled after (Hsiung et al., >>2001<<). Consensus sequence definitions according to Bella et al. (2008) and Kobe and Deisenhofer (1994)." . . _:b33392233 . _:b33392347 . _:b558790762 . _:b33392295 "The combination of overgrowth and reduced spacing of the cross veins is especially reminiscent of mutations in the Ft branch of the Hippo signaling pathway (Bryant et al., >>1988<<; Mahoney et al., 1991; Clark et al., 1995; Villano and Katz, 1995; Mao et al., 2006; Matakatsu and Blair, 2008; Mao et al., 2009)." . _:b33392276 . _:b33392249 . "PMC0" . . _:b33392250 . _:b33392264 . . _:b33392321 . . _:b33392300 "of the cross veins is especially reminiscent of mutations in the Ft branch of the Hippo signaling pathway (Bryant et al., 1988; Mahoney et al., 1991; Clark et al., 1995; Villano and Katz, 1995; Mao et al., 2006; Matakatsu and Blair, >>2008<<; Mao et al., 2009)." . _:b33392281 . . _:b33392360 . _:b33392317 "Given that Ds can be drawn toward cells with greater levels of Ft (Ma et al., >>2003<<), Ds may be drawn toward Fbxl7-overexpressing cells due to the increased Ft levels." . _:b33392349 . . _:b33392328 . _:b33392359 "transmembrane proteins, and even proteins that are associated with the inner leaflet of the cell membrane are maintained at the plasma membrane by a dynamic process involving endocytosis and vesicle recycling (e.g., Schmick et al., >>2014<<)." . _:b33392242 . _:b33392293 . _:b33392241 . _:b558790721 . . _:b558790725 . _:b33392339 . _:b33392230 . _:b33392332 . _:b33392231 . _:b33392345 "Fbxl7, we examined the localization of FLAG-Fbxl7 with 59 different markers that each labeled a subpopulation of vesicles and with several proteins that have been identified as interactors of Ft in proteomic studies (Kwon et al., >>2013<<) (Supplementary file 1)." . _:b33392257 . _:b33392263 . _:b33392356 . _:b33392290 . _:b33392325 "Additionally, in a ft null background, deletion of one of the conserved blocks (block D in Figure 5A) in a ft genomic rescue transgene was shown to cause overgrowth (Pan et al., >>2013<<) albeit to a much lesser extent than for ft61 and ftsum; flies had slightly overgrown, rounder wings with decreased spacing between the crossveins (Figure 5E)." . _:b33392259 . _:b33392230 _:b33392268 . _:b33392230 _:b33392269 . _:b33392298 "combination of overgrowth and reduced spacing of the cross veins is especially reminiscent of mutations in the Ft branch of the Hippo signaling pathway (Bryant et al., 1988; Mahoney et al., 1991; Clark et al., 1995; Villano and Katz, >>1995<<; Mao et al., 2006; Matakatsu and Blair, 2008; Mao et al., 2009)." . . _:b33392230 _:b33392264 . _:b33392329 . _:b33392230 _:b33392265 . _:b33392230 _:b33392266 . _:b33392335 "Indeed ftGrv/ft8; ftmutV flies also have phenotypic abnormalities that are very similar to those of Fbxl7 mutants (Pan et al., >>2013<<)." . _:b33392236 . _:b33392230 _:b33392267 . _:b33392230 _:b33392260 . _:b558790740 . _:b33392230 _:b33392261 . _:b33392230 _:b33392262 . _:b33392281 "Other antibodies used: rat anti-Dachs (1:500, Brittle et al., 2012), rat anti-Fat (1:1600, Feng and Irvine, 2009), rat anti-Dachsous (1:5000, Yang et al., >>2002<<) rat anti-Ecad (1:100, DCAD2, DHSB, Iowa City, IA), mouse anti-FLAG (1:1000, F3165; Sigma, St." . _:b33392237 . _:b33392230 _:b33392263 . _:b33392230 _:b33392256 . _:b33392230 _:b33392257 . _:b33392230 "introduction" . _:b33392230 _:b33392258 . _:b33392238 . _:b33392230 _:b33392259 . _:b33392239 . _:b33392286 _:b33392287 . _:b33392232 . _:b33392366 . _:b33392233 . . _:b33392286 "results" . _:b558790742 . _:b33392234 . _:b33392277 "ban (\u2018bantam sensor\u2019, Brennecke et al., 2003), FRT40A, FRT82B (Xu and Rubin, >>1993<<), UAS-GFP-cindr-PC (Johnson et al., 2008), Dachs-GFP (Bosveld et al., 2012)." . _:b33392235 . _:b33392272 . _:b33392232 . _:b33392304 . _:b33392244 . _:b558790735 . _:b33392291 . _:b33392245 . _:b33392310 . _:b33392323 . _:b33392246 . _:b33392311 . _:b33392344 "This is expected, as F-box proteins that function in SCF complexes are often themselves ubiquitylated (Galan and Peter, 1999; Yen and Elledge, >>2008<<). Interestingly, Fbxl7C616Y, which is incapable of binding to Ft, is also ubiquitylated suggesting that the incorporation of Fbxl7 into an active SCF complex does not require Ft.10.7554/eLife.03383.017Figure 7.Fbxl7 does not affect Dachs" . _:b33392286 _:b33392316 . _:b33392352 "This point of convergence likely involves D since the overgrowth of ft mutant tissue can be suppressed completely by eliminating D function (Cho et al., >>2006<<). Indeed, it has previously been suggested that Ft regulates growth by restricting the levels of apical D, and regulates PCP by influencing the planar asymmetry of apical D (Rogulja et al., 2008; Pan et al., 2013)." . _:b33392286 _:b33392317 . _:b33392357 . _:b33392286 _:b33392318 . . _:b33392247 . _:b33392286 _:b33392319 . _:b33392286 _:b33392312 . _:b33392286 _:b33392313 . _:b33392298 . _:b33392286 _:b33392314 . _:b33392240 . _:b33392286 _:b33392315 . _:b33392286 _:b33392308 . _:b33392286 _:b33392309 . _:b33392286 _:b33392310 . _:b33392241 . _:b33392286 _:b33392311 . _:b33392342 . _:b33392233 "The Hippo signaling pathway has emerged as a key regulator of organ size (reviewed by Pan, 2010; Halder and Johnson, 2011; Tapon and Harvey, >>2012<<). While most components of this pathway were originally discovered using genetic screens in Drosophila, mammalian orthologs of those genes perform similar functions. Additionally, mutations in several components of the pathway have been" . . _:b33392251 "The strong overgrowth elicited by ft mutations can be completely suppressed by loss of D function (Cho et al., >>2006<<). Additionally, PCP defects in ft mutants are partially rescued by loss of D (Mao et al., 2006). D localizes to the apical membrane where, in cells of the wing disc, it localizes preferentially to the distal edge of the cell (Mao et al.," . _:b33392286 _:b33392304 . _:b33392286 _:b33392305 . _:b33392286 _:b33392306 . _:b33392364 "Cindr and its mammalian orthologues Cin85 and CD2AP are thought to regulate interactions between membrane proteins and actin cytoskeleton (Haglund et al., 2002; Petrelli et al., 2002; Soubeyran et al., 2002; Johnson et al., >>2011<<, 2012). D is an atypical myosin with a predicted actin binding domain in its conserved head domain. Therefore, the vesicles which Fbxl7 associates with D and Cindr may be linked to the actin cytoskeleton. In addition, our finding of" . _:b33392242 . _:b33392286 _:b33392307 . _:b33392286 _:b33392300 . _:b33392286 _:b33392301 . _:b33392286 _:b33392302 . _:b33392243 . . _:b33392286 _:b33392303 . _:b33392286 _:b33392296 . . _:b33392286 _:b33392297 . _:b33392286 _:b33392298 . _:b33392252 . _:b33392286 _:b33392299 . _:b33392286 _:b33392292 . _:b33392286 _:b33392293 . _:b33392286 _:b33392294 . _:b33392253 . _:b33392286 _:b33392295 . _:b33392286 _:b33392288 . _:b33392271 "Additional stocks used were: FRT82B dcole88 (Jursnich et al., >>1990<<), P[acman]-Fat+; P[acman]-Fat\u0394D, P[acman]-Fat\u0394F (Pan et al., 2013), Diap1 3.5-GFP (Zhang et al., 2008), FRT42D fjN7, FRT40A ftGrv, FRT40A ds38k, UAS-Fat (Simon, 2004), ykiB5 (Huang et al., 2005), Tub-EGFP." . _:b33392286 _:b33392289 . _:b33392286 _:b33392290 . _:b558790718 . _:b33392254 . _:b558790750 . _:b33392286 _:b33392291 . _:b33392270 _:b33392284 . _:b558790741 . _:b33392270 _:b33392285 . _:b33392251 . _:b558790707 . _:b33392255 . _:b33392286 _:b33392344 . _:b33392270 _:b33392280 . _:b33392270 _:b33392281 . _:b33392286 _:b33392345 . "10.7554%2FeLife.03383.022" . _:b33392248 . _:b33392270 _:b33392282 . _:b33392270 _:b33392283 . _:b33392270 _:b33392276 . _:b33392286 _:b33392340 . _:b33392270 _:b33392277 . _:b33392286 _:b33392341 . _:b33392286 _:b33392342 . _:b33392249 . _:b33392270 _:b33392278 . _:b33392270 _:b33392279 . _:b33392286 _:b33392343 . _:b33392270 _:b33392272 . _:b33392286 _:b33392336 . _:b33392270 _:b33392273 . _:b33392362 . _:b33392359 . _:b558790715 . _:b33392286 _:b33392337 . _:b558790771 . _:b33392250 . _:b33392286 _:b33392338 . _:b33392270 _:b33392274 . _:b33392270 _:b33392275 . _:b33392286 _:b33392339 . _:b33392286 _:b33392332 . _:b33392363 . _:b33392286 _:b33392333 . _:b33392286 _:b33392334 . _:b33392251 . _:b33392270 _:b33392271 . _:b33392286 _:b33392335 . _:b33392286 _:b33392328 . _:b33392286 _:b33392329 . _:b33392243 . _:b33392286 _:b33392330 . _:b33392260 . _:b33392286 _:b33392331 . _:b33392286 _:b33392324 . _:b33392286 _:b33392325 . _:b33392286 _:b33392326 . _:b33392261 . . _:b33392286 _:b33392327 . _:b33392286 _:b33392320 . _:b33392286 _:b33392321 . _:b33392286 _:b33392322 . _:b33392262 . _:b33392286 _:b33392323 . _:b33392263 . _:b33392269 "Also, the palmitoyltransferase approximated (App) is needed for D localization to the membrane (Matakatsu and Blair, >>2008<<). However, for each of these proteins, their role in mediating the regulation of D levels or asymmetry by Ft is not well understood." . . . _:b33392341 "D is localized at higher levels at the distal edge of the cell (Mao et al., 2006; Brittle et al., >>2012<<) where it is likely stabilized by physical interaction with the cadherin Ds (Bosveld et al., 2012)." . _:b33392256 . _:b33392257 . _:b33392246 . _:b33392258 . _:b33392308 . _:b558790752 . _:b33392259 . . . _:b33392268 . . _:b33392269 . _:b558790744 . . _:b33392309 "In cells of the wing imaginal disc, Ft is preferentially expressed on the proximal side of cells and Ds and D on the distal surface (Ambegaonkar et al., >>2012<<; Brittle et al., 2012). We generated small clones that expressed FLAG-Fbxl7, which enabled us to examine the borders between FLAG-Fbxl7-expressing cells and wild-type cells." . _:b33392270 . _:b558790729 . _:b33392330 "2013), regions associated with the major growth suppressive function of Fat (red) (HM, Bossuyt et al., 2013; Hippo-N, Hippo-C, Matakatsu and Blair, 2012; H2, Zhao et al., 2013), region required for Dco binding (green) (Sopko et al., >>2009<<), mutV region (orange) (Pan et al., 2013), Su(DN) region (purple) (Matakatsu and Blair, 2012), and two point mutations, ftsum (Bossuyt et al., 2013) and ft61 (this study)." . . _:b33392349 "One or more regions between amino acids 4834 and 4899 in full-length Ft appear responsible for Ft\u2019s ability to regulate Hippo signaling (labeled HM in Figure 7M) (Matakatsu and Blair, 2012; Bossuyt et al., 2013; Zhao et al., >>2013<<). Several mutations within this region compromise this function of Ft and cause massive tissue overgrowth (Bossuyt et al., 2013). Intriguingly, an allele of ft, ft61, which harbors such a mutation, showed neither an effect on the" . _:b33392271 . _:b33392275 . _:b558790764 . _:b33392264 . _:b558790765 . _:b33392236 "The Ft protein localizes to the cell membrane just apical to the adherens junctions (Ma et al., >>2003<<). It has a large extracellular domain composed of 34 cadherin domains as well as 4 EGF-like domains and 2 laminin G domains (Mahoney et al., 1991) that binds to another large cadherin, Dachsous (Ds) (Clark et al., 1995), on adjacent cells" . _:b558790766 . _:b33392268 "PCP (Fanto et al., 2003), the novel protein Lowfat that regulates Ft protein levels (Mao et al., 2009), and the casein kinase I protein Discs overgrown (Dco) that phosphorylates the Ft ICD (Feng and Irvine, 2009; Sopko et al., >>2009<<). Also, the palmitoyltransferase approximated (App) is needed for D localization to the membrane (Matakatsu and Blair, 2008)." . _:b558790767 . _:b33392314 . _:b558790760 . _:b33392265 . _:b558790761 . _:b33392279 "ban (\u2018bantam sensor\u2019, Brennecke et al., 2003), FRT40A, FRT82B (Xu and Rubin, 1993), UAS-GFP-cindr-PC (Johnson et al., 2008), Dachs-GFP (Bosveld et al., >>2012<<)." . _:b558790753 . _:b558790762 . . _:b558790763 . _:b558790756 . _:b33392266 . _:b558790757 . . . _:b558790758 . _:b558790759 . _:b558790787 . _:b33392267 . _:b558790752 . _:b558790753 . _:b558790754 . _:b558790755 . . _:b33392276 . _:b558790780 . _:b558790781 . _:b33392341 . _:b33392312 "These changes in Fbxl7 localization could simply be a consequence of the more diffuse localization of Ft that is observed in ds clones (Strutt and Strutt, >>2002<<; Ma et al., 2003; Mao et al., 2009)." . _:b558790782 . _:b558790783 . _:b558790776 . _:b33392277 . _:b558790777 . _:b558790778 . . _:b558790779 . _:b33392235 . _:b33392278 . _:b558790734 . _:b558790772 . _:b558790773 . _:b33392343 . _:b558790774 . _:b558790775 . _:b33392346 "discussion" . _:b33392279 . _:b558790768 . _:b558790769 . _:b558790770 . _:b33392272 "Additional stocks used were: FRT82B dcole88 (Jursnich et al., 1990), P[acman]-Fat+; P[acman]-Fat\u0394D, P[acman]-Fat\u0394F (Pan et al., >>2013<<), Diap1 3.5-GFP (Zhang et al., 2008), FRT42D fjN7, FRT40A ftGrv, FRT40A ds38k, UAS-Fat (Simon, 2004), ykiB5 (Huang et al., 2005), Tub-EGFP." . _:b558790771 . _:b33392297 . _:b558790732 . _:b33392272 . _:b558790733 . _:b558790734 . _:b558790735 . _:b558790728 . _:b33392273 . _:b33392286 . _:b558790729 . _:b33392307 "This non-autonomous increase in Yki activity is similar to that seen when Ft is overexpressed (Matakatsu and Blair, >>2012<<) or at boundaries of differential Ds or Fj activity (Willecke et al., 2008)." . . . _:b558790770 . _:b558790730 . _:b33392254 "D localizes to the apical membrane where, in cells of the wing disc, it localizes preferentially to the distal edge of the cell (Mao et al., 2006; Mao et al., >>2011<<; Ambegaonkar et al., 2012; Bosveld et al., 2012; Brittle et al., 2012)." . _:b558790731 . _:b558790724 . _:b33392274 . _:b558790725 . _:b33392274 "Additional stocks used were: FRT82B dcole88 (Jursnich et al., 1990), P[acman]-Fat+; P[acman]-Fat\u0394D, P[acman]-Fat\u0394F (Pan et al., 2013), Diap1 3.5-GFP (Zhang et al., 2008), FRT42D fjN7, FRT40A ftGrv, FRT40A ds38k, UAS-Fat (Simon, >>2004<<), ykiB5 (Huang et al., 2005), Tub-EGFP.ban (\u2018bantam sensor\u2019, Brennecke et al., 2003), FRT40A, FRT82B (Xu and Rubin, 1993), UAS-GFP-cindr-PC (Johnson et al., 2008), Dachs-GFP (Bosveld et al., 2012)." . _:b558790726 . _:b558790727 . _:b558790774 . _:b558790720 . _:b33392275 . _:b558790721 . . _:b558790722 . _:b558790782 . _:b558790723 . _:b558790748 . _:b33392284 . _:b558790749 . _:b558790750 . _:b558790751 . . _:b33392285 . _:b33392270 . _:b558790744 . _:b558790745 . _:b558790746 . _:b558790747 . _:b558790740 . _:b558790741 . _:b33392333 . _:b33392331 . _:b33392286 . . _:b33392295 . _:b558790742 . . _:b558790743 . _:b558790736 . _:b33392287 . _:b558790737 . _:b558790738 . _:b558790739 . _:b33392280 . _:b558790755 . . _:b33392281 . _:b33392334 . _:b33392301 . _:b33392361 . _:b33392282 . _:b33392335 . _:b33392247 . _:b33392283 . . _:b33392320 . _:b558790749 . _:b558790716 . _:b33392292 . _:b558790717 . _:b558790718 . _:b558790719 . _:b33392247 "First, Ft influences the protein levels of Warts (Wts), a kinase that regulates the activity and subcellular location of the pro-growth transcriptional co-activator Yorkie (Yki) (Cho et al., 2006; Rauskolb et al., >>2011<<). Additionally, mutations in ft disrupt the localization of Expanded (Ex), a FERM-domain protein that functions upstream of Hippo (Hpo) (Bennett and Harvey, 2006; Silva et al., 2006; Willecke et al., 2006), though other studies suggest Ft" . _:b33392293 . _:b558790712 . _:b558790784 . _:b33392324 . _:b558790713 . _:b558790714 . . _:b558790715 . _:b558790708 . _:b33392294 . _:b558790709 . _:b33392329 "with mammalian Fat4 (blue, A\u2013F) (defined by Pan et al., 2013), regions associated with the major growth suppressive function of Fat (red) (HM, Bossuyt et al., 2013; Hippo-N, Hippo-C, Matakatsu and Blair, 2012; H2, Zhao et al., >>2013<<), region required for Dco binding (green) (Sopko et al., 2009), mutV region (orange) (Pan et al., 2013), Su(DN) region (purple) (Matakatsu and Blair, 2012), and two point mutations, ftsum (Bossuyt et al., 2013) and ft61 (this study)." . . _:b558790710 . . _:b558790711 . _:b558790785 . _:b33392295 . _:b558790704 . _:b558790705 . . _:b558790706 . _:b558790707 . _:b33392288 . _:b33392325 . _:b33392327 . _:b33392289 . _:b33392326 . _:b33392326 "(A) Protein model of the intracellular domain of Fat showing the transmembrane domain (TM), regions conserved with mammalian Fat4 (blue, A\u2013F) (defined by Pan et al., >>2013<<), regions associated with the major growth suppressive function of Fat (red) (HM, Bossuyt et al., 2013; Hippo-N, Hippo-C, Matakatsu and Blair, 2012; H2, Zhao et al., 2013), region required for Dco binding (green) (Sopko et al., 2009)," . _:b33392346 . _:b33392290 . . _:b33392365 . _:b33392261 "It has been proposed that Ft restricts growth by negatively regulating the levels of D at the apical membrane and that it regulates the D-dependent PCP functions by maintaining D asymmetry (Rogulja et al., >>2008<<)." . _:b33392291 . _:b33392322 . _:b33392287 . _:b33392300 . _:b33392237 . _:b33392301 . _:b33392315 "Fj is required for normal localization of Ds and Ft (Strutt and Strutt, >>2002<<; Ma et al., 2003)." . . _:b33392348 . _:b33392319 "The apical localization of Fbxl7 was not obviously changed in clones of the dco3 allele that is unable to phosphorylate Ft (Figure 4H\u2013H\u2033) (Sopko et al., >>2009<<). While Dco is capable of binding to Fbxl7 as assessed by co-immunoprecipitation from S2 cells (Figure 4J), the apical localization of Fbxl7 was still observed in clones of the null dco allele, dcole88 (Figure 4I\u2013I\u2033), thus indicating that" . _:b33392302 . _:b33392277 .