The Spectrin cytoskeleton regulates the Hippo signalling pathway

Article23 February 2015Open Access The Spectrin cytoskeleton regulates the Hippo signalling pathway Georgina C Fletcher Georgina C Fletcher Epithelial Biology Laboratory, Cancer Research UK – London Research Institute, London, UK Search for more papers by this author Ahmed Elbediwy Ahmed Elbediwy Epithelial Biology Laboratory, Cancer Research UK – London Research Institute, London, UK Search for more papers by this author Ichha Khanal Ichha Khanal Epithelial Biology Laboratory, Cancer Research UK – London Research Institute, London, UK Search for more papers by this author Paulo S Ribeiro Paulo S Ribeiro Apoptosis and Cell Proliferation Laboratory, Cancer Research UK – London Research Institute, London, UK Barts Cancer Institute, Queen Mary University of London, London, UK Search for more papers by this author Nic Tapon Corresponding Author Nic Tapon Apoptosis and Cell Proliferation Laboratory, Cancer Research UK – London Research Institute, London, UK Search for more papers by this author Barry J Thompson Corresponding Author Barry J Thompson Epithelial Biology Laboratory, Cancer Research UK – London Research Institute, London, UK Search for more papers by this author Georgina C Fletcher Georgina C Fletcher Epithelial Biology Laboratory, Cancer Research UK – London Research Institute, London, UK Search for more papers by this author Ahmed Elbediwy Ahmed Elbediwy Epithelial Biology Laboratory, Cancer Research UK – London Research Institute, London, UK Search for more papers by this author Ichha Khanal Ichha Khanal Epithelial Biology Laboratory, Cancer Research UK – London Research Institute, London, UK Search for more papers by this author Paulo S Ribeiro Paulo S Ribeiro Apoptosis and Cell Proliferation Laboratory, Cancer Research UK – London Research Institute, London, UK Barts Cancer Institute, Queen Mary University of London, London, UK Search for more papers by this author Nic Tapon Corresponding Author Nic Tapon Apoptosis and Cell Proliferation Laboratory, Cancer Research UK – London Research Institute, London, UK Search for more papers by this author Barry J Thompson Corresponding Author Barry J Thompson Epithelial Biology Laboratory, Cancer Research UK – London Research Institute, London, UK Search for more papers by this author Author Information Georgina C Fletcher1,‡, Ahmed Elbediwy1,‡, Ichha Khanal1,‡, Paulo S Ribeiro2,3,‡, Nic Tapon 2 and Barry J Thompson 1 1Epithelial Biology Laboratory, Cancer Research UK – London Research Institute, London, UK 2Apoptosis and Cell Proliferation Laboratory, Cancer Research UK – London Research Institute, London, UK 3Barts Cancer Institute, Queen Mary University of London, London, UK ‡These authors contributed equally to this work *Corresponding author. E-mail: [email protected] *Corresponding author. Tel: +44 207 269 3353; E-mail: [email protected] The EMBO Journal (2015)34:940-954https://doi.org/10.15252/embj.201489642 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The Spectrin cytoskeleton is known to be polarised in epithelial cells, yet its role remains poorly understood. Here, we show that the Spectrin cytoskeleton controls Hippo signalling. In the developing Drosophila wing and eye, loss of apical Spectrins (alpha/beta-heavy dimers) produces tissue overgrowth and mis-regulation of Hippo target genes, similar to loss of Crumbs (Crb) or the FERM-domain protein Expanded (Ex). Apical beta-heavy Spectrin binds to Ex and co-localises with it at the apical membrane to antagonise Yki activity. Interestingly, in both the ovarian follicular epithelium and intestinal epithelium of Drosophila, apical Spectrins and Crb are dispensable for repression of Yki, while basolateral Spectrins (alpha/beta dimers) are essential. Finally, the Spectrin cytoskeleton is required to regulate the localisation of the Hippo pathway effector YAP in response to cell density human epithelial cells. Our findings identify both apical and basolateral Spectrins as regulators of Hippo signalling and suggest Spectrins as potential mechanosensors. Synopsis In Drosophila, both the apical and the basolateral Spectrin cytoskeleton are able to activate the Hippo signaling pathway in a tissue-dependent manner. In cultured human cells this depends on cell density, suggesting a role for Spectrins in mechanosensing. The Spectrin cytoskeleton is required for Hippo signalling in Drosophila. Loss of Spectrins causes mild tissue overgrowth. Apical Spectrins bind to Expanded, Merlin and Kibra. Spectrins are potential mechanosensors. Introduction The Hippo pathway transduces signals from the cell surface to the nucleus to control tissue growth and regeneration in animals (Pan, 2010; Halder & Johnson, 2011; Tapon & Harvey, 2012). Recent work implicates the Hippo pathway as a key regulator of stem cell proliferation (Camargo et al, 2007; Cai et al, 2010; Karpowicz et al, 2010; Shaw et al, 2010; Staley & Irvine, 2010; Zhang et al, 2010, 2011; Cordenonsi et al, 2011). The core of the pathway was discovered in Drosophila and includes the upstream kinase Hippo (MST1/2 in mammals) and the downstream kinase Warts (LATS1/2 in mammals), which acts to phosphorylate and inhibit the transcriptional activator Yorkie (Yki; YAP/TAZ in mammals) (Harvey et al, 2003; Udan et al, 2003; Wu et al, 2003; Huang et al, 2005). Yki then acts with the Mask (MASK1/2 in mammals) co-factor to switch the nuclear DNA-binding protein Scalloped (TEAD1-4 in mammals) from a transcriptional repressor to an activator (Wu et al, 2008; Koontz et al, 2013; Sansores-Garcia et al, 2013; Sidor et al, 2013). Several proteins can act upstream of the core kinase cascade, including the apical FERM-domain proteins Expanded (Ex; similar to both FRMD6 and AMOT proteins in humans) and Merlin (Mer; the NF2 tumour suppressor in humans), which act in parallel with activate Hippo signalling (Hamaratoglu et al, 2006; Irvine, 2012). In Drosophila wing or eye epithelia, mutation of ex is sufficient to cause mild tissue overgrowth, but ex, mer double mutants cause a much stronger overgrowth phenotype, similar to hpo or wts mutants (Hamaratoglu et al, 2006). Ex is recruited to the membrane by the transmembrane protein Crumbs (Crb), an apical polarity determinant that can form apical cell–cell junctions in epithelia (Chen et al, 2010; Ling et al, 2010; Robinson et al, 2010). Mutants in crb therefore cause a mild overgrowth phenotype in wing and eye epithelia (Chen et al, 2010; Ling et al, 2010). Mer is also recruited to apical cell–cell junctions, where it binds to the Kibra (Kib) protein (Baumgartner et al, 2010; Genevet et al, 2010; Yu et al, 2010). ex, kib or kib, mer double mutants cause a strong hpo-like overgrowth phenotype, suggesting that the three proteins act together upstream of the core kinase cascade (Baumgartner et al, 2010; Genevet et al, 2010; Yu et al, 2010). In addition, ex, kib double mutants strongly affect polarisation of Crb in the ovarian follicular epithelium and polarisation of the actin cytoskeleton for border cell migration, functions that are independent of nuclear signalling via Yki (Fletcher et al, 2012; Lucas et al, 2013). In mammalian cells in culture, the Hippo pathway responds to mechanical stimulation, being activated in densely confluent epithelial cells (such that YAP is cytoplasmic) and becoming inactivated when cells are sparse and stretched out across their substrate (such that YAP becomes nuclear) (Dupont et al, 2011; Aragona et al, 2013). A functioning F-actin cytoskeleton is required for this response to mechanical stimulation, but whether F-actin itself is the molecular mechanosensor or whether other molecules might mediate this function remains unclear (Dupont et al, 2011; Aragona et al, 2013). Interestingly, regulation of YAP by mechanical stretching and the F-actin cytoskeleton appears to be partly independent of LATS phosphorylation of YAP and likely involves a yet unidentified mechanism (Dupont et al, 2011; Aragona et al, 2013; Gaspar & Tapon, 2014). Here, we identify the Spectrin cytoskeleton as crucial upstream regulator of Yki in the developing wing, eye, follicular epithelium and border cells. We further show that human Spectrins are essential for regulation of YAP in response to cell density in human cells. Results To identify novel regulators of Hippo signalling, we performed an in vivo RNAi screen in the Drosophila wing for novel genes controlling tissue growth (M. Campos & B. J. Thompson, manuscript in preparation). In this screen, we identified the apical Spectrin cytoskeleton components α-Spectrin (α-Spec) and β-heavy Spectrin (βHSpec)—also known as Karst (Kst)—as producing moderate wing and eye overgrowth phenotypes, similar to RNAi knock-down of Crb (Fig 1A–F and Supplementary Figs S1 and S2). Spectrins are large cytoskeletal proteins that form hexagonal networks at the intracellular surface of the plasma membrane in all animal cells and have been reported to have mechanosensory properties (Bennett & Baines, 2001; Johnson et al, 2007; Bennett & Healy, 2009; Stabach et al, 2009; Meng & Sachs, 2012; Krieg et al, 2014). The Spectrin cytoskeleton is polarised in Drosophila epithelia, with dimers of α- and βH-Spec/Kst localising to the apical domain and dimers of α- and β-Spec localising to the basolateral domain (Thomas & Kiehart, 1994; Lee et al, 1997; Thomas et al, 1998; Thomas & Williams, 1999; Zarnescu & Thomas, 1999; Medina et al, 2002). Notably, RNAi knock-down of the basolateral β-Spec did not have a consistent effect on eye or wing size (Fig 1G and H and Supplementary Figs S3 and S4). Furthermore, just as crb and ex mutants are known to genetically interact with kib, knock-down of α-Spec or βH-Spec/Kst strongly enhanced the overgrowth caused by a kib null mutant in the eye (Fig 1I–R). Figure 1. The Spectrin cytoskeleton restricts tissue growth in the Drosophila eye and wing A–O. UAS.RNAi lines were driven with eyeless.Gal4 gmr.Gal4 for expression during eye development or nubbin-Gal4 for expression during wing development. (A, B) Control adult Drosophila eye (A) and wing (B). (C, D) α-spectrin RNAi results in overgrowth of the eye (C) and wing (D). (E, F) βH-spectrin/karst RNAi results in overgrowth of the eye (E) and wing (F). (G, H) β-spectrin RNAi does not affect eye size (G) or wing size (H). (I, J) kibra RNAi results in overgrowth of the eye (I) and wing (J). (K, L) α-spectrin, kibra double RNAi results in stronger overgrowth of the eye (K) and wing (L). (M, N) βH-spectrin/karst, kibra double RNAi results in stronger overgrowth of the eye (M) and wing (N). (O) Quantification of female wing sizes by pixel area, 5 wings per genotype were measured. Error bars show standard deviation. P–R. The eyeless FLP MARCM system was used to generate clonally mutant fly eyes. kibra mutant eyes (Q) overgrow slightly compared to controls (P), while kibra mutant eyes expressing α-spectrin RNAi (R) overgrow strongly compared to controls (P). Data information: Scale bars, 250 μm. Download figure Download PowerPoint Despite previous reports that apical βH-Spec/Kst interacts physically with Crb, genetic analysis of βH-spec/kst mutants indicated that it is dispensable for polarisation of Crb and for epithelial polarity in general (Thomas et al, 1998; Zarnescu & Thomas, 1999; Medina et al, 2002; Pellikka et al, 2002). Since Crb is known to regulate Hippo signalling (Chen et al, 2010; Ling et al, 2010; Robinson et al, 2010), we tested whether loss of apical Spectrins also affects Hippo signalling outputs. We examined interommatidial cells in the pupal retina and found that loss of α-Spec or βH-Spec/Kst increases cell number and this effect is magnified by concominant mutation of kib (Fig 2A–F). We also examined the expression of the key Hippo reporter gene, ex.lacZ, in wing discs expressing α-spec RNAi in the posterior compartment with hh.Gal4. We found that, compared to controls, wing discs expressing α-spec RNAi exhibit a slightly elevated level of ex.lacZ expression in the posterior compartment (Fig 2G and H). This elevation of ex.lacZ expression is similar in magnitude to that caused by kib RNAi and becomes stronger in α-spec, kib double RNAi wing discs, similar to hpo RNAi (Fig 2I–K). These results show that apical Spectrins regulate Yki activity in the Drosophila wing and eye. They also show that Spectrins act in parallel with Kibra, in the same manner as Ex (Baumgartner et al, 2010), as the double-mutant spectrin kibra or expanded kibra each cause a stronger phenotype than the single mutants alone (Baumgartner et al, 2010). Figure 2. The Spectrin cytoskeleton represses interommatidial cell number and Hippo target gene expression in parallel with Kibra A–F. The eyeless FLP MARCM system was used to generate mutant eyes that also express UAS.RNAi targeting the Spectrin cytoskeleton. Pupal retinas were examined at 42–46 h after puparium formation (APF). Cell membranes were marked with Dlg staining. (A) Control pupal retina showing cone cells surrounded by interommatidial cells. (B) kibra mutant pupal retina displaying additional interommatidial cells. (C) Pupal retina expressing α-spectrin RNAi showing additional interommatidial cells. (D) kibra mutant pupal retinas expressing α-spectrin RNAi showing many additional interommatidial cells. (E, F) Clones of α-spectrin mutant cells (GFP negative) (E) and βH-spectrin/karst mutant cells (GFP negative) (F) show extra interommatidial cells. Scale bars, 20 μm. G–K. UAS.RNAi lines were driven with hh.Gal4 UAS.GFP for expression in the posterior compartment and contained the ex.lacZ reporter transgene. (G) Control wing showing a normal ex.lacZ expression pattern, which is low at the dorsal–ventral boundary but high in the proximal regions of the wing disc. (H) RNAi knock-down of α-Spectrin results in a mild up-regulation of ex.lacZ in the posterior compartment. (I) RNAi knock-down of kibra results in a mild up-regulation of ex.lacZ in the posterior compartment. (J) Dual RNAi knock-down of both α-spectrin and kibra results in enhanced up-regulation of ex.lacZ in the posterior compartment. (K) RNAi knock-down of hippo results in an up-regulation of ex.lacZ in the posterior compartment. Scale bars, 50 μm. Download figure Download PowerPoint We next investigated the mechanism by which apical Spectrins regulate Yki activity. Since Spectrins act in parallel with Kibra—similar to Ex (Baumgartner et al, 2010)—and have been shown to physically associate with Crb (Medina et al, 2002; Pellikka et al, 2002)—again similar to Ex (Chen et al, 2010; Ling et al, 2010; Robinson et al, 2010)—we tested whether Ex might interact with Spectrins. We performed co-immunoprecipitation experiments from Drosophila S2 cells expressing V5-tagged Ex and a series of constructs expressing portions of the very large βH-Spec/Kst protein. We found that Ex interacts strongly with the N-terminal region of βH-Spec/Kst (Fig 3A). Pulling down the N-terminal region of βH-Spec/Kst with Ex also co-immunoprecipitated endogenous α-Spec, which is known to form dimers with βH-Spec/Kst (Fig 3A). In vivo, we found that βH-Spec/Kst co-localises with Ex at the apical domain of wing disc epithelial cells (Fig 3B and C). α-Spec or βH-Spec/Kst is not required to localise Ex or Crb apically (Supplementary Fig S5 and data not shown). Instead, they appear to be required for normal activation of Ex signalling to Hpo and Wts, as the tissue overgrowth phenotype of α-Spec or βH-Spec/Kst knock-down is completely suppressed by overexpression of Ex (Fig 3D–I). These results show that apical Spectrins bind to and co-localise with Ex and act genetically upstream or at the level of Ex to regulate signalling to Yki. Figure 3. The apical α-βH Spectrin cytoskeleton binds to and co-localises with Expanded protein and acts genetically upstream or at the level of Expanded to control wing growth A. Co-IP of V5-tagged Expanded with the FLAG-tagged N-terminal region of Karst/βH-Spectrin, but not other Karst truncation constructs. B, C. Expanded co-localises with Karst at the apical membrane of wing imaginal disc cells (apical view, B; cross section, C). D. Control nubbin.Gal4 expressing wing. E. Overexpression of Ex activates Hippo signalling to produce a small wing. F. RNAi knock-down of α-Spectrin results in an overgrown wing. G. Overexpression of Ex blocks the effect of α-Spectrin RNAi. H. RNAi knock-down of βH-spectrin/karst results in an overgrown wing. I. Overexpression of Ex blocks the effect of βH-spectrin/karst RNAi. Data information: Scale bars, 10 μm (B, C), 250 μm (D–I). Download figure Download PowerPoint Hippo signalling has been proposed to have a possible mechanosensory role in the Drosophila wing disc, where a pattern of stretching and compression of cells at their apical surfaces correlates with the pattern of Yki activity as measured with ex.lacZ (Fig 4A and B; Aegerter-Wilmsen et al, 2007; Legoff et al, 2013; Mao et al, 2013; Schluck et al, 2013). We found that this pattern of stretching and compression influences the intensity of Crb and apical Spectrin staining in cells (Fig 4A–F). Thus, Crb and βH-Spec/Kst are concentrated at the junctions of small compressed cells and diluted at the junctions of stretched cells in a manner that inversely correlates with ex.lacZ expression. This correlation suggests a potential model of mechanosensory regulation of Yki activity via Spectrin-dependent clustering of Crb complexes (Fig 4G). According to this model, stretching of cells would exert force upon the apical Spectrin cytoskeleton that would de-cluster Crb complexes and therefore reduce Hpo and Wts activation and increase Yki activity (Fig 4G; see also Discussion). Figure 4. The apical α-βH Spectrin cytoskeleton may be mechanosensory in the wing imaginal disc A. Schematic diagram of central compression and circumferencial stretching in the third instar wing pouch. B–D. Third instar wing imaginal disc stained for beta-galactosidase expressed from the expanded.lacZ reporter gene (B), Crb (C) and Kst-YFP (D). Quantification of line intensity shown below. Note inverse correlation of Crb and Kst-YFP with ex.lacZ. Scale bars, 50 μm. E, F. Zoom of Kst-YFP cells under compression (E) and under stretch (F). Scale bars, 10 μm. G. Schematic diagram of the effect of force on the apical spectrin cytoskeleton leading to declustering of Crumbs complexes and reduced Hippo signalling. Download figure Download PowerPoint To test this model, we aimed to induce clustering of Crb complexes by overexpression of a form of Crb whose intracellular domain was replaced with GFP (CrbExTM-GFP; Fig 5A) (Pellikka et al, 2002; Thompson et al, 2013). Overexpression of CrbExTM-GFP during wing development with nub.Gal4 resulted in a small wing phenotype, highly similar to overexpression of Wts (Fig 5B–D). Furthermore, the overgrowth phenotype caused by RNAi knock-down of apical Spectrins was completely suppressed by co-expression of either CrbExTM-GFP or Wts (Fig 5E–J). CrbExTM-GFP appears to act upstream of Wts, because the tissue undergrowth phenotype induced by CrbExTM-GFP is suppressed by RNAi knock-down of Wts (Fig 5K and L). These results are consistent with the notion that clustering of Crb complexes induces Hippo signalling to inhibit tissue growth, lending support to a model of mechanosensation involving clustering of Crb complexes. However, other interpretations and models of mechanosensation are also possible. Figure 5. CrbExTM-GFP restricts tissue growth and acts upstream of Wts and in parallel with Ajuba A. Schematic diagram of clustering of Crb complexes by expression of CrbExTM-GFP. B. Control nub.Gal4 wing. C, D. Expression of UAS.CrbExTM-GFP with nub.Gal4 (C) or UAS.Wts with nub.Gal4 (D) results in a small wing. E. RNAi knock-down of α-Spectrin results in an overgrown wing. F, G. Expression of UAS.CrbExTM-GFP (F) or UAS.Wts (G) blocks the effect of α-Spectrin RNAi. H. RNAi knock-down of βH-spectrin/karst results in an overgrown wing. I, J. Expression of UAS.CrbExTM-GFP (I) or UAS.Wts (J) blocks the effect of α-Spectrin RNAi. K, L. RNAi knock-down of Wts results in a strongly overgrown wing (K) and expression of UAS.CrbExTM-GFP does not block the effect of wts RNAi (L). M, N. RNAi knock-down of Ajuba results in a small wing (M), and expression of UAS.CrbExTM-GFP enhances the effect of ajuba RNAi (N). O. Quantification of various wing sizes. Error bars show standard deviation. P, Q. Localisation of Wts-GFP (P) and E-cadherin (Q) in the third instar wing imaginal disc. R. Quantification of line intensity in (P) and (Q). No increased Wts-GFP intensity is observed in proximal regions where cells are subject to stretching. Data information: Scale bars, 250 μm (B–N), 40 μm (P, Q). Download figure Download PowerPoint One alternative model of mechanosensation involves recruitment of Warts to E-cadherin via the Ajuba protein (Rauskolb et al, 2014). This mechanism appears to be distinct from and to act in parallel with the Spectrin/Crumbs-mediated version we propose, because loss of Ajuba and overexpression of CrbExTM-GFP have an additive effect in suppressing tissue growth (Fig 5M–O). Furthermore, we do not observe increased recruitment of Warts-GFP to E-cadherin in response to tissue stretching in the wing imaginal disc (Fig 5P–R), suggesting that this alternative model does not explain the physiological control of Hippo signalling in this context. A second alternative model of mechanosensation involves activation of the JNK pathway by forces (Codelia et al, 2014). However, blocking JNK signalling in Drosophila does not affect tissue growth, and there is no evidence for physiological regulation of JNK activation by forces in the wing imaginal disc. A third alternative model of mechanosensation involves the actin cytoskeleton, which can directly influence the nuclear localisation of the Yki homologues YAP and TAZ in mammalian cell culture independently of MST and LATS kinases (Dupont et al, 2011; Aragona et al, 2013). Current evidence suggests that all effects of the actin cytoskeleton on Yki activity appear to be mediated via Hpo and Wts (Sansores-Garcia et al, 2011), although further work is needed to rigorously test whether Wts-independent regulation of Yki also occurs in Drosophila (Gaspar & Tapon, 2014). Thus, we currently favour the view that apical Spectrins act with Crb complexes to help sense forces by activating Hpo-Wts signalling during Drosophila wing and eye development. We next tested whether loss of Spectrins can produce an ex-like phenotype in other tissue contexts. In the ovarian follicular epithelium, Ex is known to act in parallel with Kibra to regulate polarisation of Crb, such that ex, kib double-mutant cells accumulate Crb in vesicles (Fletcher et al, 2012). This role of Ex and Kibra does not require more downstream signalling components such as wts (Fletcher et al, 2012). We found that βH-Spec/Kst co-localises with Ex at the apical domain of follicle cells, while Kibra is present both apically and in the cytoplasm in a punctate pattern that co-localises with the Exocyst complex—with which Kibra has been shown to interact (Rosse et al, 2009) (Supplementary Fig S6A–C). We found that loss of apical Spectrins alone does not affect Crb, but loss of apical Spectrins in combination with mutation of kib or sec15 (encoding an Exocyst component) causes a strong accumulation of Crb in vesicles (Supplementary Fig S6D–M). Thus, apical Spectrins are involved in Ex-mediated regulation of Crb polarisation in follicle cells. We noted that α-spec RNAi in kib mutant clones caused a strong overproliferation and multilayering phenotype in follicle cells (Supplementary Fig S6M). We therefore tested whether Spectrins are required for regulation of Yki activity in the ovarian follicular epithelium. Yki is known to promote early proliferation and suppress apoptosis in follicle cells until stage 6, during which time it expresses the transcription factor Cut (Huang & Kalderon, 2014). After stage 6, Cut expression is silenced and cells arrest proliferation and switch to an endocycle. When Hippo signalling is disrupted after stage 6, Cut is known to be re-expressed in a group of posterior follicle cells that continue to proliferate (Meignin et al, 2007; Polesello & Tapon, 2007; Genevet et al, 2010; Yu et al, 2010). Surprisingly, we found that mutation of either βH-spec/kst or crb is not sufficient to drive early overproliferation (Fig 6A–D) or to induce Cut in late-stage posterior follicle cells (Fig 6G–I). Multilayering only occurs in around 30% of crb mutant clones, while mutation of α-spec or β-spec leads to early overproliferation and multilayering in 95% of clones (Fig 6E and F) and also leads to re-expression of Cut in the posterior follicle cells (Fig 6J and K). Furthermore, another Hippo target gene, ex.lacZ, is also ectopically expressed in posterior follicle cells mutant for α-spec or β-spec but not in βH-spec/kst or crb mutant clones (Fig 6L–P). Notably, mutation of β-spec causes a disruption of membrane tension such that the normally regular hexagonal shape of follicle cells becomes disorganised (Fig 6Q–S). These findings indicate that the basolateral α-β Spectrin cytoskeleton controls membrane tension and Hippo signalling to the nucleus in the follicular epithelium. Figure 6. Basolateral α-β Spectrins are required for Hippo signalling and membrane tension in the Drosophila follicular epithelium A. Control egg chamber stained for DAPI to mark nuclei. B. Overexpression of Yki3SA stimulates follicle cell proliferation (MARCM clone, GFP positive). C–F. Mutation of βH-spectrin/karst (C) or crb (D) does not increase follicle cell proliferation (GFP negative clone), while mutation of α-spectrin (E) or β-spectrin (F) stimulates follicle cell proliferation (GFP negative clone). G. Control egg chamber stained for Cut, a marker of proliferating cells that is normally down-regulated after stage 6 of oogenesis. H–K. Mutation of βH-spectrin/karst (H) or crb (I) does not increase Cut expression in follicle cells (GFP negative clone), while mutation of α-spectrin (J) or β-spectrin (K) stimulates Cut expression in posterior follicle cells (GFP negative clone). L. Control egg chamber showing ex.lacZ reporter gene expression. M–P. Mutation of βH-spectrin/karst (M) or crb (N) does not increase ex.lacZ expression in follicle cells (GFP negative clone), while mutation of α-spectrin (O) or β-spectrin (P) stimulates ex.lacZ expression in posterior follicle cells (GFP negative clone). Q. Wild-type follicle cells showing normal hexagonal packing of epithelial membranes. R. Mutation of β-spectrin disrupts the normal hexagonal packing, suggesting a role in maintaining membrane tension. S. Clone of β-spectrin mutant cells (GFP negative) showing differential membrane tension at the interface with wild-type cells (GFP positive). Data information: Scale bars, 25 μm (A–R), 10 μm (S). Download figure Download PowerPoint A small group of anterior follicle cells, called border cells, are known to delaminate from the epithelium at stage 8 of development and migrate across the egg chamber to reach the oocyte at the posterior by stage 10. We previously showed that upstream Hippo pathway components have a key role in promoting border cell migration by regulating the actin cytoskeleton (Lucas et al, 2013). We found that α-spec RNAi knock-down in kib mutant border cell clusters causes a strong delay in reaching the oocyte by stage 10 of oogenesis (Supplementary Fig S7A–E). α-spec RNAi knock-down alone, or mutation of kib alone, causes a milder phenotype (Supplementary Fig S7E). Interestingly, RNAi knock-down of βH-spec/kst did not cause a phenotype, alone or in combination with kib (Supplementary Fig S7E). Accordingly, we found that βH-Spec/Kst is not actually expressed in border cells, while β-Spec localises around the entire plasma membrane with α-Spec (Supplementary Fig S7F–I). These results indicate that α-β Spectrin dimers regulate Hippo signalling during border cell migration. The Hippo pathway was recently shown to have a key role in regulating stem cell proliferation in the Drosophila intestine (Karpowicz et al, 2010; Shaw et al, 2010; Staley & Irvine, 2010). RNAi knock-down of wts or overexpression of yki in enterocytes with the myo1A.G4 driver leads to induction of stem cell proliferation (Karpowicz et al, 2010; Shaw et al, 2010; Staley & Irvine, 2010). However, the upstream regulators that control Hippo signalling in the intestine have not been identified. We therefore examined the requirement for Crb and Spectrins in the adult fly intestine. We found that RNAi of crb or apical βH-spec/kst did not lead to stem cell overproliferation (Fig 7A–C). However, RNAi silencing of α-spec or β-spec does produce a strong stem cell proliferation response, similar to overexpression of Yki (Fig 7D–G). Furthermore, a Hippo pathway reporter gene DIAP1-HRE-GFP is activated in the overproliferating stem cells (Fig 7H–K). These results indicate that the basolateral α-β Spectrin cytoskeleton, rather than the apical α-βH-Spectrin cytoskeleton, is crucial to promote Yki activity in enterocytes. Figure 7. Basolateral α-β Spectrins are required to restrict cell proliferation in the Drosophila intestinal epithelium A. Control myo1A.Gal4 UAS.GFP adult midgut stained for phospho-histone H3 to mark mitotic stem cells. B–E. RNAi knock-down of βH-spectrin/karst (B) or crumb