Crumbs organizes the transport machinery by regulating apical levels of PI(4,5)P2 in Drosophila

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Version of Record published: November 27, 2019 (This version)
Accepted Manuscript published: November 7, 2019 (Go to version)
Accepted: October 25, 2019
Received: August 6, 2019

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Jini Sugatha, Amulya Priya ... Sunando Datta

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Abstract

An efficient vectorial intracellular transport machinery depends on a well-established apico-basal polarity and is a prerequisite for the function of secretory epithelia. Despite extensive knowledge on individual trafficking pathways, little is known about the mechanisms coordinating their temporal and spatial regulation. Here, we report that the polarity protein Crumbs is essential for apical plasma membrane phospholipid-homeostasis and efficient apical secretion. Through recruiting β_Heavy-Spectrin and MyosinV to the apical membrane, Crumbs maintains the Rab6-, Rab11- and Rab30-dependent trafficking and regulates the lipid phosphatases Pten and Ocrl. Crumbs knock-down results in increased apical levels of PI(4,5)P₂ and formation of a novel, Moesin- and PI(4,5)P₂-enriched apical membrane sac containing microvilli-like structures. Our results identify Crumbs as an essential hub required to maintain the organization of the apical membrane and the physiological activity of the larval salivary gland.

Introduction

Epithelia can organize as layers or tubes, which form barriers and thus separate internal biological compartments from the environment. Many epithelia are specialized for absorption or secretion by performing selective and directional transport of nutrients, enzymes and waste products, which is essential for metazoan life (Cereijido et al., 2004; Rodriguez-Boulan and Macara, 2014; Lemaitre and Miguel-Aliaga, 2013). To perform these functions, epithelial cells are highly polarized: plasma membrane proteins and lipids are distributed asymmetrically into an apical domain facing the environment or a lumen, and a basolateral domain that contacts the neighboring cell and/or a basal lamina. In addition, polarity is manifested by uneven distribution of organelles, asymmetric cytoskeleton organization and directed trafficking (Rodriguez-Boulan and Macara, 2014; Knust and Bossinger, 2002; Eaton and Martin-Belmonte, 2014). The latter is particularly obvious in secretory epithelia, for example the salivary glands, which produce vast amounts of material that is secreted into the gland lumen (Blasky et al., 2015; Iruela-Arispe and Beitel, 2013; Eaton and Martin-Belmonte, 2014; Chung et al., 2014; Miguel-Aliaga et al., 2018).

Several evolutionarily conserved proteins regulate epithelial cell polarity. These include members of the apical Crumbs- and PAR-complexes, and the basolateral Scrib-Dlg-Lgl module (reviewed in Flores-Benitez and Knust, 2016; Román-Fernández and Bryant, 2016). The Crumbs (Crb) protein has a large extracellular domain (>2000 aa), and a small intracellular domain (37 aa) (Tepass et al., 1990; Wodarz et al., 1993), which harbors two protein-protein interaction motifs, a C-terminal PDZ (Postsynaptic density/Discs large/ZO-1)-domain binding motif (PBM) and a juxtamembrane FERM (protein 4.1/ezrin/radixin/moesin)-domain binding motif (FBM). The PBM is important for cell polarity and can bind Stardust (Sdt) and Par-6 (Li et al., 2014; Roh et al., 2002; Bulgakova et al., 2008; Bachmann et al., 2001; Hong et al., 2001; Kempkens et al., 2006; Ivanova et al., 2015). The FBM can directly interact with Yurt (Yrt), Expanded (Ex) and Moesin (Moe) (Klebes and Knust, 2000; Laprise et al., 2006; Ling et al., 2010; Wei et al., 2015), FERM-proteins that act as adaptors between membrane proteins and the actin cytoskeleton (Bennett and Baines, 2001; Lemmon et al., 2002; McClatchey, 2014; Sauvanet et al., 2015). The FBM of Crb is also important for β_Heavy-Spectrin (β_H-Spec) recruitment to the apical plasma membrane, and thereby supports the polarized organization of the membrane-associated cytoskeleton (cytocortex) (Wodarz et al., 1995; Richard et al., 2009; Pellikka et al., 2002; Lee et al., 2010; Lee and Thomas, 2011; Médina et al., 2002b).

Several epithelia of crb or sdt mutant Drosophila embryos show severe polarity defects, disruption of cell-cell adhesion and loss of tissue integrity. On the other hand, over-expression of Crb in the embryonic epidermis increases the size of the apical membrane (Tepass and Knust, 1993; Grawe et al., 1996; Tepass et al., 1990; Das and Knust, 2018; Tepaß and Knust, 1990). Similar phenotypes have been reported in mouse embryos mutant for Crb2 or Crb3 (Charrier et al., 2016; Szymaniak et al., 2015; Whiteman et al., 2014; Xiao et al., 2011; Ramkumar et al., 2016). In addition, Drosophila Crb has been associated with other functions, which are independent of its roles in epithelial integrity, such as regulation of tissue growth via the Hippo pathway, regulation of Notch signaling (Das and Knust, 2018; Nemetschke and Knust, 2016; Perez-Mockus et al., 2017; Herranz et al., 2006), as well as photoreceptor morphogenesis and survival under light stress (reviewed in Pocha and Knust, 2013; Bulgakova and Knust, 2009; Genevet and Tapon, 2011).

Apico-basal polarity is also essential for polarized membrane traffic. Directed trafficking depends on the phosphoinositide composition of the plasma membrane, the cytocortex and various Rab (Ras-related in brain) proteins. All of these are closely interconnected to organize and maintain the identity of apical and basolateral membranes (Weisz and Rodriguez-Boulan, 2009; Eaton and Martin-Belmonte, 2014; Blasky et al., 2015; Rodriguez-Boulan et al., 2005; Croisé et al., 2014). Epithelial cell polarity and polarized membrane traffic require differential enrichment of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) and phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P₃) in the apical and basolateral membranes, respectively (Di Paolo and De Camilli, 2006; Martin-Belmonte and Mostov, 2007). PI(4,5)P₂ levels are controlled by Pten (Phosphatase and tensin homolog deleted on chromosome ten), which converts PI(3,4,5)P₃ into PI(4,5)P₂, by the type I phosphatidylinositol 4-phosphate 5-kinase Skittles (Sktl), which produces PI(4,5)P₂ from phosphatidylinositol 4-phosphate (PI4P), and by Ocrl (Oculocerebrorenal syndrome of Lowe), which dephosphorylates PI(4,5)P₂ into PI4P (de Renzis et al., 2002; Knirr et al., 1997; Maehama et al., 2004; Claret et al., 2014; Gervais et al., 2008; Worby and Dixon, 2014; Balakrishnan et al., 2015; Weixel et al., 2005). Pten activity is antagonistic to that of the type IA phosphatidylinositol three kinase (Pi3K), which is enriched at basolateral membranes and converts PI(4,5)P₂ into PI(3,4,5)P₃ (Gassama-Diagne et al., 2006; Peng et al., 2015; Balakrishnan et al., 2015; Gao et al., 2000; Goberdhan et al., 1999; Huang et al., 1999). PI(4,5)P₂ can bind to pleckstrin homology (PH)-domains of FERM proteins and β-Spectrins (Yoon et al., 1994; Harlan et al., 1995), thereby linking the plasma membrane to the cytocortex and to the trafficking machinery (Barroso-González et al., 2009; Ramel et al., 2013; Beck and Nelson, 1998; Holleran and Holzbaur, 1998; Kang et al., 2009). Moreover, PI(4,5)P₂ is directly implicated in the regulation of exocytosis (Milosevic et al., 2005; Gong et al., 2005; Massarwa et al., 2009; Rousso et al., 2013) and in all forms of endocytosis (Antonescu et al., 2011; Mayinger, 2012; Jost et al., 1998).

Here, we studied the functions of Crb in a differentiated, highly polarized secretory epithelium, namely the salivary gland (SG) of the Drosophila larva, to decipher its possible role in polarized trafficking. We identified Crb as a novel regulator of apical secretion and maintenance of the apical microvilli in SG cells. We show that loss of Crb in SGs disrupts the apical cytocortex, apical secretion and the apical trafficking machinery, including the organization of Rab6-, Rab11- and Rab30-positive apical compartments, and the localization of their effector Myosin V (MyoV) (Lindsay et al., 2013). Our results show that Crb controls the apical secretion machinery via regulation of phosphoinositide metabolism. Loss of Crb increases apical levels of PI(4,5)P₂, a phenotype that requires the activity of Pten, and impairs the function of the apical secretory machinery. These defects are accompanied by the formation of a novel apical membrane compartment, which emerges as a solitary intracellular sac of PI(4,5)P₂- and phospho-Moe-enriched apical membrane containing microvilli. This compartment is reminiscent to intracellular vacuolar structures found in patients with MVID (microvillus inclusion disease), a fatal genetic disease characterized by lack of microvilli on the surface of enterocytes (www.omim.org/entry/251850). We conclude that Crb acts as an apical hub to couple phospholipid metabolism and cytoskeleton scaffolds with apical membrane traffic. Our work sheds light on the mechanism behind the determination of the apical membrane by Crb and its possible implications in different pathologies.

Results

The Crb complex is dispensable for maintenance of apico-basal polarity in larval salivary glands (SGs)

To investigate the role of the Crb protein complex in a differentiated secretory epithelium, we silenced Crb or its binding partner Sdt in the larval SG by RNAi-mediated knock-down (KD) using the SG-specific driver fkh-GAL4 (Zhou et al., 2001). We took advantage of the fact that this strategy does not affect embryonic development (data not shown). The larval SG consists of two tubes composed of columnar epithelial cells, each with a central lumen (Figure 1A). Strikingly, although the KD of Crb effectively reduces apical levels of Crb, Sdt and DPatj (Figure 1B–C’ and Figure 1—figure supplement 1C,D,Q), it does not affect the overall morphology of SGs, as determined by phalloidin staining (Figure 1—figure supplement 1A,B). Yet, the SGs lacking Crb are shorter when compared to their control counterparts (Figure 1—figure supplement 1R, Figure 1—figure supplement 1—source data 1). Similar results were observed upon RNAi-mediated KD of Sdt (Figure 1—figure supplement 1R–X). Interestingly, KD of Crb or Sdt does not alter the polarized distribution of any canonical apical or basolateral polarity marker tested, including Bazooka (Baz, Figure 1D,E), aPKC (Figure 1—figure supplement 1E,F), Par-6 (Figure 1—figure supplement 1G,H and Y,Z), Disc large (Dlg, Figure 1F,G and Figure 1—figure supplement 1AA, BB), Yurt (Yrt) (Figure 1—figure supplement 1I,J) and Coracle (Cora, Figure 1—figure supplement 1K,L). Taken together, these results show that the Crb protein complex is dispensable for maintenance of tissue integrity and overall epithelial cell polarity of larval SGs.

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Crb is required for efficient apical secretion in SG cells.

(A) Scheme indicating the anatomic location of the SG in the larval stage. (**B-G**) Localization of Crb (**B,C**), Sdt (**B’,C’**), Baz (**D,E**) and Dlg (**F,G**) in control (**B,B’,D,F**, *fkh>/+*) and Crb KD (**C,C’,E,G**, *fkh >UAS crb^RNAi*) animals. H. Pupariation efficiency of controls (black and blue) and larvae with reduced levels of Crb (magenta) at 29 °C. Error bars indicate the standard error of the mean, n indicates number of traced individual larvae of the corresponding genotypes in three independent experiments. (**I,J**) Localization of the apical transmembrane protein Cadherin99C in SGs from control (I) and Crb KD (J) animals. (**K,L**) Localization of the secreted apical cargo SerpCBD-GFP in live SGs of control (K, *fkh >UAS* SerpCBD-GFP) and Crb KD (L, *fkh >UAS crb^RNAi; UAS-SerpCBD-GFP*) animals. Arrows indicate the apical plasma membrane. Arrowheads mark the lateral plasma domain. Dotted lines indicate the basal membrane. Scale bar in A indicates10 µm applies to all panels. (**M, M**) Plotted is the fluorescence intensity (arbitrary units) of SerpCBD-GFP along the apical-to-basal direction in live SGs of control (black, *fkh >UAS* SerpCBD-GFP) and Crb KD (magenta, *fkh >UAS crb^RNAi; UAS-SerpCBD-GFP*). Error bars indicate the standard error of the mean, n indicates number of glands from the corresponding genotypes.

Figure 1—source data 1 Dataset for tracking of larval development.: https://cdn.elifesciences.org/articles/50900/elife-50900-fig1-data1-v2.xlsx
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Figure 1—source data 2 Dataset for SerpCBD-GFP fluorescence intensity in control glands.: https://cdn.elifesciences.org/articles/50900/elife-50900-fig1-data2-v2.xlsx
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Figure 1—source data 3 Dataset for SerpCBD-GFP fluorescence intensity in Crb KD glands.: https://cdn.elifesciences.org/articles/50900/elife-50900-fig1-data3-v2.xlsx
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The Crb protein complex is required for proper apical secretion in larval SGs

Because depletion of the Crb protein complex does not affect the overall polarity or integrity of the larval SGs, we analyzed whether it plays any role in maintaining their physiological functions. SGs of feeding larvae produce saliva required to digest food, whereas in later stages they produce and secrete predominantly glue proteins required to attach the pupae to surfaces (Thomopoulos, 1988; Chung et al., 2014; Maruyama and Andrew, 2012; Csizmadia et al., 2018; Gregg et al., 1990; Fraenkel and Brookes, 1953). Thus, we speculated that any defect in saliva secretion could result in less food intake and hence delayed larval development. In fact, when compared to control larvae, the time necessary to reach the pupal stage is prolonged upon depletion of Crb (Figure 1H, Figure 1—source data 1) or Sdt (Figure 1—figure supplement 1KK, Figure 1—figure supplement 1—source data 2).

To test whether the delay in pupation correlates with defects in apical membrane transport, we analyzed the localization of Cadherin99C (Cad99C), an apical transmembrane protein involved in regulation of microvillar length (Chung and Andrew, 2014), and CD8-RFP, a heterologous transmembrane protein normally targeted to the apical membrane (Xu et al., 2002; Lee and Luo, 1999). We found that upon silencing of Crb or Sdt, Cad99C and CD8-RFP do not localize properly at the apical membrane but instead localize in intracellular vesicles (Figure 1I,J and Figure 1—figure supplement 1CC,DD,M,N and EE,FF).

To evaluate apical secretion, we analyzed the expression of Sgs3-GFP. However, the glue proteins are not expressed at the feeding stage we study here (beginning of the 3^rd instar) but almost 2 days later (Tran and Ten Hagen, 2017). Indeed, at the stage of glue secretion, vesicle delivery appears normal in Crb-deficient SGs (Videos 1 and 2) (Tran et al., 2015). Furthermore, several proteins that are known to be apically secreted in other tubular epithelia, like Piopio, Vermiform and UAS-driven secreted proteins (cherry-sec, GFP-tagged wheat germ agglutinin) (Jaźwińska et al., 2003; Luschnig et al., 2006; Brankatschk and Eaton, 2010) were not suitable for our studies since they could not be detected in the lumen of wild-type feeding larval SGs (not shown). Therefore, we used the chitin-binding domain of Serpentine tagged with GFP (UAS-SerpCBD-GFP), a well-established marker to evaluate apical secretion (Luschnig et al., 2006; Kakihara et al., 2008; Förster et al., 2010; Petkau et al., 2012; Dong et al., 2013; Dong et al., 2014; Bätz et al., 2014). Notably, while SerpCBD-GFP is barely detectable upon overexpression in control glands, loss of Crb or Sdt results in an obvious intracellular retention of SerpCBD-GFP at the apical aspect (Figure 1K–M, Figure 1—source data 2 and 3; and Figure 1—figure supplement 1 GG, HH, and LL, Figure 1—figure supplement 1—source data 3 and 4). In support of the idea that Crb is necessary for efficient apical secretion, we also found that glycoprotein secretion is impaired upon loss of Crb or Sdt, as revealed by intracellular retention of peanut-agglutinin-GFP (PNA-GFP, Figure 1—figure supplement 1O,P and II,JJ), which can bind to glycoproteins produced by the SGs (Korayem et al., 2004; Theopold et al., 2001; Tian and Ten Hagen, 2007). Taken together, these results show that the Crb protein complex is required for proper apical membrane protein delivery and protein secretion in SGs of feeding larvae.

Video 1

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posterframe for video — Fusion of a glue vesicle followed by expulsion of the cargo Sgs3-GFP into the lumen SG lumen of control (*fkh>+*, top) and Crb KD (*fkh >UAS crb^RNAi*, bottom) animals.

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The Crb protein complex is dispensable for maintenance of cell-cell junctions in larval SGs

Impaired apical secretion after KD of Crb could be related to defects in cell-cell junctions. In particular, the pleated septate junctions (pSJs) are involved in apical secretion in the embryonic tracheae (Wang et al., 2006; Laprise et al., 2010; Nelson et al., 2010). Therefore, we examined the SGs by transmission electron microscopy (TEM). We did not find any abnormalities in the localization of the zonula adherens (ZA) of Crb-deficient SG cells (Figure 1—figure supplement 2A’,C’ arrowheads).

In contrast to ZA, pSJs are morphologically abnormal in SGs of Crb KD animals, showing many interruptions (Figure 1—figure supplement 2C, green highlight) and disorganized regions (Figure 1—figure supplement 2D). In contrast, control SG cells, pSJs run uniformly along the lateral membrane with few interruptions (Figure 1—figure supplement 2A,B). Defects in pSJs were corroborated by reduced immunostaining of some pSJ components, including Sinuous (Sinu, Figure 1—figure supplement 2E,F), Kune-kune (Kune, Figure 1—figure supplement 2G,H), while others, such as Fasciclin3 (Fas3, Figure 1—figure supplement 2I,J), Dlg (Figure 1F,G), Lachesin-GFP and Nervana2-GFP (not shown) were not affected. Given the defects observed in pSJs, we analyzed their permeability by monitoring any luminal appearance of fluorescently labeled 10 kDa-Dextran ex vivo (Lamb et al., 1998). Interestingly, KD of Crb does not increase dye penetration into the lumen when compared to control glands (Figure 1—figure supplement 2K–L’), suggesting that the epithelium is tight. In contrast, KD of Fas3-GFP, used as a positive control, enhances the diffusion of 10 kDa-Dextran into the gland lumen (Figure 1—figure supplement 2M,M’).

Taken together, these results suggest that loss of Crb does not affect adherens junctions or the epithelial barrier function of SGs.

Crb regulates apical membrane organization via the apical cytocortex

Crb recruits Moesin (Moe) and β_H-Spectrin (β_H-Spec, encoded by the gene karst -kst) to the apical membrane (Richard et al., 2009; Lee et al., 2010; Lee and Thomas, 2011; Médina et al., 2002b; Kerman et al., 2008), where they mediate interactions between transmembrane proteins and the apical cytocortex (reviewed in Fehon et al., 2010; Baines et al., 2014). Therefore, we analyzed whether Crb KD affects the organization of the apical cytocortex in SG cells, and if so, whether this relates to the defects in apical secretion.

We found that KD of Crb decreases apical levels of F-actin (Figure 2A–C, Figure 2—source data 1) and β_H-Spec (Figure 2D–F, Figure 2—source data 2). Similarly, silencing a knock-in Crb tagged with GFP on the extracellular domain, Crb-GFP-A (Huang et al., 2009), using fkh >gfp^RNAi as an alternative approach for the KD of the Crb protein complex (Figure 2—figure supplement 1A–D) also decreases apical levels of F-actin (Figure 2—figure supplement 1E,F). Moreover, KD of Crb-GFP-A induces accumulation of Moe, as well as its active form phospho-Moe, into a single sac per cell localized right below the apical domain (Figure 2G,H, arrows, Video 3 and not shown). These sacs are also positive for the apical transmembrane protein Stranded at second tagged with YFP (Firmino et al., 2013) (Sas-YFP, Figure 2I,J) suggesting that they have an apical plasma membrane identity. On the other hand, KD of Crb has no evident effects on the organization of α-Tubulin or α-Spectrin (Figure 2—figure supplement 1G–J). These results show that Crb is required to maintain the organization of the apical cytocortex and the morphology of the apical membrane in larval SGs.

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Crb is necessary to specifically maintain the apical cytoskeleton and the morphology of the apical membrane.

(**A-F**) Localization and quantification of F-actin (phalloidin staining, **A-C**) and β_H-Spec (**D-F**) in control (**A,D**, *fkh>/+*) and Crb KD (**B,E**, *fkh >UAS crb^RNAi*) SGs. Violin graphs (**C,F**) show the fluorescence intensity (apical vs lateral ratio) indicating the mean and quartiles for F-actin (C, n = 36 cells for control and 28 cells for Crb KD) and β_H-Spec (F, n = 44 cells for control and 40 cells for Crb KD). Statistical significance was analyzed in an unpaired two-tailed t-test. (**G-H**) Localization of phospho-Moe in control (G, *Crb-GFP, fkh>/+*) and Crb KD (H, *Crb-GFP, fkh >UAS gfp^RNAi*) SGs. (**I,J**) Localization of the apical protein Stranded at second (Sas-YFP) in live SGs of control (I, *fkh>/+*) and Crb KD (J, *Crb-GFP, fkh >UAS gfp^RNAi*) animals. Shown are single optical slices and maximal projections of half of the z-stack (half SG-tube). Arrows point to the apical domain of the cell. Dotted lines indicate the basal membrane. Scale bar in (A) displays 10 µm and applies to panes (**A-J**). (**K-L’**) TEM images of SGs prepared using the high-pressure freezing technique, visualizing the apical aspect of SG cells of control (**K,K’**, *fkh>/+*) and Crb KD (**L,L’**, *fkh >UAS crb^RNAi*) animals. The brackets in **K,L’** indicate the apical microvilli. Asterisks in (L’) mark large intracellular vesicles found in Crb-deficient glands. Arrowheads in L’ indicate microvilli found inside vesicles. Scale bars in (**K,L**) indicate 5 µm and in (**K’,L’**) indicate 1 µm. (**M, M**) Mean number of microvilli following along the apical membrane over a distance of 1 µm, adjacent to the membrane and 1 µm above the apical membrane in SG cells of control (*fkh>/+*) and Crb KD (*fkh >UAS crb^RNAi*) animals. The heatmap indicates the scale bar for the number of microvilli/µm.

Figure 2—source data 1 Dataset for phalloidin fluorescence intensity.: https://cdn.elifesciences.org/articles/50900/elife-50900-fig2-data1-v2.xlsx
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Figure 2—source data 2 Dataset for β_H-Spec fluorescence intensity.: https://cdn.elifesciences.org/articles/50900/elife-50900-fig2-data2-v2.xlsx
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Figure 2—source data 3 Dataset for microvilli quantifications.: https://cdn.elifesciences.org/articles/50900/elife-50900-fig2-data3-v2.xlsx
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Video 3

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To examine in more detail the morphology of the apical aspect of Crb-deficient cells, we prepared SGs for TEM analysis by employing the high-pressure freezing technique. This technique immobilizes complex macromolecular assemblies in their native state and helps to preserve cytoskeleton-rich structures like microvilli (Studer et al., 2008). Strikingly, cells from Crb-depleted SGs display intracellular vesicles containing microvilli (Figure 2L’, arrowheads and Figure 2—figure supplement 2), which seem to correspond to the Sas-YFP positive sacs described above (Figure 2J). In fact, we also observed cases of intracellular sacs whose membrane were continuous with the apical membrane (Figure 2—figure supplement 2A,A’). Moreover, in Crb-deficient SGs, the density of apical microvilli is dramatically reduced (Figure 2K’,L’, brackets, and M, Figure 2—source data 3). The number of microvilli per micron adjacent to the apical plasma membrane is 8.0 ± 1.219 in control vs. 4.125 ± 1.446 in Crb-deficient cells (mean ± SD, p<0.0001, n = 8). This difference is even bigger when measured at 1 μm above the plasma membrane, 7.75 ± 1.222 in control vs. 2.850 ± 1.441 in Crb-deficient cells (mean ± SD, p<0.0001, n = 8), indicating that microvilli are also shorter in Crb-deficient cells. In addition, Crb-deficient SG cells exhibit large intracellular vesicles not present in control SGs, which probably correspond to enlarged lysosomes (asterisks in Figure 2L’ and in Figure 2—figure supplement 2B’C’; see also Figure 1—figure supplement 2C blue highlight). Indeed, live imaging of SGs incubated with Lysotracker showed that KD of Crb or Sdt increases lysosomal activity (Figure 2—figure supplement 3). This suggests that lysosomal activity increases due to impaired secretion upon loss of Crb.

Since these apical membrane invaginations are enriched in PI(4,5)P₂ (described below), we refer to them as PAMS: phospho-Moe and PI(4,5)P₂-enriched apical membrane sacs. Given that silencing of Crb reduces apical β_H-Spec, we analyzed the effect of β_H-Spec KD on PAMS formation. Indeed, loss of β_H-Spec (Figure 2—figure supplement 4A,B) prompts formation of PAMS marked by phospho-Moe (Figure 2—figure supplement 4C,D). Moreover, in SGs deficient in β_H-Spec, Crb remains apical and additionally localizes to the PAMS (Figure 2—figure supplement 4E,F). These results indicate that Crb localizes to the apical domain independently of β_H-Spec while β_H-Spec requires Crb to be organized at the apical cytocortex.

Taken together, these results indicate that Crb is essential to maintain the proper amount and organization of the apical membrane by stabilizing the apical cytocortex.

Crb regulates the apical membrane organization via MyosinV

The PAMS described above are reminiscent to microvilli-containing vesicles found in samples from MVID (microvillus inclusion disease) patients, which is linked to mutations in the MYO5b gene (Müller et al., 2008). Similar inclusions are found in animal models of MVID (Sidhaye et al., 2016). MyosinV (MyoV) is a processive motor that transports cargos along F-actin (Reck-Peterson et al., 2000) and is a component of the apical secretory machinery in epithelia (Massarwa et al., 2009; Reck-Peterson et al., 2000; Li et al., 2007; Pocha et al., 2011a). Moreover, in photoreceptor cells, Crb regulates apical transport of Rhodopsin-1 by interacting with MyoV (encoded by the gene didum) (Pocha et al., 2011a). Therefore, we analyzed whether Crb regulates MyoV in the SGs. Indeed, the KD of Crb decreases apical MyoV (Figure 3A,B,D, Figure 3—source data 1 and 2). Importantly, overexpression of MyoV-GFP in Crb-deficient glands does not rescue its apical localization (Figure 3—figure supplement 1A–C, Figure 3—figure supplement 1—source data 1 and 2). Furthermore, KD of β_H-Spec also decreases apical MyoV (Figure 3A,C,D, Figure 3—source data 1 and 3) as well as apical secretion as revealed by the apical retention of SerpCBD-GFP (Figure 3—figure supplement 2A–C, Figure 3—figure supplement 2—source data 1 and 2). This suggests that β_H-Spec acts downstream of Crb to maintain apical MyoV.

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MyoV KD induces the intracellular extension of the apical membrane and disrupts apical secretion.

(**A-C**) Single optical slices and maximal projection of half of the z-stack (half SG-tube) showing the localization of MyoV in fixed SGs of control (A, *fkh>/+*), Crb KD (B, *fkh >UAS crb^RNAi*) and β_H-Spec KD (C, *fkh >UAS kst^RNAi*) animals. (**D, D**) Plotted is the intensity (arbitrary units) of MyoV detected by immunofluorescence along the apical-to-basal direction in SGs of control (black, *fkh>/+*), Crb KD (magenta, *fkh >UAS crb^RNAi*) and β_H-Spec (green, *fkh >UAS kst^RNAi*) animals. Error bars indicate the standard error of the mean, n indicates number of glands from the corresponding genotypes. (**E-J**) Maximal projection of half of the z-stack (half SG-tube) showing the localization of Crb (**E,F**), Phospho-Moe (**G,H**) and Sas-YFP in SGs of control (**E,G,I**, *fkh>/+*) and MyoV KD (**F,H,J**, *fkh >UAS didum^RNAi*) animals. (**K,L**) Localization of SerpCBD-GFP in live SGs of control (K, *fkh >UAS* SerpCBD-GFP) and MyoV KD (L, *fkh >UAS didum^RNAi; UAS-SerpCBD-GFP*) animals. Arrows point to the apical and dotted lines indicate the basal membrane. Scale bars in (**A,E,K**) indicate 10 µm. (**M, M**) Plotted is the fluorescence intensity (arbitrary units) of SerpCBD-GFP along the apical-to-basal direction in live SGs of control (black, *fkh >UAS* SerpCBD-GFP), and MyoV KD (magenta, *fkh >UAS didum^RNAi; UAS-SerpCBD-GFP*) animals. Error bars indicate the standard error of the mean, n indicates number of glands from the corresponding genotypes.

Figure 3—source data 1 Dataset for MyosinV fluorescence intensity in control glands.: https://cdn.elifesciences.org/articles/50900/elife-50900-fig3-data1-v2.xlsx
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Figure 3—source data 2 Dataset for MyosinV fluorescence intensity in Crb KD glands.: https://cdn.elifesciences.org/articles/50900/elife-50900-fig3-data2-v2.xlsx
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Figure 3—source data 3 Dataset for MyosinV fluorescence intensity in βH-Spec KD glands.: https://cdn.elifesciences.org/articles/50900/elife-50900-fig3-data3-v2.xlsx
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Figure 3—source data 4 Dataset for SerpCBD-GFP fluorescence intensity in control glands.: https://cdn.elifesciences.org/articles/50900/elife-50900-fig3-data4-v2.xlsx
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Figure 3—source data 5 Dataset for SerpCBD-GFP fluorescence intensity in MyoV KD glands.: https://cdn.elifesciences.org/articles/50900/elife-50900-fig3-data5-v2.xlsx
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To examine the role of MyoV in apical secretion and PAMS formation, we silenced MyoV expression in the SGs using a specific RNAi (didum^RNAi). Analysis of Crb, phospho-Moe and Sas-YFP in MyoV-deficient SGs shows that while these proteins localize apically, they are also found in PAMS (Figure 3E–J). Additionally, live imaging of SGs expressing Sas-YFP shows large vesicles inside the cell (Figure 3J, arrowhead), which resemble similar structures seen in an organoid model for MVID established from mouse intestinal cells with impaired apical transport (Mosa et al., 2018). Indeed, we found that MyoV KD impairs secretion of SerpCBD-GFP, which in turn accumulates at the apical aspect of MyoV-deficient SG cells (Figure 3K–M, Figure 3—source data 4 and 5). These results suggest that formation of PAMS can be a consequence of defects in the apical secretory machinery.

Together, our results indicate that loss of Crb disrupts the apical β_H-Spec cytocortex. As a consequence, the apical localization of MyoV is reduced, apical secretion is impaired, and apical membrane morphology is defective, resulting in PAMS formation.

Crb is a novel regulator of the apical Rab machinery in larval SGs

Other works have provided genetic evidence that links the presence of microvilli-containing inclusions to defects in the apical Rab trafficking machinery (Feng et al., 2017; Knowles et al., 2015; Knowles et al., 2014; Sato et al., 2007). The Rab protein family is a major regulator of intracellular membrane traffic routes (Wandinger-Ness and Zerial, 2014; Pfeffer, 2013) and MyoV is known to interact with Rab6 and Rab11 (Lindsay et al., 2013; Li et al., 2007; Iwanami et al., 2016), which play an important role in apical membrane trafficking and recycling (Khanal et al., 2016; Iwanami et al., 2016; Chung and Andrew, 2014; Li et al., 2007; Satoh et al., 2005; Pelissier et al., 2003). Therefore, to evaluate the effects of Crb depletion on the Rab machinery, we took advantage of the recently published library of Rab proteins endogenously tagged with YFP (Dunst et al., 2015). We knocked-down Crb in larval SG cells and systematically screened the expression of all Rab proteins (Figure 4—figure supplement 1). Strikingly, we found that loss of Crb affects the localization of a subset of Rab proteins, namely Rab6-YFP, Rab11-YFP and Rab30-YFP. Specifically, the apically localized pools of these Rab proteins are reduced (Figure 4A–F’, and Videos 4–6), while the basal pools are not affected significantly. The effects on this subset of Rab proteins are specific, as Crb KD does not alter the organization of other Rab proteins, like Rab1-YFP (Figure 4G–H’, Video 7, and Figure 4—figure supplement 1). Similar results were obtained in Sdt KD glands (data not shown). Importantly, total protein levels of these Rab proteins do not change significantly upon Crb KD (Figure 4I).

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Crb organizes the apical Rab machinery in larval SG cells.

(**A-H’**) Confocal images of SGs to localize endogenously expressed Rab-YFP proteins. Rab6-YFP (**A-B’**), Rab11-YFP (**C-D’**), Rab30-YFP (**E-F’**) and Rab1-YFP (**G-H’**) in control (**A,C,E,G**, *fkh>/+*) and Crb KD (**B,D,F,H,** *fkh >UAS crb^RNAi*) SGs. Dotted-line squares in A-H indicate the area blown-up to the right of the respective panel (**A’-H’**). Arrows point to the apical pool of Rab6-YFP (A’), Rab11-YFP (C’) and Rab30-YFP (E’). Arrowheads mark the intracellular vesicular localization of Rab6-YFP (**A’,B’**) and Rab1-YFP (**G’,H’**). Scale bar (A) indicates 10 µm. (**I, I**) Western blot of endogenously expressed Rab-YFP proteins. Rab1-YFP, Rab6-YFP, Rab11-YFP, and Rab30-YFP in control (*fkh>/+*) and Crb KD (*fkh >UAS crb^RNAi*) SGs, indicated as *crb^RNAi* – or +, respectively. Membranes were probed for tubulin (loading control) and for GFP; arrowheads point to Rab-YFP proteins.

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As shown above, KD of β_H-Spec affects MyoV localization and apical secretion similarly to Crb KD. Therefore, we tested the effects of β_H-Spec KD on the localization of Rab6-YFP, Rab11-YFP, Rab30-YFP and Rab1-YFP. Strikingly, KD of β_H-Spec only removes the apical pools of Rab6-YFP and Rab11-YFP (Figure 4—figure supplement 2A–D), while the apical Rab30-YFP and the intracellular Rab1-YFP compartments are not affected (Figure 4—figure supplement 2E–H). Thus, the apical localization of Rab6 and Rab11 require a functional apical cytocortex.

To examine whether the reduction in Rab6-YFP or Rab11-YFP relates to the formation of PAMS, we silenced them individually using a gfp^RNAi and analyzed CD8-RFP localization. CD8-RFP accumulates intracellularly and localizes to the PAMS in Crb- and Sdt-deficient SGs (Figure 1—figure supplement 1M,N and EE,FF, and not shown). We found that KD of Rab6-YFP severely affects the morphology of the SGs and produces intracellular accumulation of CD8-RFP in large vesicles (Figure 4—figure supplement 3A–B’’), which agrees with the general requirement of Rab6 in secretion (Homma et al., 2019). KD of Rab11-YFP also affects the morphology of the SGs, although a single lumen is still patent (Figure 4—figure supplement 3D’, asterisk). More importantly, loss of Rab11 results in formation of PAMS in larval SG cells (Figure 4—figure supplement 3D’’, arrows). Hence, defects in the apical secretory machinery can induce the formation of PAMS.

Together, our results show that Crb is a novel regulator of apically localized Rab6-YFP, Rab11-YFP and Rab30-YFP. Moreover, β_H-Spec acts downstream of Crb to organize the apical localization of Rab6-YFP and Rab11-YFP. Therefore, the stabilization of β_H-Spec by Crb is essential to organize aspects of the apical Rab machinery for efficient apical secretion in larval SGs.

Crb regulates apical membrane levels of PI(4,5)P₂

As we describe above, depletion of Crb, Sdt, β_H-Spec or MyoV induces accumulation of phospho-Moe in a subapical structure that we termed PAMS. Phospho-Moe can bind to PI(4,5)P₂ via its PH-domain (Yonemura et al., 2002; Fiévet et al., 2007; Fehon et al., 2010; Roch et al., 2010) and the phosphoinositide composition of a membrane regulates Rab protein activity, as well as the localization of cytoskeleton proteins (Wandinger-Ness and Zerial, 2014; Tan et al., 2015; Mayinger, 2012; Liem, 2016; Bennett and Healy, 2009; Fehon et al., 2010). Therefore, we explored whether loss of Crb modulates the phosphoinositide composition of the apical membrane. For this, we monitored PI(4,5)P₂ localization by employing a well-established reporter containing the PI(4,5)P₂-specific PH-domain of phospholipase Cδ fused to GFP (PLCδ-PH-EGFP) (Gervais et al., 2008; Rousso et al., 2013; Balla et al., 1998; Várnai and Balla, 1998; Rescher et al., 2004).

Live imaging of larval SGs shows that PI(4,5)P₂ is enriched in the apical membrane (Figure 5B,H, Figure 5—source data 1), as previously observed in late 3^rd instar SGs (Rousso et al., 2013). Importantly, quantification of PLCδ-PH-EGFP fluorescence intensity of Crb-deficient SGs shows an increase in apical levels of PI(4,5)P₂ (Figure 5C,H, Figure 5—source data 2). Additionally, PI(4,5)P₂ localizes in the PAMS (Figure 5C), which are also positive for phospho-Moe (Video 8). Similar results were observed in Sdt KD glands (Figure 5—figure supplement 1A,B).

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Crb organizes the apical secretory machinery by negatively regulating Pten A.

(A) Simplified scheme of PI(4,5)P₂ biosynthesis. (**B-G**) Maximal projection of half of the z-stack (half SG-tube) showing the localization of PI(4,5)P₂ (PLCδ-PH-EGFP reporter) in live SGs of control (B, *fkh >UAS-PLCδ-PH-EGFP*), Crb KD (C, *fkh >UAS crb^RNAi; UAS-PLCδ-PH-EGFP*), Pten KD (D, *fkh >UAS pten^RNAi; UAS-PLCδ-PH-EGFP*), double KD of Crb and Pten (E, *fkh >UAS crb^RNAi, UAS-pten^RNAi; UAS-PLCδ-PH-EGFP*), Pi3K92E KD (F, *fkh >UAS-pi3k92E^RNAi; UAS-PLCδ-PH-EGFP*) and double KD of Crb and Pi3K92E (G, *fkh >UAS crb^RNAi, UAS-pi3k92E^RNAi; UAS-PLCδ-PH-EGFP*) animals. (**H, H**) Plotted is the fluorescence intensity (arbitrary units) of PLCδ-PH-EGFP along the apical-to-basal axis in live SGs of the genotypes indicated in (**B-G**), respectively. Error bars indicate the standard error of the mean, n indicates number of glands for the corresponding genotype. (**I-K**) Localization and quantification of over-expressed Pten2-GFP in SGs of control (I, *fkh >UAS-Pten2-GFP*) and Crb KD (J, *fkh >UAS crb^RNAi; UAS-Pten2-GFP*) animals. Violin graph (K) indicates the fluorescence intensity (apical vs lateral ratio) indicating the mean and quartiles (n = 28 cells for control and 36 cells for Crb KD). Statistical significance was analyzed in an unpaired two-tailed t-test. (**L-N**) Localization and quantification of Ocrl-RFP fluorescence intensity detected along the apical-to-basal axis in live SGs of control (black, *fkh>/+*) and Crb KD (magenta, *fkh >UAS crb^RNAi*) animals. Error bars indicate the standard error of the mean, n indicates number of glands of the corresponding genotypes. (**O-Q**) Localization and quantification of PLCδ-PH-EGFP fluorescence intensity detected along the apical-to-basal axis in live SGs of control (black, *fkh>/+*) and Ocrl KD (orange, *fkh >UAS ocrl^RNAi*) animals. Error bars indicate the standard error of the mean, n indicates the number of glands of the corresponding genotypes. Arrows point to the apical membrane domain. Arrowheads point to the lateral membrane. Dotted lines indicate the basal membrane. Scale bars in (**B,I,L,O**) indicate 10 µm.

Figure 5—source data 1 Dataset for PLCδ-PH-EGFP fluorescence intensity in control glands (corresponding to panel H).: https://cdn.elifesciences.org/articles/50900/elife-50900-fig5-data1-v2.xlsx
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Figure 5—source data 2 Dataset for PLCδ-PH-EGFP fluorescence intensity in Crb KD glands (corresponding to panel H).: https://cdn.elifesciences.org/articles/50900/elife-50900-fig5-data2-v2.xlsx
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Figure 5—source data 3 Dataset for PLCδ-PH-EGFP fluorescence intensity in Pten KD glands (corresponding to panel H).: https://cdn.elifesciences.org/articles/50900/elife-50900-fig5-data3-v2.xlsx
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Figure 5—source data 4 Dataset for PLCδ-PH-EGFP fluorescence intensity in glands with double KD of Crb and Pten (corresponding to panel H).: https://cdn.elifesciences.org/articles/50900/elife-50900-fig5-data4-v2.xlsx
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Figure 5—source data 5 Dataset for PLCδ-PH-EGFP fluorescence intensity in Pi3K92E KD glands (corresponding to panel H).: https://cdn.elifesciences.org/articles/50900/elife-50900-fig5-data5-v2.xlsx
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Figure 5—source data 6 Dataset for PLCδ-PH-EGFP fluorescence intensity in glands with double KD of Crb and Pi3K92E (corresponding to panel H).: https://cdn.elifesciences.org/articles/50900/elife-50900-fig5-data6-v2.xlsx
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Figure 5—source data 7 Dataset for Pten2-GFP fluorescence intensity (corresponding to panel K).: https://cdn.elifesciences.org/articles/50900/elife-50900-fig5-data7-v2.xlsx
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Figure 5—source data 8 Dataset for Ocrl-RFP fluorescence intensity in control glands (corresponding to panel N).: https://cdn.elifesciences.org/articles/50900/elife-50900-fig5-data8-v2.xlsx
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Figure 5—source data 9 Dataset for Ocrl-RFP fluorescence intensity in Crb KD glands (corresponding to panel N).: https://cdn.elifesciences.org/articles/50900/elife-50900-fig5-data9-v2.xlsx
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Figure 5—source data 10 Dataset for PLCδ-PH-EGFP fluorescence intensity in control glands (corresponding to panel Q).: https://cdn.elifesciences.org/articles/50900/elife-50900-fig5-data10-v2.xlsx
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Figure 5—source data 11 Dataset for PLCδ-PH-EGFP fluorescence intensity in Ocrl KD glands (corresponding to panel Q).: https://cdn.elifesciences.org/articles/50900/elife-50900-fig5-data11-v2.xlsx
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Figure 5—source data 12 Dataset for number of PAMS and diameter of PAMS.: https://cdn.elifesciences.org/articles/50900/elife-50900-fig5-data12-v2.xlsx
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To analyze whether β_H-Spec or MyoV participate in the accumulation of PI(4,5)P₂, we analyzed the distribution of PLCδ-PH-EGFP upon β_H-Spec or MyoV depletion. Indeed, KD of β_H-Spec or MyoV induces accumulation of PI(4,5)P₂ in the PAMS (Figure 5—figure supplement 1C,D), suggesting that loss of β_H-Spec and MyoV facilitates the increase of apical PI(4,5)P₂ levels and formation of PAMS.

We noted that PAMS are very heterogenous structures that are poorly preserved during fixation for immunohistochemistry. Therefore, we made use of live imaging to assess the frequency and morphology of the PAMS in the different genetic backgrounds. We used the signal from PLCδ-PH-EGFP and DE-cadherin-mTomato to measure the apical membrane area and volume (see Materials and methods). Our measurements show that KD of Crb, β_H-Spec or MyoV do not significantly change the amount of apical membrane surface or its volume, except for Crb-deficient cells, which have a slightly increased volume (Figure 5—figure supplement 1E, Figure 5—figure supplement 1—source data 1). We found that, when PAMS appear (% of cells with PAMS: 0% in control n = 322 cells; 46.7% in Crb KD n = 417 cells, 49.3% in β_H-Spec KD n = 503 cells; and 41,9% in MyoV KD n = 393 cells), there is a single sac per cell, which localizes toward the center of the apical domain. The PAMS diameter varies between 1.737 μm to 11.52 μm (mean ± SD: 5.325 ± 1.552 μm in Crb KD, 4.718 ± 1.382 μm in β_H-Spec KD, 5.012 ± 1.544 μm in MyoV KD; Figure 5—source data 12), suggesting that they could be dynamic. However, following up on single sacs by live imaging for 20 min revealed that these structures are rather steady (Video 9). Nevertheless, PAMS are not present in late 3^rd instar SGs of wandering larvae (Figure 5—figure supplement 1F,G). Taken together these results indicate that Crb is essential to control the levels of PI(4,5)P₂ at the apical membrane. Moreover, our results suggest that at least part of this control is exerted by organizing β_H-Spec and MyoV at the apical aspect.

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Crb controls apical membrane homeostasis by regulating phosphoinositide metabolism

To understand how the loss of Crb results in accumulation of PI(4,5)P₂, we explored the involvement of Pten, Pi3K, Sktl and Ocrl, key enzymes regulating PI(4,5)P₂ levels (Figure 5A). Expression of pten^RNAi (Ramachandran et al., 2009) in Crb KD glands effectively suppresses both the accumulation of PI(4,5)P₂ as measured by PLC-PH-EGFP fluorescence, and PAMS formation (Figure 5D,E,H, Figure 5—source data 3 and 4), while expression of pi3K92E^RNAi enhances the accumulation of PI(4,5)P₂ and PAMS formation (Figure 5F,G,H, Figure 5—source data 5 and 6). The latter also results in smaller glands (Figure 5—figure supplement 1H, Figure 5—figure supplement 1—source data 2), as expected due to the role of Pi3K in cell growth (Huang et al., 1999; Goberdhan et al., 1999; Gao et al., 2000; Scanga et al., 2000). Interestingly, KD of Sktl, another enzyme producing PI(4,5)P₂, is less effective in suppressing PAMS upon Crb KD than knocking-down Pten (Figure 5—figure supplement 1I–M, Figure 5—figure supplement 1—source data 3–6). To corroborate the importance for Pten to mediate the phenotype induced by loss of Crb, we found that over-expression of Pten2 induces accumulation of PI(4,5)P₂ and formation of PAMS (Figure 5—figure supplement 2A,B), while over-expression of Sktl results in strong defects in SG morphology (Figure 5—figure supplement 2A,C). Moreover, ex vivo incubation of SGs with VO-OHpic, a chemical inhibitor of Pten activity (Mak et al., 2010), eliminates the PAMS from Crb-deficient cells (Figure 5—figure supplement 1N–R, Figure 5—figure supplement 1—source data 7–10). Thus, our findings suggest that Pten is the main source of PI(4,5)P₂ involved in the formation of the PAMS upon Crb depletion.

Since apical Pten is important for restricting PI(3,4,5)P₃ to the basolateral membrane (Worby and Dixon, 2014; Shewan et al., 2011), we asked whether KD of Crb could affect PI(3,4,5)P₃ levels and Pten localization. We evaluated PI(3,4,5)P₃ levels using a probe containing the PH-domain of cytohesin tagged with GFP (Pinal et al., 2006). The signal of this probe at the plasma membrane is very weak and quantification of the fluorescence intensity revealed no significant change in the PI(3,4,5)P₃ apical-to-lateral ratio in Crb KD glands (Figure 5—figure supplement 2D–F, Figure 5—figure supplement 2—source data 1). Immunostainings to detect endogenous Pten were unsuccessful in our hands, therefore we expressed a UAS-transgene encoding the Pten2 isoform fused to GFP, which can rescue pupal eye development of Pten mutants (Pinal et al., 2006). Pten2-GFP over-expressed in larval SGs localizes to the apical domain in addition to the nucleus (Figure 5I,J). Interestingly, quantification of the Pten2-GFP fluorescence intensity revealed a decrease in the apical-to-lateral ratio in Crb and Sdt KD glands (Figure 5K and data not shown, Figure 5—source data 7), suggesting that Crb is required to ensure Pten levels at the apical membrane (Figure 5I,J, arrowheads). However, it is important to note that no PAMS were found in glands overexpressing Pten2-GFP, which is in contrast to the ones overexpressing Pten2 without a GFP tag (Figure 5—figure supplement 2A,B). Thus, the GFP tag could partially impair the phosphatase activity or expression levels could be lower than those achieved with Pten2 over-expression.

Besides Pten, Ocrl regulates PI(4,5)P₂ levels by dephosphorylating PI(4,5)P₂ into PI4P (Balakrishnan et al., 2015). Live imaging of Ocrl-RFP (knock-in allele) revealed its localization at the apical aspect in SG cells (Figure 5L). Moreover, KD of Crb severely decreases the apical localization of Ocrl (Figure 5M,N, Figure 5—source data 8 and 9). To evaluate the effect of Ocrl loss on PI(4,5)P₂ levels, we silenced the expression of Ocrl using a specific RNAi and quantified the fluorescence intensity of PLCδ-PH-EGFP. KD of Ocrl modestly increases the apical levels of PI(4,5)P₂ (Figure 5O–Q, Figure 5—source data 10 and 11), yet this is not accompanied by formation of PAMS.

Together, these results show that apical accumulation of PI(4,5)P₂ and formation of PAMS induced by the loss of Crb, seem to result from a combined effect of increased Pten activity and loss of Ocrl from the apical membrane upon loss of Crb.

Efficient apical secretion requires the control of PI(4,5)P₂ metabolism by Crb

To assess whether the secretion defects are a consequence of altered phosphoinositide metabolism we analyzed the secretion of SerpCBD-GFP and the organization of the apical Rab machinery. Live imaging analysis revealed that apical secretion of SerpCBD-GFP in Crb-deficient SGs is restored upon concomitant KD of Pten (Figure 6A–D,G, Figure 6—source data 1 to 4), while KD of Pi3K92E alone, or in combination with Crb KD, induces a stronger apical retention of SerpCBD-GFP than the one observed in Crb-deficient SGs (Figure 6B,E–G, Figure 6—source data 5 and 6). Similar results were obtained using the probe for glycoproteins PNA-GFP (data not shown). Similarly, KD of Pten efficiently suppresses the loss of the apical pools of Rab11-YFP (Figure 6H–K,N, Figure 6—source data 7 to 10) and Rab30-YFP (Figure 6O–R,U, Figure 6—source data 13 to 16) observed upon Crb depletion. Interestingly, KD of Pi3K92E in control cells induces loss of apical Rab11-YFP (Figure 6L,N, Figure 6—source data 11 and 12), but has no effect on Rab30-YFP localization (Figure 6S,U, Figure 6—source data 17 and 18). Additionally, over-expression of Pten2 in the SGs induces the loss of apical pools of Rab11-YFP and Rab30-YFP (Figure 6—figure supplement 1A–D). This is in accordance with apical PI(4,5)P₂ levels regulating apical Rab proteins negatively. Unfortunately, the effects of Pten KD or over-expression on the apical pool of Rab6-YFP in the absence of Crb could not be studied due to lethality of the larvae. Thus, Crb function is required to organize the apical cortex and to control the phosphoinositide metabolism, which in turn regulates the apical Rab protein machinery (Rab11-YFP, Rab30-YFP and possibly Rab6-YFP).

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Control of apical secretion and localization of Rab11 and Rab30 by Crb requires Pten.

(**A-F**) Maximal projection of 6.7µm through the SG lumen showing the localization of SerpCBD-GFP in live SGs of control (A, *fkh >UAS* SerpCBD-GFP), Crb KD (B, *fkh >UAS crb^RNAi; UAS-SerpCBD-GFP*), Pten KD (C, *fkh >UAS pten^RNAi; UAS-SerpCBD-GFP*), double KD of Crb and Pten KD (D, *fkh >UAS crb^RNAi, UAS-pten^RNAi; UAS-SerpCBD-GFP*), Pi3K92E KD (E, *fkh >UAS-pi3k92E^RNAi; UAS-SerpCBD-GFP*), and double KD of Crb and Pi3K92E (F, *fkh >UAS crb^RNAi, UAS-pi3k92E^RNAi; UAS-SerpCBD-GFP*), respectively. (**H-M**) Localization of endogenously expressed Rab11-YFP in live SGs. Shown are control (H, *Rab11-YFP, fkh>/+*), Crb KD (I, *Rab11-YFP, fkh >UAS crb^RNAi*), Pten KD (J, *Rab11-YFP, fkh >UAS pten^RNAi*), double KD of Crb and Pten (K, *Rab11-YFP, fkh >UAS crb^RNAi, UAS-pten^RNAi*), Pi3K92E KD (L, *Rab11-YFP, fkh >UAS-pi3k92E^RNAi*), and double KD of Crb and Pi3K92E (M, *Rab11-YFP, fkh >UAS crb^RNAi, UAS-pi3k92E^RNAi*) animals, respectively. (**O-T**) Localization of endogenously expressed Rab30-YFP in live SGs. Shown are control (O, *Rab30-YFP, fkh>/+*), Crb KD (P, *Rab30-YFP, fkh >UAS crb^RNAi*), Pten KD (Q, *Rab30-YFP, fkh >UAS pten^RNAi*), double KD of Crb and Pten (R, *Rab30-YFP, fkh >UAS crb^RNAi, UAS-pten^RNAi*), Pi3K92E KD (S, *Rab30-YFP, fkh >UAS-pi3k92E^RNAi*), and double KD of Crb and Pi3K92E (T, *Rab30-YFP, fkh >UAS crb^RNAi, UAS-pi3k92E^RNAi*) animals, respectively. Arrows point to the apical, and dotted lines to the basal membrane domain. Scale bar in (A) indicates 10 µm and applies to all panels. (**G,N,U**) Plotted is the fluorescence intensity (arbitrary units) of SerpCBD-GFP (G), Rab11-YFP (N) and Rab30-YFP (U), respectively, along the apical-to-basal axis in live SGs of the indicated genotypes. Error bars indicate the standard error of the mean, n indicates number of glands of the corresponding genotypes. (V) Violin graph of estimated food intake in control (first column), Crb KD (second column), Pten KD (third column), double KD of Crb and Pten (fourth column), Pi3K92E KD (fifth column), and double KD of Crb and Pi3K92E (sixth column) larvae. The dotted line indicates the mean value of the control. 60 larvae of the corresponding genotype were pooled in each biological replica. 10 biological replicas were analyzed distributed in three independent experiments. Statistical significance was tested in a one-way analysis of variance (ANOVA) followed by a Dunnett’s multiple-comparison test. (W) Pupariation efficiency of control (black, *fkh>/+*), Crb KD (magenta, *fkh >UAS crb^RNAi*), Pten KD (green, *fkh >UAS pten^RNAi*), double KD of Crb and Pten KD (yellow, *fkh >UAS crb^RNAi, UAS-pten^RNAi*), Pi3K92E KD (blue, *fkh >UAS-pi3k92E^RNAi*), and double KD of Crb and Pi3K92E (, *fkh >UAS crb^RNAi, UAS-pi3k92E^RNAi*) animals. Error bars indicate the standard error of the mean, n indicates number of traced individual larvae of the corresponding genotypes in at least 15 independent experiments.

Figure 6—source data 1 Dataset for SerpCBD-GFP fluorescence intensity in control glands.: https://cdn.elifesciences.org/articles/50900/elife-50900-fig6-data1-v2.xlsx
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Figure 6—source data 2 Dataset for SerpCBD-GFP fluorescence intensity in Crb KD glands.: https://cdn.elifesciences.org/articles/50900/elife-50900-fig6-data2-v2.xlsx
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Figure 6—source data 3 Dataset for SerpCBD-GFP fluorescence intensity in Pten KD glands.: https://cdn.elifesciences.org/articles/50900/elife-50900-fig6-data3-v2.xlsx
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Figure 6—source data 4 Dataset for SerpCBD-GFP fluorescence intensity in glands with double KD of Crb and Pten.: https://cdn.elifesciences.org/articles/50900/elife-50900-fig6-data4-v2.xlsx
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Figure 6—source data 5 Dataset for SerpCBD-GFP fluorescence intensity in Pi3K92E KD glands.: https://cdn.elifesciences.org/articles/50900/elife-50900-fig6-data5-v2.xlsx
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Figure 6—source data 6 Dataset for SerpCBD-GFP fluorescence intensity in glands with double KD of Crb and Pi3K92E.: https://cdn.elifesciences.org/articles/50900/elife-50900-fig6-data6-v2.xlsx
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Figure 6—source data 7 Dataset for Rab11-YFP fluorescence intensity in control glands.: https://cdn.elifesciences.org/articles/50900/elife-50900-fig6-data7-v2.xlsx
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Figure 6—source data 8 Dataset for Rab11-YFP fluorescence intensity in Crb KD glands.: https://cdn.elifesciences.org/articles/50900/elife-50900-fig6-data8-v2.xlsx
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Figure 6—source data 9 Dataset for Rab11-YFP fluorescence intensity in Pten KD glands.: https://cdn.elifesciences.org/articles/50900/elife-50900-fig6-data9-v2.xlsx
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Figure 6—source data 10 Dataset for Rab11-YFP fluorescence intensity in glands with double KD of Crb and Pten.: https://cdn.elifesciences.org/articles/50900/elife-50900-fig6-data10-v2.xlsx
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Figure 6—source data 11 Dataset for Rab11-YFP fluorescence intensity in Pi3K92E KD glands.: https://cdn.elifesciences.org/articles/50900/elife-50900-fig6-data11-v2.xlsx
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Figure 6—source data 12 Dataset for Rab11-YFP fluorescence intensity in glands with double KD of Crb and Pi3K92E.: https://cdn.elifesciences.org/articles/50900/elife-50900-fig6-data12-v2.xlsx
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Figure 6—source data 13 Dataset for Rab30-YFP fluorescence intensity in control glands.: https://cdn.elifesciences.org/articles/50900/elife-50900-fig6-data13-v2.xlsx
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Figure 6—source data 14 Dataset for Rab30-YFP fluorescence intensity in Crb KD glands.: https://cdn.elifesciences.org/articles/50900/elife-50900-fig6-data14-v2.xlsx
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Figure 6—source data 15 Dataset for Rab30-YFP fluorescence intensity in Pten KD glands.: https://cdn.elifesciences.org/articles/50900/elife-50900-fig6-data15-v2.xlsx
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Figure 6—source data 16 Dataset for Rab30-YFP fluorescence intensity in glands with double KD of Crb and Pten.: https://cdn.elifesciences.org/articles/50900/elife-50900-fig6-data16-v2.xlsx
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Figure 6—source data 17 Dataset for Rab30-YFP fluorescence intensity in Pi3K92E KD glands.: https://cdn.elifesciences.org/articles/50900/elife-50900-fig6-data17-v2.xlsx
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Figure 6—source data 18 Dataset for Rab30-YFP fluorescence intensity in glands with double KD of Crb and Pi3K92E.: https://cdn.elifesciences.org/articles/50900/elife-50900-fig6-data18-v2.xlsx
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Figure 6—source data 19 Dataset for food intake estimations.: https://cdn.elifesciences.org/articles/50900/elife-50900-fig6-data19-v2.xlsx
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Figure 6—source data 20 Dataset for tracking of larval development.: https://cdn.elifesciences.org/articles/50900/elife-50900-fig6-data20-v2.xlsx
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To assess the physiological relevance of Crb in SG secretion, we evaluated the larval food intake and tracked the pupariation time (Deshpande et al., 2014). We found that KD of Crb in the SGs, as well as KD of Pi3K92E, slightly reduces the amount of food intake (Figure 6V, Figure 6—source data 19), yet this reduction is not statistically significant (one-way ANOVA followed by Tukey's multiple comparisons test). Interestingly, concomitant KD of Crb and Pten significantly increases the larval food intake when compared to controls (Figure 6V). Importantly, these trends are reflected in the pupariation rate (Figure 6W, Figure 6—source data 20). Hence, while animals with SG-specific depletion of Crb take longer to pupariate than control animals (Figure 6W), those with additional Pten KD pupariate faster than those with Crb KD alone. Moreover, the pupariation of Pi3K92E KD animals is similar to the one of Crb-deficient animals, while concomitant KD of Crb and Pi3K92E delays the pupariation even more. Taken together, our results demonstrate that Crb is essential for apical membrane homeostasis, apical secretion and physiological function of larval SGs.

Discussion

In this work we identified unknown roles of Crb in constitutive apical secretion of larval SGs. Defects in apical secretion upon KD of Crb are not due to an overall disruption of epithelial cell polarity. Our results point to two major components acting downstream of Crb that regulate secretion. i) We found that the Crb complex is essential for Rab6-, Rab11- and Rab30-dependent, apical membrane transport machinery by ensuring the apical pools of these Rab proteins. This suggests that Crb maintains the active pool of these Rab proteins at the apical domain, as inactive GDP-bound Rab proteins associate with chaperone-like molecules, called GDP dissociation inhibitors (GDIs), and diffuse into the cytosol (Goody et al., 2005; Grosshans et al., 2006; Müller and Goody, 2018). ii) We show that Crb restricts the levels of PI(4,5)P₂ on the apical membrane by regulating apical activity and apical localization of Pten and Ocrl, respectively. As a consequence, Crb controls the size and organization of the apical membrane and efficient apical secretion, processes that are mediated in part by β_H-Spec and MyoV. From this we conclude that the Crb protein complex functions as an apical hub that interconnects and regulates these cellular machineries, which, in turn, are essential to maintain the physiological activity of the SGs (Figure 7).

Figure 7

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Crb-dependent regulation of apical secretion in SG cells Schematic representation of Crb-dependent regulation of apical secretion in SG cells.

Under physiological conditions (left image), Crb mediates the apical localization of Moesin and β_H-Spec, which link the Crb protein (blue) to the apical F-actin cytoskeleton (black ribbon). This Crb-cytocortex complex is necessary for organization of the apical Rab-dependent traffic machinery (depicted as Rab vesicles in yellow). Under these conditions Crb negatively regulates the activity of Pten via β_H-Spec and MyoV. The precise molecular interactions involved in the negative regulation of Pten are not defined (see Discussion for details). The absence of Crb in the SG cells disrupts the efficient apical secretion (right image). The defects in apical secretion are a consequence of the disruption of the apical cytocortex (actin, β_H-Spec), the loss of MyoV and the excessive production of PI(4,5)P₂ (red dots) which require the activity of Pten. The loss of Ocrl form the apical membrane could also contribute to the increase in PI(4,5)P₂ apical levels. Another consequence is the formation of a novel apical membrane sac enriched in PI(4,5)P₂ (PAMS), Moe (green rectangles) and apical transmembrane proteins (not depicted).

The roles of Crb in the regulation of constitutive saliva secretion

The late 3^rd instar Drosophila SG has been extensively studied as a model for regulated exocytosis during the burst of glue granule secretion, which occurs at the onset of metamorphosis (reviewed in Tran and Ten Hagen, 2017). Here, we studied the roles of Crb in the regulation of constitutive saliva secretion in SGs at the beginning of the 3^rd instar, while larvae are still feeding. At this stage there is a minimal synthesis of glue proteins (Kodani, 1948; Rizki, 1967; Beckendorf and Kafatos, 1976; Korge, 1977; Zhimulev et al., 1981), while salivary glycoproteins are actively secreted into the lumen (Thomopoulos, 1988; Chung et al., 2014; Maruyama and Andrew, 2012; Csizmadia et al., 2018; Gregg et al., 1990; Fraenkel and Brookes, 1953).

Loss of Crb or Sdt in SG cells results in hampered delivery of apical transmembrane proteins (Cad99C and CD8-RFP) as well as apical accumulation of secretion reporters (SerpCBD-GFP and PNA-GFP), which suggests at least two interpretations. Loss of Crb 1) hampers secretion, so that protein transport is jammed at the apical aspect, or 2) secretion is normal but endocytosis at the apical surface is strongly enhanced resulting in an immediate re-internalization of the secreted cargo. Loss of apical Rab6, Rab11, Rab30 and MyoV upon Crb KD supports the first interpretation. MyoV is a component of the apical secretory machinery (Massarwa et al., 2009; Reck-Peterson et al., 2000; Li et al., 2007) and known interactor of Crb, Rab6, Rab11 and possibly Rab30 (Lindsay et al., 2013; Li et al., 2007; Iwanami et al., 2016; Pocha et al., 2011a). Both Rab6 and Rab11 are known to facilitate apical transport and recycling (Khanal et al., 2016; Iwanami et al., 2016; Chung and Andrew, 2014; Li et al., 2007; Satoh et al., 2005; Pelissier et al., 2003), while Rab30 is suggested to be associated with the Golgi apparatus (Kelly et al., 2012). However, in larval SG cells Rab30 shows no co-localization with Golgi markers (Dunst et al., 2015) but instead localizes in a subapical pool. Interestingly, Rab30 was found as a potential MyoV-binding partner but later dismissed due to experimental threshold settings (Lindsay et al., 2013). Thus, although the functions of Rab30 in Drosophila are less clear (Thomas et al., 2009), our results suggest that active Rab30 contributes to MyoV-dependent transport.

A role of Crb in apical secretion rather than in apical endocytosis is further supported by our observations that the distribution of Rab proteins involved in endocytosis, namely Rab5, Rab7 and Rab21, is not affected by the loss of Crb (Simpson et al., 2004; Chavrier et al., 1990). This is also consistent with earlier observations that crb loss of function does not result in an overall increase in endocytosis in the eye imaginal disc epithelium (Richardson and Pichaud, 2010). Nevertheless, we cannot exclude the contribution of endocytosis completely, as inhibition of dynamin-dependent endocytosis seems to ameliorate the secretion phenotype of Crb-deficient glands, yet it does not block the formation of PAMS (data not shown). Moreover, by using dominant active or inactive forms of Rab5 (Zhang et al., 2007), we obtained inconsistent results (data not shown), probably due to pleiotropic effects of these versions of Rab5, which tend to titer effectors shared with other Rab proteins (Pylypenko et al., 2017; Müller and Goody, 2018). Although loss of β_H-Spec function has been linked to increased endocytosis in some Drosophila epithelia (Williams et al., 2004; Pellikka et al., 2002; Richard et al., 2009; Phillips and Thomas, 2006), or Crb mobility in the embryonic epidermis (Bajur et al., 2019) data presented here support the conclusion that in larval SGs Crb predominantly regulates apical membrane traffic and secretion, though we cannot completely rule out a minor contribution of endocytosis to the phenotypes observed.

We show that Crb is necessary to maintain the apical localization of MyoV. As mentioned above, MyoV is an interactor of Rab6, Rab11 and possibly Rab30 (Lindsay et al., 2013). This suggests that Crb can directly organize the apical secretion machinery by modulating the localization of MyoV. Additionally, our results also suggest that stabilization of β_H-Spec by Crb is important for organizing the apical Rab proteins. It is known that Crb regulates the actin cytoskeleton, and is necessary for recruitment of β_H-Spec and Moe to the apical cytocortex (Flores-Benitez and Knust, 2015; Tsoumpekos et al., 2018; Salis et al., 2017; Röper, 2012; Sherrard and Fehon, 2015; Loie et al., 2015; Das and Knust, 2018; Médina et al., 2002a; Wei et al., 2015; Wodarz et al., 1995). Unlike in other epithelia (Wodarz et al., 1995; Pellikka et al., 2002; Médina et al., 2002b; Richard et al., 2009), depletion of β_H-Spec in SGs does not result in loss of Crb or Sdt from the apical domain, but rather hampers apical secretion and induces the loss of Rab6- and Rab11-positive apical compartments. Therefore, the normal apical secretory activity of the SGs requires the Crb-dependent stabilization of β_H-Spec and MyoV at the apical cytocortex.

Crb organizes the apical trafficking machinery by controlling apical PI(4,5)P₂ levels

Our results suggest that Crb is required to maintain the apical localization of Ocrl and to negatively regulate the activity of Pten, both key regulators of PI(4,5)P₂ levels (Worby and Dixon, 2014; Balakrishnan et al., 2015). Hence, apical PI(4,5)P₂ levels increase upon loss of Crb. Concomitantly, PI(4,5)P₂ as well as phospho-Moe and apical transmembrane proteins are found in a singular apical membrane extension, dubbed PAMS. Chemical inhibition or genetic ablation of Pten in Crb-deficient glands not only suppresses the formation of PAMS, but also restores the apical pools of Rab11, Rab30, apical secretion, larval food intake and timely pupariation. The relevance of PI(4,5)P₂ levels for proper secretion is highlighted by recent results demonstrating that the activity of Drosophila Crag (a Rab10 GEF) and Stratum (a Rab8 GEF) is regulated by the levels of PI(4,5)P₂ (Devergne et al., 2014; Devergne et al., 2017). For example, in the follicle epithelium, reduction of PI(4,5)P₂ levels results in defective secretion of basal membrane proteins, which then accumulate at the apical membrane (Devergne et al., 2014; Devergne et al., 2017). Moreover, recent work showed that another phosphoinositide species, PI(3,4)P₂, and the enzyme producing it, SHIP1, are key determinants of apical identity in a model of lumen formation (Román-Fernández et al., 2018). PI(3,4)P₂ was found to be an essential component of the pre-apical membrane and of Rab11a-positive recycling endosomes containing apical proteins that cluster together during de novo formation of the lumen. Indeed, perturbing PI(3,4)P₂ levels disrupts polarization through subcortical retention of vesicles at apical membrane initiation sites (Román-Fernández et al., 2018). Therefore, the control of PI(4,5)P₂ levels by Crb might impact the apical secretory machinery by altering the localization of specific effectors (GEFs or GAPs) of Rab6, Rab11 and Rab30.

Crb-mediated regulation of Pten partially depends on the organization of the apical cytocortex, but the precise molecular mechanism remains to be elucidated. So far, based on co-immunoprecipitation assays (data not shown) or on previous mass-spectrometry data (Pocha et al., 2011b) no direct interactions between Crb and Pten could be established. Yet, since apical localization of Baz, a binding partner of Pten (von Stein et al., 2005), is not affected by the loss of Crb, we suggest that the Baz-Pten interaction does not depend on Crb. On the other hand, KD of MyoV or β_H-Spec induces the formation of PAMS. Therefore, we favor the hypothesis that regulation of Pten might be mediated by β_H-Spec and MyoV acting downstream of Crb. Interestingly, an interaction between β_H-Spec and Pten was found by tandem affinity purification assays (Vinayagam et al., 2016), but whether this interaction regulates Pten activity was not analyzed. Furthermore, inhibition of MyoV-based transport increases the cell size of neurons, which mimics the PTEN-loss of function (van Diepen et al., 2009). Indeed, using immunoprecipitation and FRET analysis, it was shown that mammalian PTEN can interact directly with the MyoV C-terminal cargo-binding domain, yet the consequences of this interaction on PTEN activity or its localization were not evaluated (van Diepen et al., 2009). Therefore, it is plausible that Pten activity in the larval SGs can be regulated by interactions with MyoV and β_H-Spec. It is well-known that Pten regulation is very complex. Mammalian PTEN, for example, has more than 20 different sites, which can be subject to post-translational modifications (Worby and Dixon, 2014; Gorbenko and Stambolic, 2016). Therefore, it is likely that Crb can impinge on Pten activity via several different mechanisms, which can even be tissue- or developmental stage-specific. Indeed, it is well established that Crb as well as other polarity proteins have tissue specific functions, regulating cell signaling, cytoskeleton dynamics, cell division and cell adhesion as well as tissue growth and morphogenesis (reviewed in Flores-Benitez and Knust, 2016; Tepass, 2012). But even in one tissue like the larval SGs, Crb may control apical trafficking via additional mechanisms independent of Pten. For example, loss of apical Rab30 upon Crb KD is independent of β_H-Spec, suggesting that Crb can organize the apical trafficking machinery by additional effectors.

PAMS – membrane entities dependent on PI(4,5)P₂ levels

It is well-known that Crb is a key determinant of the apical membrane and that over-expression of Crb in Drosophila embryos expands the apical membrane (Wodarz et al., 1995), Our findings on the functional link between Crb and Pten now provide a possible mechanism by which Crb exerts this function. In this context, the formation of the PAMS, apical membrane invaginations containing microvilli enriched in PI(4,5)P₂, phospho-Moe and apical transmembrane proteins (Sas, Crb and CD8-RFP), offer an attractive model to study the regulation of apical membrane organization by Crb and Pten. Recently published data implicate PTEN in the regulation of apical membrane size. By using intestinal epithelial Ls174T:W4 cells in culture, Bruurs et al., showed that loss of PTEN results in formation of a larger brush border. In contrast, in mouse small intestinal organoids no change was observed (Bruurs et al., 2018), indicating that these effects can be tissue specific. Pten activity is necessary for the morphogenesis of rhabdomeres, a specialized apical membrane domain composed of a tightly packed stack of microvilli in Drosophila photoreceptors (Pinal et al., 2006). Indeed, Crb overexpression in Drosophila photoreceptor cells increases the amount of apical membrane (Pellikka et al., 2002; Muschalik and Knust, 2011). Therefore, it will be interesting to test whether this is mediated by Pten or by changes in intracellular trafficking.

It is important to note that the PAMS do not represent an expansion of the apical membrane upon loss of Crb (Figure 5—figure supplement 1E). On the contrary, based on our measurements of microvilli density (Figure 2M) and the disruption of Cad99C localization, the net effect of Crb KD is a reduction in the amount of apical membrane. Considering that one microvillus has a surface of approx. 0.55 μm² (roughly calculated from our EM images the height≈2.5 μm and radius≈35 nm), 80 microvilli are found along 10 μm of apical membrane thus ‘contain’ approx. 44 μm² of plasma membrane. Therefore, the reduction in microvilli number in Crb-deficient cells (without considering the reduction in their height) indicates that approx. 20 μm² of plasma membrane is found along the same length of the apical membrane, which is a loss of >50% of the apical membrane. Moreover, this loss of microvilli might be related to the defects in Cad99C localization upon loss of Crb, as Cad99C is important in maintenance of microvillar length (Chung and Andrew, 2014). Therefore, our results support the conclusion that Crb regulates the apical membrane architecture by maintaining the lipid homeostasis and the organization of the apical cytocortex. Upon loss of Crb, the collapse of the cytocortex together with the increase in PI(4,5)P₂ lead to the formation of PAMS and destabilization of the microvilli. Therefore, to understand how Crb regulates the proper proportions of apical vs. basolateral membranes, future studies need to address the biogenesis of the PAMS and whether their formation occurs at the expense of the basolateral membrane.

Possible implications in human pathology

Defects in the membrane trafficking machinery are linked to a plethora of different pathologies, including immune syndromes, deafness, neuronal degeneration and cancer (reviewed in Seabra et al., 2002; Holthuis and Menon, 2014; Krzewski and Cullinane, 2013; Bronfman et al., 2007). The PAMS in Crb-deficient SG cells have striking similarities to the inclusion bodies observed in MVID patients carrying mutations in MYO5b (Müller et al., 2008; Ruemmele et al., 2010) or those found in animal models of MVID, like zebrafish mutant for myosin Vb (Sidhaye et al., 2016) and mice mutant for Rab8a and Rab11a (Feng et al., 2017; Sato et al., 2007). Moreover, recent data obtained in an intestinal organoid model of microvillus inclusion formation showed that these inclusion bodies are dynamic. Within hours, these inclusions can form and detach from the plasma membrane or collapse (Mosa et al., 2018). Furthermore, disruption of MyoVB, Rab8a, Rab11a, Syntaxin three and Syntaxin binding protein 2, all lead to defects similar to the ones observed in MVID enterocytes (Sidhaye et al., 2016; Feng et al., 2017; Vogel et al., 2017; Schneeberger et al., 2015; Mosa et al., 2018). Therefore, it is tempting to speculate that up-regulation of Pten activity could contribute to the pathogenesis of MVID.

Retinal degeneration is another pathological condition often caused by compromised trafficking machinery. Mutations in human CRB1 induce retinal degeneration (Richard et al., 2006b; Bulgakova and Knust, 2009), similar as mutations in Drosophila crb (Johnson et al., 2002; Pocha et al., 2011a; Pellikka et al., 2002; Izaddoost et al., 2002; Chartier et al., 2012; Spannl et al., 2017) or overexpression of dominant negative versions (Pellikka and Tepass, 2017). Indeed, disruption of many of the proteins regulated by Crb in the SGs, including MyoV and Pten, can affect eye development, trafficking of Rh1 and ultimately photoreceptor survival in the fly (Pocha et al., 2011a; Pellikka et al., 2002; Richard et al., 2009; Karagiosis and Ready, 2004; Iwanami et al., 2016; Pinal et al., 2006; Satoh et al., 2016). Thus, it will be interesting to analyze whether Crb regulates the apical trafficking machinery in photoreceptor cells by modulating the phosphoinositide metabolism and how this is related to the pathogenesis of retinal degeneration.

In conclusion, data presented here reveal a role for the Crb complex beyond its canonical function as a polarity determinant in differentiating epithelial cells and show that Crb can fine-tune the morphology and the molecular composition of the apical domain in a mature epithelium. In the future it will be interesting to explore whether the functional interactions described here are unique to early Drosophila SG cells or represent a conserved module also acting in other Crb-expressing epithelia.

Materials and methods

Fly stocks

Request a detailed protocol

Fly stocks (see Table 1) were maintained at room temperature (RT) on standard food. We employed the UAS-GAL4 system (Elliott and Brand, 2008) to drive the expression of different UAS-transgenes specifically in the salivary gland with the fkh-GAL4 driver (Henderson and Andrew, 2000). For detailed descriptions of the genotypes used in each figure see Table 2. The stocks in which the UAS-RNAi lines were recombined with the fkh-GAL4 driver together with the temperature sensitive repressor GAL80[ts] were maintained and expanded at 18°C. For experiments (see example in Figure 8), the crosses driving the different UAS-RNAi lines, and their corresponding controls, were done and maintained at 25°C. Eggs were collected overnight and then transferred to 29°C for approx. 48 hr. After this period, the feeding third instar larvae (not yet wandering) were collected for salivary gland dissections.

Table 1

List of fly stocks used in this study.

Designation	Genotype (as reported in FlyBase when available)	Description
Balancer	w[1118]; In(2LR)Gla, wg[Gla-1]/CyO, P{w[+mC]=GAL4 twi.G}2.2, P{w[+mC]=UAS-2xEGFP}AH2.2	Balancer for 2nd chromosome; BSC 6662
Balancer	w[1118]; Dr[Mio]/TM3, P{w[+mC]=GAL4 twi.G}2.3, P{UAS-2xEGFP}AH2.3, Sb[1] Ser[1]	Balancer for 3rd chromosome; BSC 6663
Balancer	w[*]; ry[506] Dr[1]/TM6B, P{w[+mC]=Dfd-EYFP}3, Sb[1] Tb[1] ca[1]	Balancer for 3rd chromosome; BSC 8704
crb^RNAi	w[1118]; P{GD14463}v39177	Expresses the RNAi against crb under the control of UAS sequences; VDRC 39177
sdt^RNAi	w[1118]; P{GD9163}v23822	Expresses the RNAi against sdt under the control of UAS sequences; VDRC 23822
sdt^RNAi	y[1] sc[*] v[1]; P{y[+t7.7] v[+t1.8]=TRiP.HMS01652}attP40	Expresses dsRNA for RNAi of sdt (FBgn0261873) under UAS control. BSC 37510
gfp^RNAi	y[1] sc[*] v[1]; P{y[+t7.7] v[+t1.8]=VALIUM20 EGFP.shRNA.3}attP40	Expresses small hairpin RNA under the control of UAS for RNAi of EGFP and EYFP as well as fusion proteins containing these fluors, BSC 41559
gfp^RNAi	y[1] sc[*] v[1]; P{y[+t7.7] v[+t1.8]=VALIUM20 EGFP.shRNA.3}attP2	Expresses small hairpin RNA under the control of UAS for RNAi of EGFP and EYFP as well as fusion proteins containing these fluors, BSC 41560
moe^RNAi	w[1118]; P{GD5211}v37917	Expresses the RNAi against moe under the control of UAS sequences; VDRC 37917
kst^RNAi	y[1] v[1]; P{y[+t7.7] v[+t1.8]=TRiP.GLC01654}attP40	Expresses dsRNA for RNAi of kst (FBgn0004167) under UAS control, BSC 50536
ocrl^RNAi	y[1] sc[*] v[1] sev[21]; P{y[+t7.7] v[+t1.8]=TRiP.HMS01201}attP2/TM3, Sb[1]	Expresses dsRNA for RNAi of Ocrl (FBgn0023508) under UAS control in the VALIUM20 vector. BSC 34722
GAL80ts	w[*]; P{w[+mC]=tubP-GAL80[ts]}7	Expresses temperature-sensitive GAL80 under the control of the alphaTub84B promoter; outcrossed from BSC 7018
Dicer	w[1118]; P{w[+mC]=UAS-Dcr-2.D}2	Expresses Dicer-2 under UAS control, BSC 24650
myoV^RNAi	y[1] sc[*] v[1]; P{y[+t7.7] v[+t1.8]=TRiP.HMC03900}attP40	Expresses dsRNA for RNAi of didum (FBgn0261397) under UAS control; BSC 55740
pten^RNAi	y[1] w[1118]; P{w[+mC]=UAS Pten.dsRNA.Exel}2	Expresses a snapback transcript for RNAi of Pten under the control of UAS. BSC 8549
pten^RNAi	w[1118]; P{w[+mC]=UAS Pten.dsRNA.Exel}3	Expresses a snapback transcript for RNAi of Pten under the control of UAS. BSC 8550
pi3k92E^RNAi	y[1] sc[*] v[1]; P{y[+t7.7] v[+t1.8]=TRiP.HMC05152}attP40	Expresses dsRNA for RNAi of Pi3K92E (FBgn0015279) under UAS control. BSC 61182
pi3k92E^RNAi	y[1] sc[*] v[1]; P{y[+t7.7] v[+t1.8]=TRiP .GL00311}attP2	Expresses dsRNA for RNAi of Pi3K92E (FBgn0015279) under UAS control. BSC 35798
sktl^RNAi	y[1] sc[*] v[1]; P{y[+t7.7] v[+t1.8]=TRiP .GL00072}attP2	Expresses dsRNA for RNAi of sktl (FBgn0016984) under UAS control. BSC 35198
SerpCBD-GFP	w[*];; UAS-SerpCBD-GFP	Expresses the N-terminus of Serp including the signal peptide and chitin binding domain (CBD) fused to GFP (Luschnig et al., 2006), kindly provided by S. Luschning
MyosinV-GFP	w[*];; UAS-didum-GFP	Expresses full length didum (amino acids 1–1792) tagged at the C-terminal end with EGFP (Krauss et al., 2009), kindly provided by A. Ephrussi
Sas-Venus	w[*];; tub::Sas-Venus	Stranded at Second fused with Venus under tubulin promoter on 3rd chromosome (Firmino et al., 2013)
PNA-GFP	w[*]; M{w[+mC]=UAS PNA.GFP}ZH-86Fb	Expresses GFP-tagged peanut agglutinin under UAS control. BSC 55247
CD8-RFP	w[*]; P{y[+t7.7] w[+mC]=10XUAS-IVS-mCD8::RFP}attP2	Expresses mCD8-tagged RFP under the control of 10 UAS sequences. BSC 32218
PI(4,5)P₂ sensor	y[1] w[*]; P{w[+mC]=UAS-PLCdelta-PH-EGFP}3	Expresses GFP-tagged pleckstrin homology domain from human PLCδ. BSC 39693
PI(3,4,5)P₃ sensor	w[*];; tub::GPR1-PH-EGFP	Expresses GFP-tagged pleckstrin homology domain from cytohesin/GRP1 (Pinal et al., 2006), kindly provided by F. Pichaud
Pten2-GFP	w[*]; UAS-Pten2-GFP	Expresses Pten2 isoform GFP-tagged under the control of UAS sequences (Pinal et al., 2006), kindly provided by F. Pichaud
Pten2	w[*]; UAS-Pten2	Expresses the Pten2 isoform under the control of UAS sequences (von Stein et al., 2005), kindly provided by A. Wodarz
fkhGAL4	w[*]; fkh-GAL4	On 3rd chromosome, expresses GAL4 under the control of the fkh promoter (Henderson and Andrew, 2000), kindly provided by K. Röpper
Fas3-GFP	w[*]; P{w[+mC]=PTT-GA}Fas3[G00258]	Fas3 fused with GFP protein trap. BSC 50841
DE-cad-GFP	w*;DE-cad::GFP	DE-cadherin fused with GFP knock-in allele; homozygous viable (Huang et al., 2009), kindly provided by Y. Hong
DE-cad-mTomato	w*;DE-cad::mTomato	DE-cadherin fused with mTomato knock-in allele; homozygous viable (Huang et al., 2009), kindly provided by Y. Hong
Crb-GFP	w*;;crb::GFP-A	Crumbs fused with GFP knock-in allele; homozygous viable (Huang et al., 2009), kindly provided by Y. Hong
Lac-GFP	w*; lac::GFP	Protein trap line: lachesin fused with GFP under endogenous promoter on 2nd chromosome; homozygous viable (kindly provided by the Klämbt Protein trap consortium)
Nrv2-GFP	w*; nrv2::GFP	Protein trap line: nervana2 fused with GFP under endogenous promoter on 2nd chromosome; homozygous viable (kindly provided by the Klämbt Protein trap consortium)
Ocrl-RFP	TI{T-STEP.TagRFP-T}Ocrl[KI] w[*]	A T-STEP cassette was knocked into Ocrl to tag the endogenous protein with TagRFP-T. BSC 66529
Dlg-mTagRFP	Dlg-mTagRFP	On X chromosome, expresses Dlg-mTagRFP under the control of a ubiquitous promoter (Pinheiro et al., 2017), kindly provided by Y. Bellaïche
Rab-YFP	Rab-YFP	endogenously YFP::tagged Rab protein library generated in Dunst et al. (2015)
BSC - Bloomington Drosophila stock Center

VDRC - Vienna Drosophila Resource Center.

Table 2

List of detailed genotypes analyzed in each figure.

Figure 1
B,B'	w; UAS-crb[RNAi]/+*
C,C'	w; UAS-crb[RNAi]/+; fkh-GAL4/+*
D	w; Rab30-YFP, UAS-crb[RNAi]/+*
E	w; Rab30-YFP, UAS-crb[RNAi]/+; fkh-GAL4/+*
F	w; UAS-crb[RNAi]/+; Rab11-YFP/+*
G	w; UAS-crb[RNAi]/+; Rab11-YFP/fkh-GAL4*
I	w;; fkhGAL4, ubiGAL80[ts]*
J	w; UAS-crb[RNAi]; fkhGAL4, ubiGAL80[ts]*
K	w;; fkhGAL4, UAS-SerpCBD-GFP-GFP/+*
L	w; UAS-crb[RNAi]/+; fkhGAL4, UAS-SerpCBD-GFP-GFP/+*
Figure 1—figure supplement 1
A,C,E,G,I,K	w; UAS-crb[RNAi]/+*
B,D,F,H,J,L	w; UAS-crb[RNAi]/+; fkh-GAL4/+*
M	w;; UAS-CD8-RFP/fkhGAL4*
N	w; UAS-crb[RNAi]/+; UAS-CD8-RFP/fkhGAL4*
O	w;; UAS-PNA-GFP/fkhGAL4 ubiGAL80[ts]*
P	w;; UAS-crb[RNAi]/+; UAS-PNA-GFP/fkhGAL4 ubiGAL80[ts]*
Q: Control	w; UAS-crb[RNAi]/+; Rab11-YFP/+*
Q: Crb KD	w; UAS-crb[RNAi]/+; Rab11-YFP/fkh-GAL4*
S,U,U’,W,Y	w;; UAS-std[RNAi]/+*
T,V,V’,X,Z	w; UAS-sdt[RNAi]/fkh-GAL4*
AA	w;; Rab11-YFP, UAS-sdt[RNAi]/Rab11-YFP*
BB	w;; Rab11-YFP, UAS-sdtRNAi/Rab11-YFP, fkhGAL4*
CC	w;; fkhGAL4, ubiGAL80[ts]/+*
DD	w; UAS-sdt[RNAi]; fkhGAL4, ubiGAL80[ts]/+*
EE	w;; fkhGAL4, UAS-CD8-RFP/+*
FF	w;; fkhGAL4, UAS-CD8-RFP/UAS-sdt[RNAi]*
GG	w;; fkhGAL4, UAS-SerpCBD-GFP/+*
HH	w;; fkhGAL4, UAS-SerpCBD-GFP/UAS-sdt[RNAi]*
II	w;; fkhGAL4, UAS-PNA-GFP/+*
JJ	w;; fkhGAL4, UAS-PNA-GFP/UAS-sdt[RNAi]*
Figure 1—figure supplement 2
A,A',B	w; UAS-crb[RNAi]/+; Rab11-YFP/+*
C,C’,D'	w; UAS-crb[RNAi]/+; Rab11-YFP/fkh-GAL4*
E,G,I	w; UAS-crb[RNAi]/+*
F,H,J	w; UAS-crb[RNAi]/+; fkh-GAL4/+*
K,K'	w; Fas3-GFP/Fas3-GFP; fkhGAL4/+*
L,L'	w; Fas3-GFP/Fas3-GFP, UAS-crb[RNAi]; fkhGAL4/+*
M,M'	w; Fas3-GFP/Fas3-GFP; fkhGAL4/UAS-gfp[RNAi]*
Figure 2
A,D	w; UAS-crb[RNAi]/+*
B,E	w; UAS-crb[RNAi]/+; fkh-GAL4/+*
G	w; UAS-gfp[RNAi]/+; crb-GFP-A/crb-GFP-A*
H	w; UAS-gfp[RNAi]/+; crb-GFP-A/crb-GFP-A, fkh-GAL4*
I	w;; fkhGAL4, ubiGAL80[ts]/tub::Sas-Venus*
J	w; UAS-crb[RNAi]; fkhGAL4, ubiGAL80[ts]/tub::Sas-Venus*
K,K'	w;; fkhGAL4, UAS-PLCdelta-PH-EGFP/+*
L,L'	w; UAS-crb[RNAi]/+; fkhGAL4, UAS-PLCdelta-PH-EGFP/+*
Figure 2—figure supplement 1
A,C,E	w; UAS-gfp[RNAi]/+; crb-GFP-A/crb-GFP-A*
B,D,F	w; UAS-gfp[RNAi]/+; crb-GFP-A/crb-GFP-A, fkh-GAL4*
G,I	w; UAS-crb[RNAi]/+*
H,J	w; UAS-crb[RNAi]/+; fkh-GAL4/+*
Figure 2—figure supplement 2
A-C'	w; UAS-crb[RNAi]/+; fkhGAL4, UAS-PLCdelta-PH-EGFP/+*
Figure 2—figure supplement 3
A	w; UAS-crb[RNAi]/+*
B	w; UAS-crb[RNAi]/+; fkh-GAL4/+*
C	w; UAS-sdt[RNAi]/+*
D	w; UAS-sdt[RNAi]/+; fkh-GAL4/+*
Figure 2—figure supplement 4
A,C,E	w;; fkhGAL4, ubiGAL80[ts]*
B,D,F	w; UAS-kst[RNAi]; fkhGAL4, ubiGAL80[ts]*
Figure 3
A	w;; fkhGAL4, ubiGAL80[ts]*
B	w; UAS-crb[RNAi]; fkhGAL4, ubiGAL80[ts]*
C	w; UAS-kst[RNAi]; fkhGAL4, ubiGAL80[ts]*
E,G	w;; fkhGAL4, ubiGAL80[ts]/+*
F,H	w; UAS-didum[RNAi]/+; fkhGAL4, ubiGAL80[ts]/+*
I	w;; fkhGAL4, ubiGAL80[ts]/tub::Sas-Venus*
J	w; UAS-didum[RNAi]/+; fkhGAL4, ubiGAL80[ts]/tub::Sas-Venus*
K	w;; fkhGAL4, UAS-SerpCBD-GFP/+*
L	w; UAS-didum[RNAi]/+; fkhGAL4, UAS-SerpCBD-GFP/+*
Figure 3—figure supplement 1
A	w;; fkhGAL4, ubiGAL80[ts]/UAS-MyoV-GFP*
B	w; UAS-crb[RNAi]/+; fkhGAL4, ubiGAL80[ts]/UAS-MyoV-GFP*
Figure 3—figure supplement 2
A	w;; fkhGAL4, ubiGAL80[ts]/UAS-SerpCBD-GFP*
B	w; UAS-kst[RNAi]; fkhGAL4, ubiGAL80[ts]/UAS-SerpCBD-GFP*
Figure 4
A	w; Rab6-YFP, UAS-crb[RNAi]/+*
B	w; Rab6-YFP, UAS-crb[RNAi]/+; fkh-GAL4/+*
C	w;; Rab11-YFP, fkhGAL4, ubiGAL80[ts]/Rab11-YFP*
D	w; UAS-crb[RNAi]/+; Rab11-YFP, fkhGAL4, ubiGAL80[ts]/Rab11-YFP*
E	w; Rab30-YFP/Rab30-YFP; fkhGAL4, ubiGAL80[ts]/+*
F	w; UAS-crb[RNAi], Rab30-YFP/Rab30-YFP; fkhGAL4, ubiGAL80[ts]/+*
G	w; UAS-crb[RNAi]/+; Rab1-YFP/+*
H	w; UAS-crb[RNAi]/+; Rab1-YFP/fkh-GAL4*
I: Rab1 Control	w; UAS-crb[RNAi]/+; Rab1-YFP/+*
I: Rab1 Crb KD	w; UAS-crb[RNAi]/+; Rab1-YFP/fkh-GAL4*
I: Rab6 Control	w; Rab6-YFP, UAS-crb[RNAi]/+*
I: Rab6 Crb KD	w; Rab6-YFP, UAS-crb[RNAi]/+; fkh-GAL4/+*
I: Rab11 Control	w; UAS-crb[RNAi]/+; Rab11-YFP/+*
I: Rab11 Crb KD	w; UAS-crb[RNAi]/+; Rab11-YFP/fkh-GAL4*
I: Rab30 Control	w; UAS-crb[RNAi], Rab30-YFP/+;*
I: Rab30 Crb KD	w; UAS-crb[RNAi], Rab30-YFP/+; fkhGAL4/+*
Figure 4—figure supplement 1
Rab1 Control	w;; Rab1-YFP/fkhGAL4*
Rab1 Crb KD	w;UAS-crb[RNAi]/+; Rab1-YFP/fkhGAL4*
Rab2 Control	w; Rab2-YFP/+; fkhGAL4/+*
Rab2 Crb KD	w; Rab2-YFP, UAS-crb[RNAi]/+; fkhGAL4/+*
Rab4 Control	w; Rab4-YFP/+; fkhGAL4/+*
Rab4 Crb KD	w; Rab4-YFP, UAS-crb[RNAi]/+; fkhGAL4/+*
Rab5 Control	w; Rab5-YFP/+; fkhGAL4/+*
Rab5 Crb KD	w; Rab5-YFP, UAS-crb[RNAi]/+; fkhGAL4/+*
Rab6 Control	w; Rab6-YFP/+; fkhGAL4/+*
Rab6 Crb KD	w; Rab6-YFP, UAS-crb[RNAi]/+; fkhGAL4/+*
Rab7 Control	w;; Rab7-YFP/fkhGAL4*
Rab7 Crb KD	w;UAS-crb[RNAi]/+; Rab7-YFP/fkhGAL4*
Rab8 Control	w;; Rab8-YFP/fkhGAL4*
Rab8 Crb KD	w;UAS-crb[RNAi]/+; Rab8-YFP/fkhGAL4*
Rab10 Control	w Rab10-YFP/+;; fkhGAL4/+*
Rab10 Crb KD	w Rab10-YFP/+; UAS-crb[RNAi]/+; fkhGAL4/+*
Rab11 Control	w;; Rab11-YFP/fkhGAL4*
Rab11 Crb KD	w;UAS-crb[RNAi]/+; Rab11-YFP/fkhGAL4*
Rab18 Control	w Rab18-YFP/+;; fkhGAL4/+*
Rab18 Crb KD	w Rab18-YFP/+; UAS-crb[RNAi]/+; fkhGAL4/+*
Rab21 Control	w Rab21-YFP/+;; fkhGAL4/+*
Rab21 Crb KD	w Rab21-YFP/+; UAS-crb[RNAi]/+; fkhGAL4/+*
Rab35 Control	w Rab35-YFP/+;; fkhGAL4/+*
Rab35 Crb KD	w Rab35-YFP/+; UAS-crb[RNAi]/+; fkhGAL4/+*
Rab39 Control	w Rab39-YFP/+;; fkhGAL4/+*
Rab39 Crb KD	w Rab39-YFP/+; UAS-crb[RNAi]/+; fkhGAL4/+*
Rab40 Control	w Rab40-YFP/+;; fkhGAL4/+*
Rab40 Crb KD	w Rab40-YFP/+; UAS-crb[RNAi]/+; fkhGAL4/+*
Figure 4—figure supplement 2
A	w; Rab6-YFP/Rab6-YFP; fhkGAL4/+*
B	w; Rab6-YFP, UAS-kst[RNAi]/Rab6-YFP; fhkGAL4/+*
C	w;; Rab11-YFP, fkhGAL4, ubiGAL80[ts]/Rab11-YFP*
D	w; UAS-kst[RNAi]/+; Rab11-YFP, fkhGAL4, ubiGAL80[ts]/Rab11-YFP*
E	w; Rab30-YFP/Rab30-YFP; fhkGAL4/+*
F	w; Rab30-YFP, UAS-kst[RNAi]/Rab30-YFP; fhkGAL4/+*
G	w;; Rab1-YFP/fkhGAL4, ubiGAL80[ts]*
H	w; UAS-kst[RNAi]/+; Rab1-YFP/fkhGAL4, ubiGAL80[ts]*
Figure 4—figure supplement 3
A-A’’	w; Rab6-YFP/Rab6-YFP; fhkGAL4, UAS-CD8-RFP/+*
B-B’’	w; Rab6-YFP/Rab6-YFP, UAS-gfp[RNAi]; fhkGAL4, UAS-CD8-RFP/+*
C-C’’	w;; Rab11-YFP, fkhGAL4, UAS-CD8-RFP/Rab11-YFP*
D-D’’	w; UAS-gfp[RNAi]/+; Rab11-YFP, fkhGAL4, UAS-CD8-RFP/Rab11-YFP*
Figure 5
B	w;; fkhGAL4, UAS-PLCdelta-PH-EGFP/+*
C	w; UAS-crb[RNAi]/+; fkhGAL4, UAS-PLCdelta-PH-EGFP/+*
D	w;; fkhGAL4, UAS-PLCdelta-PH-EGFP/UAS-pten[RNAi]*
E	w; UAS-crb[RNAi]/+; fkhGAL4, UAS-PLCdelta-PH-EGFP/UAS-pten[RNAi]*
F	w;; fkhGAL4, UAS-PLCdelta-PH-EGFP/UAS-pi3k92E[RNAi]*
G	w; UAS-crb[RNAi]/+; fkhGAL4, UAS-PLCdelta-PH-EGFP/UAS-pi3k92E[RNAi]*
I	w;; UAS-pten2-GFP/fkh-GAL4, ubiGAL80[ts]*
J	w; UAS-crb[RNAi]/+; UAS-pten2-GFP/fkh-GAL4, ubiGAL80[ts]*
L	Ocrl-RFP, w/+;; fkh-GAL4, ubiGAL80[ts]/+*
M	Ocrl-RFP, w; UAS-crb[RNAi]/+; fkh-GAL4, ubiGAL80[ts]/+*
O	w;; fkhGAL4, UAS-PLCdelta-PH-EGFP/+*
P	w;; fkhGAL4, UAS-PLCdelta-PH-EGFP/UAS-ocrl[RNAi]*
Figure 5—figure supplement 1
A	w; DE-cad-mTomato/+; UAS-PLCdelta-PH-EGFP/fkhGAL4, ubiGAL80[ts]*
B	w;; fkhGAL4, UAS-PLCdelta-PH-EGFP/UAS-sdt[RNAi]*
C	w; UAS-kst[RNAi]/DE-cad-mTomato; fkhGAL4, ubiGAL80[ts]/UAS-PLCdelta-PH-EGFP*
D	w; UAS-didum[RNAi]/+; fkhGAL4 UAS-PLCdelta-PH-EGFP/+*
F	w;; UAS-PLCdelta-PH-EGFP/fkh-GAL4, ubiGAL80[ts]*
G	w; UAS-crb[RNAi]/+; UAS-PLCdelta-PH-EGFP/fkh-GAL4, ubiGAL80[ts]*
I	w;; fkhGAL4, UAS-PLCdelta-PH-EGFP/+*
J	w; UAS-crb[RNAi]/+; fkhGAL4, UAS-PLCdelta-PH-EGFP/+*
K	w;; fkhGAL4, UAS-PLCdelta-PH-EGFP/UAS-sktl[RNAi]*
L	w; UAS-crb[RNAi]/+; fkhGAL4, UAS-PLCdelta-PH-EGFP/UAS-sktl[RNAi]*
N,P	w;; fkhGAL4, ubiGAL80[ts]/UAS-PLCdelta-PH-EGFP*
O,Q	w; UAS-crb[RNAi]/+; fkhGAL4, ubiGAL80[ts]/UAS-PLCdelta-PH-EGFP*
Figure 5—figure supplement 2
A	w;; fkhGAL4, UAS-PLCdelta-PH-EGFP/+*
B	w;; fkhGAL4, UAS-PLCdelta-PH-EGFP/UAS-pten2*
C	UAS-Sktl w/+;; fkhGAL4, UAS-PLCdelta-PH-EGFP/+*
D	w;; fkhGAL4, ubiGAL80[ts]/tub::GPR1-PH-EGFP*
E	w; UAS-crb[RNAi]/+; fkhGAL4, ubiGAL80[ts]/tub::GPR1-PH-EGFP*
Figure 6
A	w;; fkhGAL4, UAS-SerpCBD-GFP/+*
B	w; UAS-crb[RNAi]/+; fkhGAL4, UAS-SerpCBD-GFP/+*
C	w;; fkhGAL4, UAS-SerpCBD-GFP/UAS-pten[RNAi]*
D	w; UAS-crb[RNAi]/+; fkhGAL4, UAS-SerpCBD-GFP/UAS-pten[RNAi]*
E	w;; fkhGAL4, UAS-SerpCBD-GFP/UAS-pi3k92E[RNAi]*
F	w; UAS-crb[RNAi]/+; fkhGAL4, UAS-SerpCBD-GFP/UAS-pi3k92E[RNAi]*
H	w;; Rab11-YFP, fkhGAL4, ubiGAL80[ts]/Rab11-YFP*
I	w; UAS-crb[RNAi]/+; Rab11-YFP, fkhGAL4, ubiGAL80[ts]/Rab11-YFP*
J	w; UAS-pten[RNAi]/+; Rab11-YFP, fkhGAL4, ubiGAL80[ts]/Rab11-YFP*
K	w; UAS-crb[RNAi]/UAS-pten[RNAi]; Rab11-YFP, fkhGAL4, ubiGAL80[ts]/Rab11-YFP*
L	w; UAS-pi3k92E[RNAi]/+; Rab11-YFP, fkhGAL4, ubiGAL80[ts]/Rab11-YFP*
M	w; UAS-crb[RNAi]/UAS-pi3k92E[RNAi]; Rab11-YFP, fkhGAL4, ubiGAL80[ts]/Rab11-YFP*
O	w; Rab30-YFP/Rab30-YFP; fkhGAL4, ubiGAL80[ts]/+*
P	w; UAS-crb[RNAi], Rab30-YFP/Rab30-YFP; fkhGAL4, ubiGAL80[ts]/+*
Q	w; Rab30-YFP/Rab30-YFP; fkhGAL4, ubiGAL80[ts]/UAS-pten[RNAi]*
R	w; UAS-crb[RNAi], Rab30-YFP/Rab30-YFP; fkhGAL4, ubiGAL80[ts]/UAS-pten[RNAi]*
S	w; Rab30-YFP/Rab30-YFP; fkhGAL4, ubiGAL80[ts]/UAS-pi3k92E[RNAi]*
T	w; UAS-crb[RNAi], Rab30-YFP/Rab30-YFP; fkhGAL4, ubiGAL80[ts]/UAS-pi3k92E[RNAi]*
Figure 6—figure supplement 1
A	w;; Rab11-YFP, fkhGAL4, ubiGAL80[ts]/Rab11-YFP*
B	w;; Rab11-YFP, fkhGAL4, ubiGAL80[ts]/Rab11-YFP, UAS-pten2*
C	w; Rab30-YFP/Rab30-YFP; fkhGAL4, ubiGAL80[ts]/+*
D	w; Rab30-YFP/Rab30-YFP; fkhGAL4, ubiGAL80[ts]/UAS-pten2*

Immunostaining of salivary glands

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For all experiments, and in order to always compare equal timepoints of larval development, control and experimental genotypes were collected under the same conditions (see example in Figure 8). After growing at 29°C for approx. 50 hr, the salivary glands of non-wandering third instar larvae were dissected in ice cold Grace’s medium (Thermo Fisher Scientific). Corresponding control and experimental glands were mounted together directly on a slide (previously coated with embryo-glue; Figard and Sokac, 2011) and then fixed. In this way, all staining-conditions were always identical for controls and experimental samples. Depending on the antigen (see Table 3), fixation was done in 100% methanol at −20°C for 5 min or in 6% formaldehyde in Grace’s medium at RT for 15 min. For microtubule staining (Riparbelli et al., 1993), fixation was done in 100% methanol for 10 min followed by 5 min in acetone both at −20°C. Samples were washed at least 5 times with 0.1% Triton X-100 in 1xPBS (PBT) and blocked in 5% normal goat serum (NGS) in PBT (blocking solution) for 30 min at 4°C. Primary antibody staining was done in blocking solution over night at 4°C. Samples were washed at least 5 times with PBT before incubation with the appropriate secondary antibody in blocking solution for two hours at RT and washed again 5 times with PBT. The samples were covered with Vectashield (Vector Laboratories) and visualized using a Zeiss LSM 880 Airy upright single photon point scanning confocal system (ZEISS Microscopy, Jena, Germany) with a Zeiss iLCI Plan-Neofluar 63 × 1.3 Imm Korr DIC objective. In all cases, for any given marker, images were acquired under the same settings for laser power, PMT gain and offset. Maximal projections, merging and LUT-pseudocolor assignment were performed using Fiji (Schindelin et al., 2012). Image montage was done in Adobe Photoshop CS5 version 12.1 and when brightness, contrast and levels were adjusted, the modifications were linear and equally applied to the whole set of images. IMARIS 7.6 software was used to render the Video 8. Unless otherwise is stated, images are representative of at least three independent experiments, with at least three technical replicates in each experiment.

Table 3

List of antibodies and probes employed.

	Dilution	Fixation	Source
DAPI	1:200000	FA	Invitrogen Cat. D1306
Phalloidin Alexa Flour 488, 555	1:2000	FA	Invitrogen Cat. A12379, A34055
Alexa Flour 488-, 568- and 647 -conjugated	1:1000 - 1:2000		Invitrogen
Mouse antibodies
Anti-α-Spectrin	1:100	MeOH	DSHB 3A9
Anti-Coracle	1:200	MeOH	DSHB C566.9
Anti-Disc large	1:500	MeOH	DSHB 4F3
Anti-FasIII	1:4	MeOH	DSHB 7G10
Anti-αTubulin	1:2000	MeOH/Acetone	MPI-CBG Antibody facility, P. Keller
Rabbit antibodies
Anti-aPKC (C-20)	1:500	MeOH	Santa Cruz Biotechnology Cat. sc-216-G
Anti-Bazooka	1:200	MeOH	kindly provided by A. Wodarz (Wodarz et al., 1999)
Anti-Stardust	1:2000	MeOH	(Berger et al., 2007)
Anti-Cadherin99C	1:250	FA	kindly provided by D. Godt (Glowinski et al., 2014)
Anti-GFP	1:1000	FA	Invitrogen A-11122
Anti-Sinuous	1:8000	MeOH	kindly provided by G.J. Beitel (Wu et al., 2004)
Anti-β_HSpectrin	1:5000	MeOH	kindly provided by G. Thomas (Thomas and Williams, 1999)
Anti-KuneKune	1:5000	MeOH	kindly provided by M. Furuse (Nelson et al., 2010)
Anti-Phospho-Ezrin (Moesin)	1:500	FA	Cell Signaling Technology Cat. 3141
Anti-Moesin (Q480)	1:400	FA	Cell Signaling Technology Cat. 3150
Anti-MyosinV	1:2000	MeOH	(Pocha et al., 2011a)
Anti-DPatj	1:1000	FA	(Richard et al., 2006a)
Rat antibodies
Anti-Yurt	1:500	MeOH	kindly provided by U. Tepass (Laprise et al., 2006)
Anti-Stardust	1:2000	FA	(Berger et al., 2007)
Chicken antibodies
Anti-GFP	1:100	FA	Abcam Cat. Ab13970
Guinea pig antibodies
Anti-Crumbs 2.8	1:500	MeOH	(Richard et al., 2006a)
Anti-Par6	1:500	FA	kindly provided by A. Wodarz (Shahab et al., 2015)
DSHB - Developmental Studies Hybridoma Bank (Iowa city, Iowa, USA)
Invitrogen, Molecular Probes (Eugene, Oregon, USA)
Santa Cruz Biotechnology, Inc (Dallas, Texas, USA)
Cell Signaling Technology (Danvers, Massachusetts, USA)
Abcam plc (Cambridge, United Kingdom)

Live imaging of salivary glands

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Collection of control and experimental larvae was done as described above. For live imaging, the salivary glands were dissected in ice-cold Grace’s medium, mounted on the bottom of a Petri dish previously coated with embryo-glue (Figard and Sokac, 2011) and imaged directly using a Zeiss LSM 880 Airy upright single photon point scanning confocal system (ZEISS Microscopy, Jena, Germany) with a Zeiss W Plan-Apochromat 40 × 1.0 objective. Excitation was performed with 488 nm for GFP or YFP from an Argon Multiline Laser, and 561 nm from a Diode Pumped Solid State (DPSS) Laser for RFP, mTomato and Dextran-Rhodamine. For time-lapse imaging of Rab-YFP proteins, 10 steps (0.67 µm/step) were acquired every 5 s for 5 min. Using FIJI software, the original stack was scaled 2X with a bicubic average interpolation, filtered with a Gaussian Blur (Sigma = 1) and animation speed set of 16 fps. Final montage and rendering were made in Photoshop CC 2018. Unless otherwise is stated, images are representative of at least three independent experiments, with at least three technical replicates in each experiment.

Image quantifications

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The distribution and intensity levels of different markers were assessed using FIJI software. A flow-diagram of the analyses as well as all values obtained can be found in the accompanying Source Data. Briefly, to obtain the apical-to-basal fluorescence intensities of a particular marker, in a single-optical slice, individual straight lines (ROIs) were made from the apical membrane towards the basal membrane. The line width was set to 18 and all lines were arranged parallel to each other. Enough lines were made to cover the whole length of the salivary gland in the field of view (>70 μm) or a minimum of five cells per gland were covered (approx. 50 μm). The intensity values along the lines were obtained using the Multi Plot measurement option of FIJI. These intensity values were averaged along the length of the gland to obtain a single intensity distribution for one gland. The values for the line length were normalized to one and divided into 20 segments. The intensity values for each of the 20 segments was averaged and used to plot the final apical-to-basal fluorescence intensities.

To evaluate the apical-to-lateral ratios of a particular marker, in a single-optical slice, using the Multi-point tool of FIJI, a total of five dots (ROIs) were equally distributed along the apical membrane and five dots along the lateral membrane. The respective mean intensity values for apical and lateral membranes were obtained, averaged and the ratio was calculated. A minimum of four cells were evaluated for each gland.

For the quantification of the apical membrane (surface and volume), we analyzed the fluorescence of PLCδ-PH-EGF, to mark the plasma membrane including the PAMS, and DE-cadherin-mTomato, to distinguish the boundaries of the apical membrane, in Z-stacks acquired by confocal microscopy as described above. The plasma membrane was manually segmented using the Segmentation Editor plugin in Fiji Software. The labeled images obtained were subsequently analyzed using the 3D Object Counter plugin to obtain the values for surface and volume.

Transmission electron microscopy (TEM) and high-pressure freezing (HPF)

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Control and experimental larvae were collected as described above. Salivary glands were dissected on ice in 1xPBS and fixed with 2.5% glutaraldehyde, 2% paraformaldehyde in 1xPBS for 2 hr at RT, washed with 1xPBS, 3 times for 5 min at RT, post-fixed with 1% osmium tetroxide, 1.5% Potassium ferricyanide in water for 1 hr at 4°C. Samples were dehydrated in serial steps (30%, 50%, 70%, 90%, and 100%) Ethanol (EtOH) 5 min/step at 4°C, infiltrated with 1:3 EPON LX112/EtOH for 1 hr, 1:1 EPON LX112/EtOH for 1 hr, 3:1 EPON LX112/EtOH 1 hr, pure EPON LX112 overnight, and pure EPON LX112 for 2 hr. The salivary glands were embedded in rubber mold and polymerized for 24 hr at 60°C. 70 nm cross sections were obtained using an ultramicrotome and were picked up with formvar coated copper slot grid. Grids were stained with 2% uranyl acetate in water for 10 min and lead citrate for 5 min at RT.

For HPF, salivary glands were dissected on ice in 1xPBS and frozen afterwards using a Leica ICE high pressure freezer (Leica Microsystems, Germany). Media of frozen samples was substituted with a cocktail containing 0.1% uranyl acetate and 4% water in acetone at −90°C. Samples were transferred into ethanol at −25°C. Then, samples were embedded into a Lowicryl HM20 resin (Polysciences, Inc, Germany) followed by UV polymerization at the same temperature. Semi-thin sections (300 nm) were cut and contrasted as described above for chemically fixed samples.

To quantify the density of microvilli, five lines, 1 μm in length each, were drawn adjacent to the apical membrane and distributed over the span of a cell. Five identical lines were drawn parallel to the first ones but at exactly 1 μm away from the first group, that is 1 μm above the apical membrane. The microvilli crossed by these lines were counted and the average per cell is presented in the Figure 2M.

Image acquisition was done using a Tecnai 12 (FEI, Thermo Fisher Scientific) with a standard single tilt holder with a TVIPS TemCam F214A (TVIPS, Gauting, Germany) digital camera at 440x for an overview of the whole salivary gland cross section, and 1200x for single-cell overview and 13000x for subcellular structures. Images are representative of 3 independent experiments, at least 3–5 different salivary glands were analyzed per genotype.

Dextran-permeability assay, lysosomal activity and treatment with inhibitors

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For Dextran permeability assays we adapted the method from Lamb et al. (1998). Briefly, the salivary glands were dissected as described above, and incubated 15 min at RT in Grace’s medium containing 40 µg/ml Dextran-Rhodamine B 10,000 MW (Molecular Probes D1824), and immediately imaged after incubation. For lysosomal activity analysis, the salivary glands were incubated 30 min at RT in Grace’s medium containing 150 nM LysoTracker Red DND-99 (Molecular Probes L7528), and immediately imaged after incubation. For the inhibition of PTEN, the salivary glands were incubated 30 min at RT with 10 µM VO-OHpic trihydrate (Santa Cruz Biotechnology sc-216061). DMSO was used as vehicle and its final concentration was 0.25 µL/mL in Grace’s medium. Images are representative of 3 independent experiments, with at least three technical replicates in each experiment.

Western blot

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Control and experimental larvae were collected as described above. At least 15 whole salivary glands were dissected per genotype on ice in 1xPBS, immediately frozen in liquid nitrogen and kept at −80°C. For protein extraction, the glands were homogenized with a plastic pestle in 1% PBT lysis buffer and pelleted at 20,000 × g for 5 min at 4°C. Protein content from recovered supernatants was measured using BCA (manufacturer protocol, Invitrogen) and equal protein amounts were loaded per lane and separated on 12.5% SDS-PAGEs. Proteins were transferred to nitrocellulose membranes, blocked with 5% milk powder in 0.1% Triton X-100 in 1xPBS and blots were probed for GFP (rabbit anti-GFP 1:2000, Molecular Probes A11122), Crb (rat anti-Crb2.8 1:1000, see supplementary Table 3) and Tubulin (mouse anti-αTubulin 1:1000, see supplementary Table 3).

Food intake assay and puparium formation rate

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For the food intake assay we adapted a protocol reported by Deshpande et al. (2014). Briefly, eggs from the appropriate genotypes were collected overnight on apple juice agar plates and transferred into normal food containing blue bromophenol (500 mg/L). As indicated in Figure 8, after 2 days of incubation at 29°C, larvae were briefly rinsed in iced cold PBS to remove attached food. Then, for each replica, 60 larvae were manually transferred into an Eppendorf tube containing 220 µL PBS + 0.1% Triton X-100 (PBST), and frozen immediately in dry ice. The samples were thawed and homogenized with a rotor pestle, centrifuged at 10 000 x g at 4°C for 10 min. The supernatant was diluted 1:2 into PBST for absorbance measurement at 680 nm. The standard curve was made by diluting 200 µL of liquefied bromophenol-containing food into 800 µL PBST, mixed in a ThermoMixer (Eppendorf, Germany) block at 900 rpm 80°C for 30 min, followed by centrifugation at 10 000 x g. The supernatant was serially diluted in PBST and the serial dilutions measured at 680 nm using a FLUOstar Omega (MBG Labtech, Germany).

Figure 8

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Schematic representation of the experimental setup.

Indicated in the workflow are the times and incubation temperatures, as well as the time for dissections.

For the assessment of the pupariation rate, eggs from the appropriate genotypes were collected for one hour on apple juice agar plates. Afterwards, 20 eggs were transferred to a new apple juice plate containing fresh yeast paste. To score the puparium formation, the plates with the embryos were incubated at 29°C for 72 hr and afterwards were assessed every 3 hr (excluding the overnight period). All newly appearing pupae were counted until all larvae had pupariated. To determine the puparium formation rate, the number of newly formed pupae at a given time point are divided by the total number of pupated animals. For the graphs of larval development speed (Figure 1H, Figure 1—figure supplement 1KK, Figure 6W) percentages were added up for the consecutive time points (also see source data).

Statistical analyses

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All statistical analyses were performed using GraphPad Prism 8. Statistical significance was calculated in unpaired t-test or a one-way analysis of variance (ANOVA) followed by a Dunnett’s multiple-comparison when experimental groups are specifically compared only to control conditions, or a Tukey’s multiple comparison test when all groups are compared to each other. P values are indicated in each corresponding graph.

Data availability

We provide as source data files all the data used for statistical analyses and generation of all graphs. These files are sorted according to the figure and the corresponding figure supplements, which correspond to Figure 1 and its supplements, Figure 2, Figure 3 and its supplements, Figure 5 and its supplements, and Figure 6.

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Decision letter

Utpal Banerjee

Senior and Reviewing Editor; University of California, Los Angeles, United States

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

Acceptance summary:

Crumbs is a widely studied molecule for its role in defining apical aspects of epithelial organisation. However, this study distinguishes itself in convincingly demonstrating that Crb (and other components of the Crb complex) are required for the apical organization of β-Spectrin and actin, which in turn are required for the recruitment of Myosin 5 and the Rabs involved in apical secretion (Rab6, Rab11 and Rab30). Combined with data that link lipid phosphatase function, these studies potentially reveal a mechanistic aspect of Crb in the organisation of the apical domains of polarized epithelia, linking Crb activity to both organization of the cytoskeleton and to lipid homeostasis. Given the importance of CRB and these processes to development and cancer, this work demands further analysis and validation.

Decision letter after peer review:

[Editors’ note: this article was originally rejected after discussions between the reviewers, but the authors submitted for reconsideration. The first decision letter after peer review is shown below.]

Thank you for submitting your work entitled "Crumbs organizes the apical transport machinery by negatively regulating Pten in Drosophila larval salivary glands" for consideration by eLife. Your article has been reviewed by a Senior Editor, a Reviewing Editor, and three reviewers. The reviewers have opted to remain anonymous.

Our decision has been reached after consultation between the reviewers. Based on these discussions and the individual reviews below, we regret to inform you that your work will not be considered further for publication in eLife at this time.

As you can see from the reviews, your manuscript generated a lot of discussion and suggestions. Following extensive post review consultation, the referees were unanimous in their view that the work is not far enough along to warrant an eLife publication at this time. Also, since the questions raised pertain as deeply to mechanistic issues, as they do to specific experiments, it was not possible to create a simple set of experiments doable over a two-month revision period that would satisfy the reviewers. This sentiment was more apparent in the discussions than even those in the reviews below. As you know, eLife policy does not recommend multiple revisions, and the reviewers (including reviewer 3, who was more positive) really felt that it will not be fair to the authors to unreasonably delay the process if you wish to submit elsewhere.

Reviewer #1:

The article by Lattner, Leng, Knust, Brankatschk and Flores-Benitez describes the role of Crumbs in organizing the apical membrane of the Drosophila salivary gland cells.

Crumbs is a known apical large transmembrane protein that interacts with Stardust and binds FERM proteins via its FERM binding domain. It also binds β-Spectrin.

In the first part of the manuscript, the authors show that in salivary glands, loss of Crumbs does not affect the localization of cytoplasmic apical markers but affect the delivery of a generic TM protein CD8-RFP. Loss of Crumbs does not affect AJ but affects a bit SJ, albeit not the extent of compromising the epithelial integrity.

However, loss of Crumbs leads to the appearance of plasma membrane apical sacs (PAMS), per apical PM. PAMS bears all the characteristics to PM including the presence of some microvilli.

Loss of Stardust leads to similar defects. Loss of β-Spectrin, also, albeit to a lesser extent. Crumbs now present in these PAMS.

Moe binds Crumbs and loss of Moe leads to similar phenotype to a certain extent. The results are not as convincing.

In the second part of the manuscript, in what appears to be a jump from one subject to another, the authors examine the localization of many Rabs and found that the localization of 3 is particularly affected in a Crumbs mutant. Namely the localization of Rab6, Rab11 and Rab30 near the apical cortex is lost and that they end up at the basal side.

In a third part, the authors address the role of PI(4,5)P₂ in the apical membrane. For this, they use a probe that specifically binds PI(4,5)P₂ and show that the probe localizes mostly to the apical membrane. In the absence of Crumbs, PI(4,5)P₂ is no longer localized apically. Inhibiting Pten pharmacologically and genetically modulates the pool of PI(4,5)P₂ at the apical membrane. Importantly, Pten depletion suppress the formation of the PAMs that are induced by Crumbs depletion.

They conclude that Crumbs through its binding to Stardust, β-Spectrin and Moesin controls integrity of the apical membrane by downregulating Pten activity. When Pten is no longer downregulated, it leads to apical membrane extension leading to PAMS.

The manuscript represents a lot of work combining quite a lot of techniques from fly genetics to light and electron microscopy. It is also well performed. As it is, however, it is hard to get a sense of what the paper is about, what the question is, what is new and important, and overall how Crumbs is required in the maintenance of the apical membrane microvilli and prevents the formation of plasma membrane sacs (PAMS). The story telling appears disconnected. The depletion of many factors leads to the formation of PAMS but the reader is not given a mechanistic sense of how these factors are connected in promoting PAMS formation.

Second, the relationship between apical secretion and PAMS formation is not clear. As a result, the reviewer does not have a sense of their role. How are they connected to apical secretion of saliva formation? True the larvae grow slower by a couple of hours but how does it matter?

The reviewer is not even sure what are the PAMS about. Are they a consequence of PM expansion? If so, why does it invaginate inward? Also, the microvilli do not form. Is it how the PM looks elongated? Why are the microvilli not forming? Are TM proteins such as Prominin not delivered properly? Is this lack of delivery related to PI(4,5)P₂ level? How does Crumbs regulates Pten activity?

Third, none of the microscopy is not quantified. Especially PAMS appear quite heterogeneous, at least from one genotype to the next. Are they all the same? How penetrant?

Last, some sections of the manuscript are written in a pushy manner and this should be remedied.

These points are detailed below:

1) The choice of apical cargo is problematic. Both rely on generic probes.

1.1) The first is CD8-RFP that is not an endogenous protein and that is by default transported to the apical membrane.

What are the dots observed in Crumbs mutant?

1.2) The second is a lectin, PNA that binds the carbohydrate sequence Gal-β(1-3)-GalNAc.

The reviewer would perhaps understand the figure better if labeled PNA is used as a labeling tool (as an antibody). Instead PNA is expressed as a GFP fusion protein under the control of UAS. As it does not have a TM segment, it would localize to the cytoplasm of the salivary gland cells.

If this is the case, the reviewer does not understand how it could bind to glycoproteins as their sugar moieties are in the lumen of organelles (Golgi) and facing the extracellular medium, so away from PNA.

As a result, the reviewer is mighty puzzled as that the dots represent in WT salivary glands Furthermore, the reviewer does not understand the PNA pattern observed in Crumbs mutant? The bright PNA patches appear to be facing the lumen of the tube. Why would glycoproteins be now present at the PM? The reviewer has no notion of what is observed in this figure in term of glycoprotein transport. Is PNA secreted at all? How?

It would not matter much but these two tools are used at many places in the manuscript and the conclusions made are strong. Therefore, they need to be documented in a much better fashion and establish good standard for using this proxy.

1.3) Furthermore, while a large part of the manuscript is performed with endogenous saliva proteins (or endogenously tagged in the genome), why did the authors not study endogenous cargo like the saliva proteins Sgs1-8 (at least some of them). Or the ORF encoded by the gene Saliva.

These are obviously apical cargos and would be greatly preferable than the artificial ones used in the manuscript. Their secretion should be observed in Crumbs mutant. This has been done indirectly in weighing the larvae that cannot forage in the food.

1.4) Also, intriguingly, Crumbs remodeling of the apical PM only occurs in foraging larvae, not wandering when they start synthesizing and secreting the glue. Yet, is Glu protein secretion impaired in Crumbs mutant?

2) Related to the choice of cargo, one clear phenotype of the loss of Crumbs is the loss of microvilli.

2.1) Is it how the PM appears elongated? By losing microvilli?

2.2) Why are the microvilli not forming? Are TM proteins such as Prominin not delivered properly? In mammals, it is clearly required for microvilli formation. Prominin and prominin-like should definitely be followed in WT and Crumbs mutants (in fact prominin appears to genetically interact with Crumbs). The prediction is that it should be delivered to the apical membrane and WT and not in Crumbs mutants.

2.3) The density of the microvilli needs to be quantified in WT and Crumbs mutants. Only then can the authors say that the decrease is dramatic.

3) The Crumbs phenotype is clearly different from embryos where Crumbs have a function in keeping the apico-basal polarity intact as well as the cell adhesion. The reviewer is confused as to why this is and what underlies the difference? Is it because of the absence of microvilli?

Can this be tested? Can other hypothesis put forward to explain the differences? What about other epithelia such as the follicle cells?

4) The PAMS are inward invaginations. But the degree to which they appear depends on the genetic background. They sometimes appear big and deep and sometimes not.

4.1) This needs to be seriously quantified for each background, for instance how many cells exhibit a shallow invagination, a deeper one and a very deep pit which clear morphological definition? For instance, some depletion in WT leads to shallow invaginations. Are those significant?

4.2) Are some PAMs found completely sealed and no longer accessible? This should also be quantified as it is much more connected to the disease.

4.3) If most of them are continuous with the PM, what is their relevance? What does it affect secretion? Or are the two phenotypes not connected? In this regard, looking at prominin or any other TM proteins involved in microvilli formation would result in a clearer understanding of the message.

5) In subsection “Crb controls the apical membrane homeostasis by negatively regulating Pten activity”, the authors state “in addition, these results show that the Crb protein complex is essential for the regulation of Pten activity in SGs.”

“….suggests that accumulation of PI(4,5)P₂ in Crb KD glands is likely due to an increase in Pten activity rather than a change in its localization.”

To state this, Pten activity should be measured directly, using extracts from WT and Crumbs mutant salivary glands with known substrates such as PI(3,4,5)P₃. A probe assessing PI(3,4,5)P₃ level should work if this cannot be done in vitro.

6) Pten is involved in many different pathways. It is a tumor suppressor and acts in negatively regulating the AKt pathway.

6.1) Is there a link between the PAMS and the other phenotype?

6.2) Is there any thoughts or data on how Crumbs regulates Pten activity? Do they form a complex

7) Rabs:

7.1) The reviewer suggests to put this part after the PI(4,5)P₂/Pten section as the transition from Myo5 to Rabs is really abrupt and does not make a lot of sense.

7.2) Why was Rab35 not considered as it also loses its apical localization in Crumbs mutant?

7.3) What is the role of these Rabs near the apical membrane? Are they active? Rabs mediate membrane traffic steps and these in particular from the TNG to the PM (Rab6) or from endosomes to the PM (Rab11), but none of this is described and tested. How does loss of Crumbs leads to their mislocalisation? Is it only PI(4,5)P₂ composition? How?

Is any of the Rab depletion lead to PAMs formation?

So overall, the manuscript is the description of a number of factors (related to the cytocortex-Rab network) whose depletion leads to PAMS. But there is very little mechanistic insights or sense of what is new.

Reviewer #2:

In this paper, the authors remove Crb (or other Crb complex protein) function in mid-third instar larval SGs and determine the consequences. What they observe is that many aspects of SG organization are unaffected; overall SG morphology, cell polarity, barrier function, as well as the localization of a number of membrane domain-specific proteins appear largely normal. Apical secretion of both an apical membrane protein (CD8-RFP) and glycoproteins (those detected by PCNA binding) are diminished, however, with the reduction in Crb or with the reduction of Sdt (a component of the Crb complex). Reduced Crb also results in decreases in apical accumulation of F-actin, β_Heavy-Spectrin and MyoV (the myosin that has been implicated in secretion). Also observed are decreases in apical localization of three Rab proteins that have previously been implicated in apical secretion: Rab6, Rab11 and Rab30. The reduction in Crb also results in the invagination of the apical domain to form structures the authors refer to as PAMS (PI(4,5)P₂ and Phospho-Moesin enriched apical membrane sacs). To make sense of the observed molecular changes, the authors also determined the effects of reducing β_Heavy-Spectrin, MyoV, and the enzymes involved in controlling PI(4,5)P₂ levels in cells: Skittles (a PIP kinase that makes PI(4,5)P₂), Pten (which dephosphorylates PI(3,4,5)P₃ to make PI(4,5)P₂) and PI3K (which phosphorylates PI(4,5)P₂ to make PI(3,4,5)P₃). The reduction in β_Heavy-Spectrin and of MyoV also diminishes secretion and promotes formation of PAMs, although not to the same level as reduction in Crb. The authors also show that reduction of Pten activity as well as increases in P13K can reduce the formation of PAMs associated with reduced Crb function, as well as restoring the apical localization of Rab6 and Rab11. Based on these experimental findings, the authors propose the following model: Crb is required to maintain the proper amount and organization of the apical domain by stabilizing the apical cytocortex (through β_Heavy-Spectrin and F-actin), that the Crb-β_Heavy-Spectrin complex facilitates apical secretion in larval SGs by maintaining apical MyoV. The authors also conclude that Crb normally limits PI(4,5)P₂ accumulation by limiting Pten activity (but not Pten accumulation since levels appear unchanged between controls and Crb knockdown).

Essential revisions:

1) Most of the phenotypes are not quantified and should be. When the authors say that PAM formation is reduced or increased or that secretion is reduced, we are relying on the limited number of images that can be shown and an impression the authors have of the data, rather than any quantification.

2) The authors make a good case that PI(4,5)P₂ is required for the formation of PAMs, but they do not make a very good case that Crb works through Pten to increase PI(4,5)P₂ levels. From the images provided, it is not at all clear that overall apical PI(4,5)P₂ levels are any higher in the Crb knockdown than in WT control SGs, although the apical distribution does seem to change – less in the PM and more in PAMs. Reducing PI(4,5)P₂ (which is known to bind and activate Moesin) does indeed appear to reduce PAM formation and increasing PI(4,5)P₂ does appear to increase PAM formation but that does not indicate that Crb normally acts to limit Pten activity; it only says that high levels of PI(4,5)P₂ are required for this loss of Crb phenotype. Indeed, the Pten overexpression phenotypes does seem to increase the apical domain area but it doesn't seem to create structures that have the same morphology as the PAMs. So, either change the conclusions/title of the manuscript or provide better evidence that PI(4,5)P₂ levels are indeed higher with loss of crb.

3) Skittles is not the only PIP kinase encoded in the Drosophila genome, perhaps explaining the relatively mild effects of skittles knockdown on the loss of Crb phenotypes. The authors should acknowledge the existence of this other protein when they note the relatively mild effects.

4) Although the authors claim that loss of Crb does not affect AJs (based on TEMs and DE-cad immunostaining), it does appear from the images that levels of DE-cad found near the apical surface are notably reduced (perhaps there is also an increase in vesicular staining of DE-cad). Thus, the authors need to look at this more carefully and indicate that there is some reduction of the AJ-localized DE-cad, or clearly demonstrate that levels are unaffected.

5) There seem to be some PAMs with microvilli-like structures and other similarly large vesicular structures with no microvilli-like structures. Are the latter only found with Crb knockdown or are they also found in controls? If they are only found in the Crb knockdown, what are they proposed to be?

6) This may be challenging to determine, but it would be helpful to know if the Crb knockdown actually changes the total amount of apical surface area. Do control SG cells have the same amount of apical area if, in the Crb knockdown cells, one were to include the apical area found within the PAMs? This is related to the observation that the pleated septate junctions in the Crb knockdown SGs seem to be much more convoluted than those in WT (Figure 1—figure supplement 2), suggesting that there might be an increase in membrane in those domains. This might suggest that with loss of Crb, some of the apical membrane is being converted to basolateral membrane.

7) Importantly, is this study showing us that Crb is doing what it has been known to do – controlling the balance of apical versus basolateral domains and providing some clues as to the mechanism or is this study really uncovering a new and unrelated role for Crb? Either way the study is interesting, but exploring the first possibility is worthwhile since it has not been clear how Crb normally controls apical specification.

Reviewer #3:

This manuscript explores the roles of the polarity protein Crumbs (Crb) in a particular type of secretory epithelium: the salivary glands of the Drosophila larva. The authors provide data consistent with a model whereby Crb regulates apical secretion by (1) maintaining the active pool of a subset of Rab proteins at the apical domain and (2) negatively modulating Pten activity, thereby modulating the lipid composition of the apical membrane. Based on a series of loss-of-function/knockdown experiments, the authors propose that both these roles are least in part mediated by the effects of Crb – complexed with β-Spectrin – on Myosin V localisation.

The manuscript is clearly written and the data is generally convincing. The following points/conclusions are only weakly supported by the data provided in the current version of the manuscript, and would benefit from a few additional experiments:

1) All conclusions re: crb loss-of-function phenotypes (e.g. "defects in apical secretion are not due to an overall disruption of cell polarity", or "this strategy does not affect embryonic development") are currently based on knockdown experiments. These may only reduce – rather than abrogate – Crb expression, and do so at a currently undefined developmental point. Clonal mutation of crb in salivary glands and/or temporally controlled knockdown experiments would allow the authors to draw stronger conclusions about the roles of Crb (or lack thereof) in establishing vs. maintaining overall epithelial polarity in the salivary gland, as well as their temporal requirement. Related to this, different fkh-Gal4s lines have been generated with distinct expression. Is the expression of the one used in this particular study really confined to salivary glands? Most lines are also expressed in other portions of the intestine.

2) The authors explore physiological aspects of Crb-mediated salivary gland secretion very superficially. This seemed to me a missed opportunity. If they wish to make any statements about Crb regulating the "physiological activity of the salivary gland" or promoting "efficient apical secretion of glycoproteins" (as currently stated in the abstract), the authors need to show effects on endogenous secretion – for example by visualizing the effect of crb knockdown on a protein normally secreted by the salivary gland. Related to this, the authors assume but do not show a reduction in food intake following salivary gland-specific crb knockdown. Is food intake reduced following crb knockdown? Does Pten knockdown rescue the feeding and developmental timing defects of crb knockdown larvae? Finally, what happens to Crb expression/localisation and/or downstream targets around the saliva to glue switch? In other words, is Crb permissive or instructive in the context of salivary gland secretion?

3) The authors should try to rule in/out increased endocytosis (rather than reduced apical secretion) as one of the reasons for the observed phenotypes; it seems to me that at least some of the Rab targets are involved in recycling as well as secretion. Also, the appearance of these novel intracellular microvilli-containing sacs and the concurrent reduced density of microvilli on the cell surface may both also be consistent with increased endocytosis; I am not an expert in this but, as far as I know, microvilli are not "trafficked" to the membrane in secretory compartments? Related to this, it may be informative to test the Rab signature of PAM sacs in both Crb/β-Spectrin knockdowns – might this shed light on the origin of these compartments?

https://doi.org/10.7554/eLife.50900.sa1

Author response

Reviewer #1:

The article by Lattner, Leng, Knust, Brankatschk and Flores-Benitez describes the role of Crumbs in organizing the apical membrane of the Drosophila salivary gland cells.

Crumbs is a known apical large transmembrane protein that interacts with Stardust and binds FERM proteins via its FERM binding domain. It also binds β-Spectrin.

In the first part of the manuscript, the authors show that in salivary glands, loss of Crumbs does not affect the localization of cytoplasmic apical markers but affect the delivery of a generic TM protein CD8-RFP. Loss of Crumbs does not affect AJ but affects a bit SJ, albeit not the extent of compromising the epithelial integrity.

However, loss of Crumbs leads to the appearance of plasma membrane apical sacs (PAMS), per apical PM. PAMS bears all the characteristics to PM including the presence of some microvilli.

Loss of Stardust leads to similar defects. Loss of β-Spectrin, also, albeit to a lesser extent. Crumbs now present in these PAMS.

Moe binds Crumbs and loss of Moe leads to similar phenotype to a certain extent. The results are not as convincing.

We have the impression that the reviewer is mistaken at this point. Although certainly an interesting idea, we did not and do not show any data for silencing of Moesin, but for silencing Myosin V. We used Moesin or phospho-Moesin as marker for the apical cortex.

In the second part of the manuscript, in what appears to be a jump from one subject to another, the authors examine the localization of many Rabs and found that the localization of 3 is particularly affected in a Crumbs mutant. Namely the localization of Rab6, Rab11 and Rab30 near the apical cortex is lost and that they end up at the basal side.

Rab6 and Rab11 have a complex cellular distribution, as these proteins localize close to the apical plasma membrane, but they are also found associated with punctae-like compartments throughout the cell (Dunst et al., 2015). In Crumbs knock-down exclusively the apical portion of Rab6, 11 and 30 is lost, without affecting their localization elsewhere in the cell. Thus, we would like to clarify that we do not observe nor stated that they “end up at the basal side”.

In a third part, the authors address the role of PI(4,5)P₂ in the apical membrane. For this, they use a probe that specifically binds PI(4,5)P₂ and show that the probe localizes mostly to the apical membrane. In the absence of Crumbs, PI(4,5)P₂ is no longer localized apically. Inhibiting Pten pharmacologically and genetically modulates the pool of PI(4,5)P₂ at the apical membrane. Importantly, Pten depletion suppress the formation of the PAMs that are induced by Crumbs depletion.

Our results show that PI(4,5)P₂ apical levels are increased in the absence of Crumbs. This was stated along the first version of the manuscript and illustrated as well (old Figure 5). However, to solidify our findings, we added the quantifications (Figure 5 of the current version) of our microscopy data.

They conclude that Crumbs through its binding to Stardust, β-Spectrin and Moesin controls integrity of the apical membrane by downregulating Pten activity. When Pten is no longer downregulated, it leads to apical membrane extension leading to PAMS.

The manuscript represents a lot of work combining quite a lot of techniques from fly genetics to light and electron microscopy. It is also well performed. As it is, however, it is hard to get a sense of what the paper is about, what the question is, what is new and important, and overall how Crumbs is required in the maintenance of the apical membrane microvilli and prevents the formation of plasma membrane sacs (PAMS). The story telling appears disconnected. The depletion of many factors leads to the formation of PAMS but the reader is not given a mechanistic sense of how these factors are connected in promoting PAMS formation.

In brief, Crb is a well characterized determinant essential for the establishment of apico/basal polarity. However, it is less clear to what extent Crb is required to maintain the apical identity in polarized cells and if so, how Crb mechanistically regulates the apical membrane domain. Here we show in Drosophila salivary gland cells that Crb negatively regulates the phosphatase Pten required to maintain the apical PI(4,5)P₂/PI(3,4,5)P₃ ratio essential for the membrane identity. Moreover, deregulated Pten activity is changing the localization of (at least) the apical Rab machinery and disrupts the link between the plasma membrane and the cytoskeleton. In consequence, the apically-targeted secretion of proteins is impaired resulting in consequences for the whole organism.

Second, the relationship between apical secretion and PAMs formation is not clear. As a result, the reviewer does not have a sense of their role. How are they connected to apical secretion of saliva formation?

The reviewer is correct, it is not easy to explain the formation of PAMS with our results at hand. PAMS are membrane sacs enriched in PI(4,5)P₂ and Phospho-Moesin that are never present in control salivary gland cells (see subsection “Crb regulates apical membrane levels of PI(4,5)P₂”). We consider PAMS as morphological manifestations of failed apical membrane transport, although we cannot completely exclude the possibility that a weakened apical cytocortex contributes to their formation. In fact, the link between PAMS, secretion and PI(4,5)P₂ levels is such that when PI(4,5)P₂ levels are lowered in Crb-depleted SGs (by knocking down Pten or by using its inhibitor VO-OHpic), the formation of PAMS is suppressed (Figure 5) as well as the secretion defects (Figure 6).

True the larvae grow slower by a couple of hours but how does it matter?

The reviewer is not even sure what are the PAMS about. Are they a consequence of PM expansion? If so, why does it invaginate inward? Also, the microvilli do not form. Is it how the PM looks elongated? Why are the microvilli not forming? Are TM proteins such as Prominin not delivered properly? Is this lack of delivery related to PI(4,5)P2 level? How does Crumbs regulates Pten activity?

Third, none of the microscopy is not quantified. Especially PAMS appear quite heterogeneous, at least from one genotype to the next. Are they all the same? How penetrant?

Last, some sections of the manuscript are written in a pushy manner and this should be remedied.

These points are detailed below:

Detailed answers to these points are given below in the specific comments.

Our experimental approach is based on the organ-specific KD of Crb. As a matter of fact, induced systemic Crb knock-down is lethal. The defects in SG secretion induced by loss of Crb do matter at the level of the whole organism, as we show by quantifying the effects on developmental speed (Figure 1H, Figure 1—figure supplement 1KK and Figure 6W) and food intake (Figure 6V). While others have suggested that SGs might be dispensable for larval survival (Jones et al., 1998), our assays are sensitive enough to report these effects. It is important to note that, in the work of Jones et al. (1998), several technical details of the assays reported in Table 2 are missing (for example, stage of the larvae when isolated, duration of the assay, and incubation conditions), but nevertheless they report a lethality of 72.8% in trans-heterozygous eyg^C1/eyg^C53ductless larvae, which highlights the importance of the SGs in larval survival.

We re-structured our manuscript to improve the delivery of our message. We addressed the role of the Rab proteins in the formation of the PAMS (Figure 4—figure supplement 3) and the relevance of proper secretion for the pupation rate and food intake (Figure 6V,W). We included an analysis of the microvilli density (Figure 2M) as well as the size and frequency of PAMS (Figure 5—figure supplement 1E and Subsection “Crb regulates apical membrane levels of PI(4,5)P₂”) and included these points along our Discussion about their significance and their relation to the secretion defects.

Instead of Prominin, we analyzed the localization of endogenous Cadherin99C, a TM protein regulating microvilli structure (Figure 1I,J and Figure1—figure supplement 1CC,DD).

We provide additional evidence that Crb regulates PI(4,5)P₂ levels by regulating Ocrl apical localization (Figure 5L-Q) and updated our Discussion about the connections between Crb, β_Heavy-Spectrin and MyosinV with Pten2, and about the role of Crb in maintaining the localization of Pten at the apical membrane (Figure 5I-K). We provide quantifications (and the corresponding source data) to most relevant microscopy data.

1) The choice of apical cargo is problematic. Both rely on generic probes.

1.1) The first is CD8-RFP that is not an endogenous protein and that is by default transported to the apical membrane.

We include the analysis of two different apical proteins. By immunolocalization of endogenous Cadherin99C and live imaging of a Venus-tagged version of Stranded at Second, we confirmed our previous results using CD8-RFP (Figure 1, Figure 1—figure supplement 1, Figure 2 and Figure 3).

What are the dots observed in Crumbs mutant?

These represent most likely endosomes and lysosomes. Similar punctae have also been observed in models for MVID (see for example Mosa et al., 2018). Other published work also suggests that impaired secretion results in enhanced lysosomal activity to degrade the accumulated material. We have included this point in subsection “Crb regulates apical membrane organization via the apical cytocortex”.

1.2) The second is a lectin, PNA that binds the carbohydrate sequence Gal-β(1-3)-GalNAc.

The reviewer would perhaps understand the figure better if labeled PNA is used as a labeling tool (as an antibody). Instead PNA is expressed as a GFP fusion protein under the control of UAS. As it does not have a TM segment, it would localize to the cytoplasm of the salivary gland cells.

If this is the case, the reviewer does not understand how it could bind to glycoproteins as their sugar moieties are in the lumen of organelles (Golgi) and facing the extracellular medium, so away from PNA.

As a result, the reviewer is mighty puzzled as that the dots represent in WT salivary glands Furthermore, the reviewer does not understand the PNA pattern observed in Crumbs mutant? The bright PNA patches appear to be facing the lumen of the tube. Why would glycoproteins be now present at the PM? The reviewer has no notion of what is observed in this figure in term of glycoprotein transport. Is PNA secreted at all? How?

It would not matter much but these two tools are used at many places in the manuscript and the conclusions made are strong. Therefore, they need to be documented in a much better fashion and establish good standard for using this proxy.

We used the PNA line listed in the Bloomington Stock Center as a tool to analyze glycoproteins. However, the FlyBase has no further information to clarify the concerns raised by the reviewer. Therefore, we opted to use other available lines to analyze apical secretion. After testing different lines, we decided to repeat all experiments using a construct containing the chitin binding domain of Serpentine tagged with GFP (UAS-SerpCBD-GFP). This construct is a well-established apical secreted cargo and has been used as a proxy to analyze apical secretion (Luschnig et al., 2006; Kakihara et al., 2008; Förster et al., 2010; Petkau et al., 2012; Dong et al., 2013; Dong et al., 2014; Bätz et al., 2014). Using this tool, we confirmed the results obtained with the PNA line, and thus exchanged all figures with the ones obtained with SerpCBD-GFP (Figure 1, Figure1—figure supplement 1, Figure 3, Figure 3—figure supplement 2, Figure 6).

1.3) Furthermore, while a large part of the manuscript is performed with endogenous saliva proteins (or endogenously tagged in the genome), why did the authors not study endogenous cargo like the saliva proteins Sgs1-8 (at least some of them). Or the ORF encoded by the gene Saliva.

These are obviously apical cargos and would be greatly preferable than the artificial ones used in the manuscript. Their secretion should be observed in Crumbs mutant. This has been done indirectly in weighing the larvae that cannot forage in the food.

As we described in the manuscript, we analyzed the salivary glands at a stage where the larvae are feeding, which is almost two days before the expression of Sgs1-8 proteins is detectable. Therefore, none of the glue proteins are useful for our analyses, and indeed, glue secretion is not affected by the loss of Crb (see Video 1 and Video 2). Regarding the suggested ORF encoded by the gene Saliva, it is important to note that the product of this gene does not form part of the saliva, but it is a transmembrane transporter of sugar that is highly expressed in the salivary gland (FlyBase). Due to this fact, it was named Saliva, but it is not predicted to be a secreted protein. See also answer to reviewer #3 point 2.

1.4) Also, intriguingly, Crumbs remodeling of the apical PM only occurs in foraging larvae, not wandering when they start synthesizing and secreting the glue. Yet, is Glu protein secretion impaired in Crumbs mutant?

Glue secretion (monitored following Sgs3-GFP) occurs normally (see Video 1 and Video 2). In fact, pupae attach properly to the walls of the vials.

2) Related to the choice of cargo, one clear phenotype of the loss of Crumbs is the loss of microvilli.

2.1) Is it how the PM appears elongated? By losing microvilli?

We have quantified the density of microvilli (Figure 2M). Unfortunately, we cannot measure the absolute amount of apical and lateral membranes, including the amount of membrane in the microvilli. For this, specific algorithms and computational analysis would be necessary, which is out of the scope of our study. As to how or why the microvilli are not forming, we have included different possibilities into the updated Discussion section.

2.2) Why are the microvilli not forming? Are TM proteins such as Prominin not delivered properly? In mammals, it is clearly required for microvilli formation. Prominin and prominin-like should definitely be followed in WT and Crumbs mutants (in fact prominin appears to genetically interact with Crumbs) The prediction is that it should be delivered to the apical membrane and WT and not in Crumbs mutants.

We appreciate the suggestions of using Prominin to analyze the microvilli phenotype, however this protein is barely expressed in the salivary glands according to FlyBase. Indeed, the genetic interaction between Prominin and Crumbs has been reported in the eye, where Prominin is very abundant. Instead, we decided to analyzed the localization of Cadherin99C, a protein that regulates microvillar length (Chung and Andrew, 2014 and references within). Our results indicate that Cad99C is not properly localized to the apical aspect of Crb deficient cells (Figure 1 and Figure 1—figure supplement 1) and we included a Discussion section about these results.

2.3) The density of the microvilli needs to be quantified in WT and Crumbs mutants. Only then can the authors say that the decrease is dramatic.

We have quantified the density of microvilli (Figure 2M).

3) The Crumbs phenotype is clearly different from embryos where Crumbs have a function in keeping the apico-basal polarity intact as well as the cell adhesion. The reviewer is confused as to why this is and what underlies the difference? Is it because of the absence of microvilli?

Can this be tested? Can other hypothesis put forward to explain the differences? What about other epithelia such as the follicle cells?

If the reviewer refers to the work by Chartier et al., 2011, which proposes that Crumbs controls epithelial integrity by inhibiting Rac1 and PI3K, it is difficult to provide a concrete answer. That work relies mostly on genetic interactions and analyses of cuticle phenotypes and did not analyze markers for PI(4,5)P₂, cytoskeleton components, intracellular trafficking or showed any EM to get an idea of the defects at the cellular level. Therefore, it is difficult to speculate what part of the mechanisms described in our work might or might not be involved in the embryonic phenotype. Also see comment in point 6.1 below.

As for the follicle cells, the interactions between Crumbs, Moesin, aPkc and the cytoskeleton have been shown to be highly dynamic, during the morphogenesis of the follicles. In this case, the role of Crb is more complex, as this protein appears to have other roles in epithelial morphogenesis, e.g. in dorsal closure in the Drosophila embryo (Flores-Benitez and Knust, 2015, eLife). Further, it is well-known that Crb, and other polarity proteins, have tissue specific functions (Tepass, 2012; Flores-Benitez and Knust, 2016) some of which are mentioned in our Introduction and along our Discussion section.

4) The PAMS are inward invaginations. But the degree to which they appear depends on the genetic background. They sometimes appear big and deep and sometimes not.

4.1) This needs to be seriously quantified for each background, for instance how many cells exhibit a shallow invagination, a deeper one and a very deep pit which clear morphological definition? For instance, some depletion in WT leads to shallow invaginations. Are those significant?

We have quantified the morphology and frequency of PAMS in the different genetic backgrounds and the results are shown in Figure 5—figure supplement 1E as well as in subsection “Crb regulates apical membrane levels of PI(4,5)P₂” We have also addressed the significance of these results in the Discussion section. It is important to note that in some of the images it may appear that the PAMS seem to have different depths, but that is also due to the perspective generated in the single optical sections and maximal projections. The morphology of the PAMS can be better appreciated in the new Video 8.

4.2) Are some PAMs found completely sealed and no longer accessible? This should also be quantified as it is much more connected to the disease.

As recent work shows, these inclusion bodies are dynamic and can form at the apical or the basolateral membrane, but also in the cytoplasm (see Mosa et al., 2018). We have included this point in the Discussion section in context with the dynamics of the formation of the inclusion bodies. See also answer to reviewer #3 point 3.

4.3) If most of them are continuous with the PM, what is their relevance? What does it affect secretion? Or are the two phenotypes not connected? In this regard, looking at prominin or any other TM proteins involved in microvilli formation would result in a clearer understanding of the message.

See answer in point 2 above.

5) In subsection “Crb controls the apical membrane homeostasis by negatively regulating Pten activity”, the authors state “in addition, these results show that the Crb protein complex is essential for the regulation of Pten activity in SGs.”

“….suggests that accumulation of PI(4,5)P₂ in Crb KD glands is likely due to an increase in Pten activity rather than a change in its localization.”

To state this, Pten activity should be measured directly, using extracts from WT and Crumbs mutant salivary glands with known substrates such as PI(3,4,5)P3. A probe assessing PI(3,4,5)P3 level should work if this cannot be done in vitro.

Although there are commercially available kits to measure PI(4,5)P₂ and PI(3,4,5)P₃ levels, they require a vast amount of sample material, which is not possible to isolate by manual dissection of the larval salivary glands. In addition, phosphoinositides are very transient molecules with short-life time ex vivo. We used a probe for quantifying PI(3,4,5)P₃ levels and the results are shown in Figure 5—figure supplement 2D-F. We quantified also the PI(4,5)P₂ levels by live imaging of glands expressing the probe for this phospholipid. Similarly, we quantified the distribution of Pten2-GFP and Ocrl-RFP (Figure 5). According to these measurements we made more conservative statements and updated our conclusions.

6) Pten is involved in many different pathways. It is a tumor suppressor and acts in negatively regulating the AKt pathway.

6.1) Is there a link between the PAMS and the other phenotype?

Pten is a major negative regulator of the insulin pathway and its activity prevents the recruitment of the protein kinaseB/AKT to the plasma membrane. We performed Western blot analyses to investigate whether AKT phosphorylation (activity) is changed in Crb KD salivary glands. We tested the specificity of our assay by over-expressing PI3K, presuming that high PI3K levels will increase PI(3,4,5)P₃ levels and activate AKT (i.e. GOF). Indeed, the PI3K gain of function increases both the salivary gland size (as expected from its role in cell growth – Scanga et al., 2000) and the amount of phosphorylated AKT (see Author response image 1, lane 1). However, wild type and Crb KD salivary gland cells do not show detectable AKT activity (lanes 3 and 4, respectively) although the enzyme is clearly present. Thus, we conclude that the PI(4,5)P₂ increase in Crb deficient salivary gland cells does not seem to affect AKT signaling significantly.

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Regarding the role of Pten as a tumor suppressor, we did not find any evidence suggesting that cell proliferation is modified by the loss of Crb, and as our results suggest that Pten activity is enhanced by the loss of Crb, it would be expected that tumorigenesis is downregulated, which is difficult to test in the context of our work, which was performed in a non-proliferating epithelium.

6.2) Is there any thoughts or data on how Crumbs regulates Pten activity? Do they form a complex

We performed immunoprecipitation experiments and found no complex containing Crumbs and Pten2. We also re-analyzed samples of mass spectrometry experiments of pull-downs from Crb and Sdt made in previous studies from our lab and did not find peptides for Pten2 or any other clear candidate that could mediate the regulation of Pten. The regulation of Pten is known to be extremely complex (see for example: PTEN Methods and Protocols, edited by Leonardo Salmena, DOI 10.1007/978-1-4939-3299-3) and we have addressed this point in the Discussion section, and provided references that identified β_Heavy-Spectrin and MyosinV as interactors of Pten.

7) Rabs:

7.1) The reviewer suggests to put this part after the PI(4,5)P₂/Pten section as the transition from Myo5 to Rabs is really abrupt and does not make a lot of sense.

Thanks for the suggestion, we made the transition clearer.

7.2) Why was Rab35 not considered as it also loses its apical localization in Crumbs mutant?

The loss of apical Rab35 is not reproducible. Therefore, the corresponding panel in Figure 4—figure supplement 1 has been replaced for a representative image where the apical localization of Rab35 is visible in the Crb deficient glands.

7.3) What is the role of these Rabs near the apical membrane? Are they active? Rabs mediate membrane traffic steps and these in particular from the TNG to the PM (Rab6) or from endosomes to the PM (Rab11), but none of this is described and tested. How does loss of Crumbs leads to their mislocalisation? Is it only PI(4,5)P2 composition? How?Is any of the Rab depletion lead to PAMs formation?

Fluorescently YFP-tagged Rab proteins that we detect are membrane associated, active Rab proteins. They are GTP-bound and regulate a plethora of membrane trafficking steps. Cytoplasmic, inactive GDP-Rabs are complexed with “escort-proteins” dubbed GDI. To do so, Rab proteins represent localized binding platforms capable to recruit specific effector proteins (Caviglia et al., 2017). Interestingly, activated Rab6, Rab11, and probably Rab30, are found to bind MyoV (Lindsay et al., 2013). Thus, Rab proteins and Crb might share direct interactors. On the other hand, Crb is directly involved in the organization of the membrane-associated apical cytocortex (see Figure 2 and Figure 2—figure supplement 1). Most vesicular transport is executed by motor proteins which are bound to the tubulin (kinesin/dynein) or actin (myosins) meshwork. In consequence, the localization of Rab proteins close to the membrane domain is dependent also on the organization of the cytoskeleton (Eaton and Martin-Belmonte, 2014).

The phosphoinositide composition of the plasma membrane is known to regulate every step of the membrane traffic machinery (Mayinger, 2012; Martin, 2015; Posor, Eichhorn-Grünig and Haucke, 2015). As we write in our Introduction, “PI(4,5)P₂ is directly implicated in the regulation of exocytosis (Milosevic et al., 2005; Gong et al., 2005; Massarwa et al., 2009; Rousso et al., 2013) and in all forms of endocytosis (Antonescu et al., 2011; Mayinger, 2012; Jost et al., 1998)”. However, as we also write in our Discussion section, we did not identify localization defects of Rab proteins involved in the endocytic route.

Regarding the role of the Rab proteins in the formation of PAMS, we show that the KD of Rab11 leads to PAMS formation, supporting the conclusion that defective apical secretion results in PAMS formation (Figure 4—figure supplement 3).

So overall, the manuscript is the description of a number of factors (related to the cytocortex-Rab network) whose depletion leads to PAMS. But there is very little mechanistic insights or sense of what is new.

The relation between Crb and the regulation of phosphoinositides, as well as the requirement of Crb to organize the apical secretion machinery have not been described before. We made this message clearer along the manuscript. Moreover, we expanded the evidence that support these conclusions by including several new experiments and quantifications that show that these interactions are important for the physiology and development of the larvae.

Reviewer #2:

In this paper, the authors remove Crb (or other Crb complex protein) function in mid-third instar larval SGs and determine the consequences. What they observe is that many aspects of SG organization are unaffected; overall SG morphology, cell polarity, barrier function, as well as the localization of a number of membrane domain-specific proteins appear largely normal. Apical secretion of both an apical membrane protein (CD8-RFP) and glycoproteins (those detected by PCNA binding) are diminished, however, with the reduction in Crb or with the reduction of Sdt (a component of the Crb complex). Reduced Crb also results in decreases in apical accumulation of F-actin, βHeavy-Spectrin and MyoV (the myosin that has been implicated in secretion). Also observed are decreases in apical localization of three Rab proteins that have previously been implicated in apical secretion: Rab6, Rab11 and Rab30. The reduction in Crb also results in the invagination of the apical domain to form structures the authors refer to as PAMS (PI(4,5)P2 and Phospho-Moesin enriched apical membrane sacs). To make sense of the observed molecular changes, the authors also determined the effects of reducing βHeavy-Spectrin, MyoV, and the enzymes involved in controlling PI(4,5)P2 levels in cells: Skittles (a PIP kinase that makes PI(4,5)P2), Pten (which dephosphorylates PI(3,4,5)P3 to PI(4,5)P2) and PI3K (which phosphorylates PI(4,5)P2 to make PI(3,4,5)P3). The reduction in βHeavy-Spectrin and of MyoV also diminishes secretion and promotes formation of PAMs, although not to the same level as reduction in Crb. The authors also show that reduction of Pten activity as well as increases in P13K can reduce the formation of PAMs associated with reduced Crb function, as well as restoring the apical localization of Rab6 and Rab11. Based on these experimental findings, the authors propose the following model: Crb is required to maintain the proper amount and organization of the apical domain by stabilizing the apical cytocortex (through βHeavy-Spectrin and F-actin), that the Crb-βHeavy-Spectrin complex facilitates apical secretion in larval SGs by maintaining apical MyoV. The authors also conclude that Crb normally limits PI(4,5)P2 accumulation by limiting Pten activity (but not Pten accumulation since levels appear unchanged between controls and Crb knockdown).Essential revisions:

1) Most of the phenotypes are not quantified and should be. When the authors say that PAM formation is reduced or increased or that secretion is reduced, we are relying on the limited number of images that can be shown and an impression the authors have of the data, rather than any quantification.

We provide quantifications and corresponding source data in the new manuscript. All statistical analyses are described in the Materials and methods section.

2) The authors make a good case that PI(4,5)P2 is required for the formation of PAMs, but they do not make a very good case that Crb works through Pten to increase PI(4,5)P2 levels. From the images provided, it is not at all clear that overall apical PI(4,5)P2 levels are any higher in the Crb knockdown than in WT control SGs, although the apical distribution does seem to change – less in the PM and more in PAMs. Reducing PI(4,5)P2 (which is known to bind and activate Moesin) does indeed appear to reduce PAM formation and increasing PI(4,5)P2 does appear to increase PAM formation but that does not indicate that Crb normally acts to limit Pten activity; it only says that high levels of PI(4,5)P2 are required for this loss of Crb phenotype. Indeed, the Pten overexpression phenotypes does seem to increase the apical domain area but it doesn't seem to create structures that have the same morphology as the PAMs. So, either change the conclusions/title of the manuscript or provide better evidence that PI(4,5)P2 levels are indeed higher with loss of crb.

We provide the quantification of PI(4,5)P₂ levels by measuring the fluorescence intensity using a well-established probe for PI(4,5)P₂ (see for example Jouette et al., 2019, eLife). Besides, by quantifying the distribution of an overexpressed Pten2 tagged with GFP we found that its distribution (the apical-to-lateral ratio) decreases in the Crb-deficient salivary glands (Figure 5I-K). Moreover, we extended our analyses to include the inositol polyphosphate 5-phosphatase Ocrl (Oculocerebrorenal syndrome of Lowe), that regulates PI(4,5)P₂ homeostasis by dephosphorylating PI(4,5)P₂. Our new results show that Ocrl localizes to the apical aspect of the salivary gland cells and that Crb is necessary for Ocrl apical localization. Accordingly, we have changed the title and conclusions of our manuscript to reflect that Crumbs is indeed involved in regulation of PI(4,5)P₂ metabolism at different levels.

3) Skittles is not the only PIP kinase encoded in the Drosophila genome, perhaps explaining the relatively mild effects of skittles knockdown on the loss of Crb phenotypes. The authors should acknowledge the existence of this other protein when they note the relatively mild effects.

Thanks for the suggestion. As noted in the previous point, we now include Ocrl in our analyses.

4) Although the authors claim that loss of Crb does not affect AJs (based on TEMs and DE-cad immunostaining), it does appear from the images that levels of DE-cad found near the apical surface are notably reduced (perhaps there is also an increase in vesicular staining of DE-cad). Thus, the authors need to look at this more carefully and indicate that there is some reduction of the AJ-localized DE-cad, or clearly demonstrate that levels are unaffected.

In fact, DE-cad levels at the AJ seem to be reduced. At the same time, the intracellular localization is increased. We think that this phenotype is a manifestation of the defects in the trafficking machinery due to the loss of Crb. KD of DE-cad-GFP (knock-in allele) using the gfp^RNAi effectively reduced the levels of DE-cad-GFP, but surprisingly, the distribution of Dlg-mTagRFP and the overall shape of the salivary glands are not affected. Therefore, we believe that the reduction of DE-cad-GFP levels at the AJ in the Crumbs deficient glands does not contribute to the defect in secretion, and that these results are redundant to the main message of our manuscript and therefore do not add more relevant information to the main conclusions. Therefore, we removed those results and relied on the EM analyses instead in order to streamline the manuscript.

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5) There seem to be some PAMs with microvilli-like structures and other similarly large vesicular structures with no microvilli-like structures. Are the latter only found with Crb knockdown or are they also found in controls? If they are only found in the Crb knockdown, what are they proposed to be?

These structures resemble lysosomes and have also been observed in models for MVID (see for example Mosa et al., 2018). Other works have also suggested that impaired secretion results in enhanced lysosomal activity to degrade the accumulated material. We have included these points in our results (Figure 2—figure supplement 3 and subsection “Crb regulates apical membrane organization via the apical cytocortex”).

6) This may be challenging to determine, but it would be helpful to know if the Crb knockdown actually changes the total amount of apical surface area. Do control SG cells have the same amount of apical area if, in the Crb knockdown cells, one were to include the apical area found within the PAMs? This is related to the observation that the pleated septate junctions in the Crb knockdown SGs seem to be much more convoluted than those in WT (Figure 1—figure supplement 2), suggesting that there might be an increase in membrane in those domains. This might suggest that with loss of Crb, some of the apical membrane is being converted to basolateral membrane.

This is a wonderful idea but extremely difficult to tackle. We have included an estimation of the microvilli density in the Crb deficient cells, which do have less microvilli when compared with the controls (Figure 2M). Also, we added a quantification of the apical surface from confocal z-stacks and EM analyses (Figure 5, Figure 5—figure supplement1E, subsection “Crb regulates apical membrane organization via the apical cytocortex”, and subsection “Crb organizes the apical trafficking machinery by controlling apical PI(4,5)P₂ levels”). Nevertheless, without a complete tomogram of the salivary gland cell, it is impossible to know the precise amount of membrane present in each domain (of course assuming that we will have developed the computational methods to extract the required information from such tomograms). Other super-resolution microscopy approaches could help to measure the amount of membrane in the different domains, but these methods are limited because of the considerable size of the salivary gland cells. Additionally, we have no way to answer whether some of the membrane contained in the large lysosomes in the Crb deficient glands was originally apical, lateral or just not trafficked to the respective domain and was targeted for degradation instead.

7) Importantly, is this study showing us that Crb is doing what it has been known to do – controlling the balance of apical versus basolateral domains and providing some clues as to the mechanism or is this study really uncovering a new and unrelated role for Crb? Either way the study is interesting, but exploring the first possibility is worthwhile since it has not been clear how Crb normally controls apical specification.

The functional relation between Crb and the regulation of phosphoinositides, as well as the requirement of Crb to organize the apical secretion machinery have not been described before. We have tried to make this message clearer along the manuscript. Moreover, we expanded the evidence that support these conclusions by including several new experiments and quantifications that show that these interactions are important for the physiology and development of the larvae. Regarding the balance of apical versus basolateral domains, to uncover the molecular mechanism(s) behind this role of Crb deserves to be addressed in a whole new project, as we do not even know whether these mechanisms are the same in different epithelia and different developmental stages. Nevertheless, we added our views on these points to the Discussion section, and we also suggest that our work could provide molecular targets for future studies understanding the physiological control of epithelial cell polarity (subsection “Crb organizes the apical trafficking machinery by controlling apical PI(4,5)P₂ levels”). Also see our answer to point 1 of reviewer #3.

Reviewer #3:

[…]

1) All conclusions re: crb loss-of-function phenotypes (e.g. "defects in apical secretion are not due to an overall disruption of cell polarity", or "this strategy does not affect embryonic development") are currently based on knockdown experiments. These may only reduce – rather than abrogate – Crb expression, and do so at a currently undefined developmental point. Clonal mutation of crb in salivary glands and/or temporally controlled knockdown experiments would allow the authors to draw stronger conclusions about the roles of Crb (or lack thereof) in establishing vs maintaining overall epithelial polarity in the salivary gland, as well as their temporal requirement. Related to this, different fkh-Gal4s lines have been generated with distinct expression. Is the expression of the one used in this particular study really confined to salivary glands? Most lines are also expressed in other portions of the intestine.

The fkh-GAL4 line used in our study drives strong expression in the salivary glands and very weakly in a portion of the hindgut. However, in crb NULL embryo mutants the hindgut is not affected (Kumichel and Knust, 2014). Therefore, our Crb KD experiments are likely not affected by any miss-expression of fkh-Gal4.

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With respect to our knock-down strategy, Crb-RNAi knock-down reduces Crb levels strongly (see Figure 1—figure supplement 1). Thus, our study is based more on a sensitized Crb background rather than a crb null-like setting. However, reduced Crb levels allow us to investigate processes regulated by this protein in more detail, as the complete loss of polarity is an extreme phenotype where it is difficult to analyze more subtle processes. To investigate how total ablation of the Crb protein regulates the morphology of salivary glands we decided to apply the deGradFP-mediated knock out approach. The long-term loss of Crb by employing the deGradFP-mediated knockout (Caussinus et al., 2012) results in polarity defects. We used the deGradFP in combination with the GAL80^ts system to control the induction of the nanobodies by shifting larvae to 29ºC. We used flies expressing Crb-GFP on the intracellular or the extracellular domain (Crb-GFP^intra or CrbGFP^extra, both knock-in alleles). In this experimental setup, we observed that Crb-GFP^intra is no longer detectable already at 4 hours of incubation at 29ºC. Importantly, even after 7 hours at 29ºC, the glands do not show any gross morphological defects. Only after 12 hours at 29ºC we found defects in the morphology of the salivary glands (bracket in Author response image 4). It is important to note that the high efficiency of the nanobodies in the salivary gland may also be due to the very low turn-over rate of Crb at the apical membrane of the larval salivary glands (Bajur and Knust, 2019). As expected, the nanobodies do not affect the localization of Crb-GFP^extra.

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To complement these observations and analyze possible polarity defects at earlier times, we used animals expressing DE-cad-mTomato (a knock-in allele). In this case we employed the deGradFPmediated knockout of Crb-GFP without the GAL80^ts. In fact, the larvae raised at 29ºC expressing the nanobodies and Crb-GFP^intra are smaller than their counterpart controls expressing the nanobodies with Crb-GFP^extra. In these animals, the glands appear severely misshaped and disorganized (not shown). However, if we maintained the larvae at 19-20ºC to limit the GAL4 activity, it was possible to obtain larvae with normal looking salivary glands. Under these conditions, we monitored the effects of the deGradFP-mediated knockout by incubating early third instar larvae (feeding animals not wandering) for 4 or 7 hours at 29ºC. As shown in the Z-max projections in Author response image 5, after 7 hours Crb-GFP^intra is not detectable at the apical membrane. Despite this, DE-cad-mTomato is still localized in a similar pattern as in the controls.

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Taken together, the complete loss of Crb in differentiated cells is fatal and a result of many cellular processes affected. Our “milder” crb phenotype allowed us to identify the subtler defects in molecular networks not linked to Crb before, yet important for the physiology of the animal.

2) The authors explore physiological aspects of Crb-mediated salivary gland secretion very superficially. This seemed to me a missed opportunity. If they wish to make any statements about Crb regulating the "physiological activity of the salivary gland" or promoting "efficient apical secretion of glycoproteins" (as currently stated in the abstract), the authors need to show effects on endogenous secretion – for example by visualizing the effect of crb knockdown on a protein normally secreted by the salivary gland. Related to this, the authors assume but do not show a reduction in food intake following salivary gland-specific crb knockdown. Is food intake reduced following crb knockdown? Does Pten knockdown rescue the feeding and developmental timing defects of crb knockdown larvae? Finally, what happens to Crb expression/localisation and/or downstream targets around the saliva to glue switch? In other words, is Crb permissive or instructive in the context of salivary gland secretion?

We analyzed whether the rescue of secretion by Pten knockdown also rescues the pupation as well as the feeding of the larvae (Figure 6). Our new results support the conclusion that defects in apical secretion in the salivary glands are mediated by an increase in PI(4,5)P₂, as Pten knockdown suppresses the delay in pupation, while the Pi3K knockdown delays pupation even more than the Crb knockdown alone (Figure 6W). Additionally, we estimated the food intake by adapting the protocol reported in Deshpande et al., 2014, a total of 3,060 larva were used for this experiment. We found that Crb knockdown tends to reduce the food intake, although not statistically significant (Figure 6V). Importantly, the Pten knockdown increases the food intake, even when Crb is silenced simultaneously (Figure6V).

Regarding the localization of Crb during the transition to glue secretion, the protein remains apical during these stages (see Author response image 6).

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We recognize that it will be ideal to follow a protein that is normally secreted by the salivary glands. Unfortunately, no endogenous cargo has been characterized at the developmental stages we performed our analyses. In fact, glue secretion occurs almost 2 days later from the stages we analyzed (Video 1 and Video 2). We revised transcriptomic data from FlyBase and found possible gene candidates that may encode secreted proteins including mucins, lysozymes and chitinases (the ORF Saliva suggested by the reviewer #1 is predicted to be a transmembrane transporter of sugar, not a secreted protein). However, none of the mucins, lysozymes and chitinases have been characterized, and there are no available antibodies or insertion lines (i.e. MiMIC) that will allow us to generate GFP tagged proteins easily. Thus, establishing, characterizing and validating these possible secreted proteins is out of the scope of our manuscript.

As noted also by the reviewer #1, the PNA-GFP probe that we used as a proxy for secretion is not well characterized. Therefore, we screened other lines used to track apical secretion and decided to replicate all experiment made with the PNA-GFP probe using instead a construct containing the chitin binding domain of Serpentine tagged with GFP (UAS-SerpCBD-GFP). This construct is a well-established apical secreted cargo and has been used as a proxy to analyze apical secretion (Luschnig et al., 2006; Kakihara et al., 2008; Förster et al., 2010; Petkau et al., 2012; Dong et al., 2013; Dong et al., 2014; Bätz et al., 2014). Using this tool, we have confirmed the results obtained with the PNA line, and thus exchanged all figures for the ones obtained with SerpCBD-GFP (Figure 1, Figure 1—figure supplement 1, Figure 3, Figure 3—figure supplement 2, Figure 6).

3) The authors should try to rule in/out increased endocytosis (rather than reduced apical secretion) as one of the reasons for the observed phenotypes; it seems to me that at least some of the Rab targets are involved in recycling as well as secretion. Also, the appearance of these novel intracellular microvilli-containing sacs and the concurrent reduced density of microvilli on the cell surface may both also be consistent with increased endocytosis; I am not an expert in this but, as far as I know, microvilli are not "trafficked" to the membrane in secretory compartments? Related to this, it may be informative to test the Rab signature of PAM sacs in both Crb/β-Spectrin knockdowns – might this shed light on the origin of these compartments?

We explored the role of endocytosis in three ways. By using the temperature sensitive allele of dynamin (Shi^ts), by ex vivo incubation of glands with Dynasore (cell-permeable inhibitor of dynamin) and by overexpressing dominant active (DA) or dominant inactive (DN) forms of Rab5.

For the first approach we generated the following flies:

- Shi^ts; UAS-crb[RNAi]

- Shi^ts;; fkh-GAL4>UAS-SerpCBD-GFP

- Shi^ts;; fkh-GAL4>UAS-PLCd-PH-EGFP

All stocks were kept at 19-20ºC, as adult’s viability decays very fast at temperatures >24ºC. To analyze the role of dynamin in the Crb phenotypes we set crosses (indicated below), and kept them at 19-20ºC. The eggs were collected overnight and then transferred to 28ºC (Shi^ts larvae die at 29ºC), where they were incubated for 2 days. It is important to note that generation of these stocks as well as testing the different iterations for this experiment required several months. The salivary glands were dissected on the third day on ice-cold Grace’s medium and then analyzed by live imaging:

For SerpCBD-GFP:

Control w*: w*;; fkh-GAL4>UAS-SerpCBD-GFP X w*/⦢

crb^RNAi w*: w*;; fkh-GAL4>UAS-SerpCBD-GFP X w*/⦢; UAS-crb[RNAi] Control Shi^ts: Shi^ts;; fkh-GAL4>UAS-SerpCBD-GFP X Shi^ts/⦢

crb^RNAi Shi^ts: Shi^ts;; fkh-GAL4>UAS-SerpCBD-GFP X Shi^ts/⦢; UAS-crb[RNAi]

For UAS-PLCd-PH-EGFP:

Control w*: w*;; fkh-GAL4>UAS-PLCd-PH-EGFP X w*/⦢

crb^RNAi w*:w*;; fkh-GAL4>UAS-PLCd-PH-EGFP X w*/⦢; UAS-crb[RNAi]

Control Shi^ts:Shi^ts;; fkh-GAL4>UAS-PLCd-PH-EGFP X Shi^ts/⦢

crbRNAi Shits: Shi^ts;; fkh-GAL4>UAS-PLCd-PH-EGFP X Shi^ts/⦢; UAS-crb[RNAi]

Representative images of the results obtained are shown in Author response image 7. In summary, the secretion defect evaluated with SerpCBD-GFP seems to be ameliorated in the Shi^ts background. However, the formation of PAMS is not completely suppressed in the Shi^ts background. Of note, these results are not modified by further incubating the dissected glands for 15 min at 37ºC (to make sure that Shi^ts is completely inhibited).

Author response image 7

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For the second approach, ex vivo incubation of glands with Dynasore, all analyses were inconsistent and no solid conclusion about the role of dynamin could be reached.

For the third approach, we analyzed the formation of PAMS induced by Crb KD in glands co-expressing Rab5 DA or Rab5 DN. However, we obtained inconsistent results, as we found enhancement or suppression of the PAMS phenotype with either Rab5 DA or Rab5 DN. This could be due to pleiotropic effects of dominant versions of Rab proteins, which tend to titer effectors and regulators shared with other Rab proteins (e.g. Rab4).

These results suggest that dynamin-mediated endocytosis could be involved in the defects observed in the Crb-deficient glands, and accordingly we added these pointS to our Discussion section. However, it is beyond the scope of this manuscript to describe the precise role of endocytosis in the phenotypes generated by loss of Crb in the salivary glands. Indeed, we think that these observations deserve a systematic analysis that will make the current manuscript too large to be comprehensible. Regarding the second part or the question, about the formation of the intracellular vesicles containing microvilli. As we describe in our manuscript, these inclusions resemble those found in the intestine of MVID patients. There are published works that provide genetic evidence supporting the hypothesis that defects in apical transport results in formation of similar microvilli-inclusion bodies (Feng et al., 2017; Knowles et al., 2014; Knowles et al., 2015; Mosa et al., 2018; Müller et al., 2008; Ruemmele et al., 2010; Sato et al., 2007; Schneeberger et al., 2015; Sidhaye et al., 2016; Weis et al., 2016).

The mechanism by which these inclusions form is unknown, but the reviewer is referred to a recent work (Mosa et al., 2018) in which intestinal organoids were used to analyze the formation of the microvillus inclusions. Using live imaging, Mosa et al. show that these inclusion bodies are dynamic, and can form inside the cytoplasm, or derive from the apical or basolateral membranes, as well as to fuse with the apical or basolateral membrane, or collapse inside the cell. The time scale for these inclusions from forming to the moment of detachment or collapse is several hours. Their results suggest that indeed, microvilli might be assembled inside the cytoplasm and trafficked to the plasma membrane. Nevertheless, we don’t think that intracellular formation of microvilli occurs in our experimental model. Instead, we have observed some large intracellular vesicles, but these are probably PAMS that detached or cleaved away from the apical membrane. However, we have been unable to follow these events in our live imaging experiments, as these may happen very sporadically and over a long period of time (hours). We have included this additional information in our Discussion section.

Finally, regarding the Rab signature of the PAMS, we could not define a Rab signature for the PAMS. As shown in the Figure 4—figure supplement 1, no specific Rab protein accumulated in a compartment similar to the one marked by the PI(4,5)P₂ reporter or phospho-Moesin.

https://doi.org/10.7554/eLife.50900.sa2

Article and author information

Author details

Johanna Lattner

Max-Planck Institute of Molecular Cell Biology and Genetics (MPI-CBG), Dresden, Germany

Contribution
Conceptualization, Formal analysis, Investigation, Visualization, Methodology, Writing—original draft, Writing—review and editing

Competing interests
No competing interests declared

"This ORCID iD identifies the author of this article:" 0000-0003-3421-9134
Weihua Leng

Max-Planck Institute of Molecular Cell Biology and Genetics (MPI-CBG), Dresden, Germany

Contribution
Investigation

Competing interests
No competing interests declared
Elisabeth Knust

Max-Planck Institute of Molecular Cell Biology and Genetics (MPI-CBG), Dresden, Germany

Contribution
Conceptualization, Resources, Funding acquisition, Writing—review and editing

Competing interests
Reviewing editor, eLife

"This ORCID iD identifies the author of this article:" 0000-0002-2732-9135
Marko Brankatschk

The Biotechnological Center of the TU Dresden (BIOTEC), Dresden, Germany

Contribution
Conceptualization, Resources, Funding acquisition, Investigation, Methodology, Writing—original draft, Project administration, Writing—review and editing

For correspondence
marko.brankatschk@tu-dresden.de

Competing interests
No competing interests declared

"This ORCID iD identifies the author of this article:" 0000-0001-5274-4552
David Flores-Benitez

Max-Planck Institute of Molecular Cell Biology and Genetics (MPI-CBG), Dresden, Germany

Contribution
Conceptualization, Data curation, Formal analysis, Supervision, Investigation, Visualization, Methodology, Writing—original draft, Project administration, Writing—review and editing

For correspondence
flores@mpi-cbg.de

Competing interests
No competing interests declared

"This ORCID iD identifies the author of this article:" 0000-0001-8244-9335

Funding

Max-Planck-Gesellschaft

Johanna Lattner
Weihua Leng
Elisabeth Knust
David Flores-Benitez

Deutsche Forschungsgemeinschaft (BR5490/2)

Marko Brankatschk

Deutsche Forschungsgemeinschaft (BR5490/3)

Marko Brankatschk

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We thank S Eaton, A Wodarz, GJ Beitel, G Thomas, M Furuse, and U Tepass for kindly providing antibodies for our studies. We thank the MPI-CBG facilities: Patrick Keller, antibody facility, for antibody production; Light Microscopy Facility, in particular Jan Peychl and Sebastian Bundschuh, for microscopy guidance; the Electron Microscopy Facility, in particular Michaela Wilsch-Bräuninger and Tobias Fürstenhaupt, for discussions and troubleshooting. Stocks were obtained from the Bloomington Drosophila Stock Center (NIH P40OD018537) and from the Vienna Drosophila Resource Center. Antibodies were obtained from the Developmental Studies Hybridoma Bank, created by the NICHD of the NIH and maintained at The University of Iowa, Department of Biology, Iowa City, IA 52242. We thank the fly keepers Sven Ssykor, Cornelia Mass and Stefan Wernicke for excellent care of our flies. MB grants BR5490/2, BR5490/3. The work was supported by the Max-Planck Society and the Deutsche Forschungsgemeinschaft. (DFG)

Senior and Reviewing Editor

Utpal Banerjee, University of California, Los Angeles, United States

Publication history

Received: August 6, 2019
Accepted: October 25, 2019
Accepted Manuscript published: November 7, 2019 (version 1)
Version of Record published: November 27, 2019 (version 2)

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This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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Crb is required for efficient apical secretion in SG cells.

Figure 1—source data 1

Figure 1—source data 2

Figure 1—source data 3

Fusion of a glue vesicle followed by expulsion of the cargo Sgs3-GFP into the lumen SG lumen of control (fkh>+, top) and Crb KD (fkh >UAS crbRNAi, bottom) animals.

Overview showing the fusion of glue vesicles followed by expulsion of Sgs3-GFP into the SG lumen of control (fkh>+, top) and Crb KD (fkh >UAS crbRNAi, bottom) animals.

Crb is necessary to specifically maintain the apical cytoskeleton and the morphology of the apical membrane.

Figure 2—source data 1

Figure 2—source data 2

Figure 2—source data 3

3D rendering of a SG from a Crb KD animal (fkh >UAS crbRNAi) probed for phospho-Moesin.

MyoV KD induces the intracellular extension of the apical membrane and disrupts apical secretion.

Figure 3—source data 1

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Figure 3—source data 5

Crb organizes the apical Rab machinery in larval SG cells.

Live imaging of endogenously expressed Rab6-YFP in SGs of control (left, Rab6-YFP, fkh>/+) and Crb KD (right, Rab6-YFP, fkh >UAS crbRNAi).

Live imaging of endogenously expressed Rab11-YFP in SGs of control (left, Rab11-YFP, fkh>/+) and Crb KD (right, Rab11-YFP, fkh >UAS crbRNAi).

Live imaging of endogenously expressed Rab30-YFP in SGs of control (left, Rab30-YFP, fkh>/+) and Crb KD (right, Rab30-YFP, fkh >UAS crbRNAi).

Live imaging of endogenously expressed Rab1-YFP in SGs of control (left, Rab1-YFP, fkh>/+) and Crb KD (right, Rab1-YFP, fkh >UAS crbRNAi).

Crb organizes the apical secretory machinery by negatively regulating Pten A.

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3D rendering of a fixed SG of a Crb KD animal expressing the PI(4,5)P2 reporter PLCδ-PH-EGFP (green) and stained for phospho-Moesin (magenta).

Live imaging of a SG of a Crb KD animal expressing the PI(4,5)P2 reporter PLCδ-PH-EGFP (fkh >UAS crbRNAi; UAS-PLCδ-PH-EGFP).

Control of apical secretion and localization of Rab11 and Rab30 by Crb requires Pten.

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Crb-dependent regulation of apical secretion in SG cells Schematic representation of Crb-dependent regulation of apical secretion in SG cells.

List of fly stocks used in this study.

List of detailed genotypes analyzed in each figure.

List of antibodies and probes employed.

Schematic representation of the experimental setup.

Author details

Johanna Lattner

Contribution

Competing interests

Weihua Leng

Contribution

Competing interests

Elisabeth Knust

Contribution

Competing interests

Marko Brankatschk

Contribution

For correspondence

Competing interests

David Flores-Benitez

Fusion of a glue vesicle followed by expulsion of the cargo Sgs3-GFP into the lumen SG lumen of control (fkh>+, top) and Crb KD (fkh >UAS crb^RNAi, bottom) animals.

Overview showing the fusion of glue vesicles followed by expulsion of Sgs3-GFP into the SG lumen of control (fkh>+, top) and Crb KD (fkh >UAS crb^RNAi, bottom) animals.

3D rendering of a SG from a Crb KD animal (fkh >UAS crb^RNAi) probed for phospho-Moesin.

Live imaging of endogenously expressed Rab6-YFP in SGs of control (left, Rab6-YFP, fkh>/+) and Crb KD (right, Rab6-YFP, fkh >UAS crb^RNAi).

Live imaging of endogenously expressed Rab11-YFP in SGs of control (left, Rab11-YFP, fkh>/+) and Crb KD (right, Rab11-YFP, fkh >UAS crb^RNAi).

Live imaging of endogenously expressed Rab30-YFP in SGs of control (left, Rab30-YFP, fkh>/+) and Crb KD (right, Rab30-YFP, fkh >UAS crb^RNAi).

Live imaging of endogenously expressed Rab1-YFP in SGs of control (left, Rab1-YFP, fkh>/+) and Crb KD (right, Rab1-YFP, fkh >UAS crb^RNAi).

3D rendering of a fixed SG of a Crb KD animal expressing the PI(4,5)P₂ reporter PLCδ-PH-EGFP (green) and stained for phospho-Moesin (magenta).

Live imaging of a SG of a Crb KD animal expressing the PI(4,5)P₂ reporter PLCδ-PH-EGFP (fkh >UAS crb^RNAi; UAS-PLCδ-PH-EGFP).