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 PIP2 and formation of a novel, Moesin- and PIP2-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.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 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 . To perfPostsynaptic 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 (Heavy-Spectrin (βH-Spec) recruitment to the apical plasma membrane, and thereby supports the polarized organization of the membrane-associated cytoskeleton (cytocortex)  .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  proteins. All of these are closely interconnected to organize and maintain the identity of apical and basolateral membranes (2) and phosphatidylinositol 3,4,5-trisphosphate P3) in the apical and basolateral membranes, respectively (2 levels are controlled by Pten (Phosphatase and tensin homolog deleted on chromosome ten), which converts PIP3 into PIP2, by the type I phosphatidylinositol 4-phosphate 5-kinase Skittles (Sktl), which produces PIP2 from phosphatidylinositol 4-phosphate (PI4P), and by Ocrl , which dephosphorylates PIP2 into PI4P P3 (2 can bind to pleckstrin homology (PH)-domains of FERM proteins and β-Spectrins nto PI4P . Pten ac3,4,5)P3 . PIpectrins , therebypectrins . Moreoveocytosis  and in aocytosis .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) 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.Here, we studied the functions of Crb in a differentiated, highly polarized secretory epithelium, namely the salivary gland (SG) of the V (MyoV) . Our resfkh-GAL4  using the SG-specific driver fkh-GAL4 . We tookfkh-GAL4 . Strikin4 , an apical transmembrane protein involved in regulation of microvillar length , and CD8rd instar) but almost 2 days later  of Crb-deficient SG cells  are involved in apical secretion in the embryonic tracheae . TherefoIn contrast to ZA, pSJs are morphologically abnormal in SGs of Crb KD animals, showing many interruptions . In contTaken together, these results suggest that loss of Crb does not affect adherens junctions or the epithelial barrier function of SGs.H-Spectrin  to the apical membrane (Crb recruits Moesin (Moe) and βmembrane , where tmembrane . TherefoH-Spec , we refer to them as PAMS: phospho-Moe and PIP2-enriched apical membrane sacs. Given that silencing of Crb reduces apical βH-Spec, we analyzed the effect of βH-Spec KD on PAMS formation. Indeed, loss of βH-Spec P βH-Spec  prompts  βH-Spec . Moreovethe PAMS . These rTaken together, these results indicate that Crb is essential to maintain the proper amount and organization of the apical membrane by stabilizing the apical cytocortex.MYO5b gene (didum)  patients, which is linked to mutations in the O5b gene . SimilarO5b gene . MyosinVO5b gene  and is aO5b gene . Moreove (didum) . Therefo (didum)  and 2. I (didum)  and 2. Fcal MyoV  and 3 ascal MyoV  and 2. TdidumRNAi). 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 P2 via its PH-domain P2-specific PH-domain of phospholipase Cδ fused to GFP (PLCδ-PH-EGFP)  .2 is enriched in the apical membrane Pmembrane , as prevstar SGs . Importar SGs P2, we analyzed the distribution of PLCδ-PH-EGFP upon βH-Spec or MyoV depletion. Indeed, KD of βH-Spec or MyoV induces accumulation of PIP2 in the PAMS P2 levels and formation of PAMS.To analyze whether βthe PAMS , suggestDE-cadherin-mTomato to measure the apical membrane area and volume . Our measurements show that KD of Crb, βH-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 , 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 P2 levels P2 and PAMS formation P2 levels . ExpressptenRNAi  in Crb Kormation  and 4, w,5P2 leveormation  and 6. Tormation , as expeormation . Interesown Pten –6. To co of PAMS , while o of PAMS . Moreove of PAMS , elimina of PAMS –10. Thus3 to the basolateral membrane P3 levels using a probe containing the PH-domain of cytohesin tagged with GFP Pmembrane , we askewith GFP . The sigD glands . Immunos mutants . Pten2-G mutants . Interes mutants , suggest mutants . Thus, t2 levels by dephosphorylating PIP2 into PI4P P2 Pnto PI4P . Live imnto PI4P . Moreovento PI4P  and 9. TIP2  and 11, 2 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.Together, these results show that apical accumulation of PIP2 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 .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  to 4, whTo assess the physiological relevance of Crb in SG secretion, we evaluated the larval food intake and tracked the pupariation time . We foun2 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 βH-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  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 . ii) We  the SGs .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  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  and knowss clear , our rescrb loss of function does not result in an overall increase in endocytosis in the eye imaginal disc epithelium 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 PIP2 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 PIP2 P2 levels disrupts polarization through subcortical retention of vesicles at apical membrane initiation sites  of Rab6, Rab11 and Rab30.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 PIP2 levels . Hence, IP2 . For examembrane . Moreoveormation . PIon sites . TherefoH-Spec induces the formation of PAMS. Therefore, we favor the hypothesis that regulation of Pten might be mediated by βH-Spec and MyoV acting downstream of Crb. Interestingly, an interaction between βH-Spec and Pten was found by tandem affinity purification assays  or on previous mass-spectrometry data  no direcn assays , but when assays . Indeed,n assays . Therefoications . Therefoications . But eveDrosophila embryos expands the apical membrane , 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 , 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  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.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.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 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  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 . 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  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 Image acquisition was done using a Tecnai 12  with a standard single tilt holder with a TVIPS TemCam F214A  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.For Dextran permeability assays we adapted the method from 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  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 , Crb . 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  percentat-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.All statistical analyses were performed using GraphPad Prism 8. Statistical significance was calculated in unpaired  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 . 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: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.Thank you for submitting your work entitled "Crumbs organizes the apical transport machinery by negatively regulating Pten in eLife at this time.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 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  really felt that it will not be fair to the authors to unreasonably delay the process if you wish to submit elsewhere.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 Reviewer #1:Drosophila salivary gland cells.The article by Lattner, Leng, Knust, Brankatschk and Flores-Benitez describes the role of Crumbs in organizing the apical membrane of the 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.2 in the apical membrane. For this, they use a probe that specifically binds PIP2 and show that the probe localizes mostly to the apical membrane. In the absence of Crumbs, PIP2 is no longer localized apically. Inhibiting Pten pharmacologically and genetically modulates the pool of PIP2 at the apical membrane. Importantly, Pten depletion suppress the formation of the PAMs that are induced by Crumbs depletion.In a third part, the authors address the role of PIPThey 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?2 level? How does Crumbs regulates Pten activity?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 PIPThird, 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 . 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.”2 in Crb KD glands is likely due to an increase in Pten activity rather than a change in its localization.”“….suggests that accumulation of PIP3. A probe assessing PIP3 level should work if this cannot be done in vitro.To state this, Pten activity should be measured directly, using extracts from WT and Crumbs mutant salivary glands with known substrates such as PIP6) 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 complex7) Rabs:2/Pten section as the transition from Myo5 to Rabs is really abrupt and does not make a lot of sense.7.1) The reviewer suggests to put this part after the PIP7.2) Why was Rab35 not considered as it also loses its apical localization in Crumbs mutant?2 composition? How?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 PIPIs 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: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 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 PIP2 levels in cells: Skittles P2), Pten P3 to make PIP2) and PI3K P2 to make PIP3). 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 PIP2 accumulation by limiting Pten activity (but not Pten accumulation since levels appear unchanged between controls and Crb knockdown).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, βEssential 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 is required for the formation of PAMs, but they do not make a very good case that Crb works through Pten to increase PIP2 levels. From the images provided, it is not at all clear that overall apical PIP2 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 PIP2 (which is known to bind and activate Moesin) does indeed appear to reduce PAM formation and increasing PIP2 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 PIP2 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 PIP2 levels are indeed higher with loss of crb.2) The authors make a good case that PIPDrosophila 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.3) Skittles is not the only PIP kinase encoded in the DE-cad immunostaining), it does appear from the images that levels of DE-cad found near the apical surface are notably reduced . 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.4) Although the authors claim that loss of Crb does not affect AJs (based on TEMs and 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 , 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: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.This manuscript explores the roles of the polarity protein Crumbs (Crb) in a particular type of secretory epithelium: the salivary glands of the 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:crb loss-of-function phenotypes  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.1) All conclusions re: 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?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 3) The authors should try to rule in/out increased endocytosis  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? 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 . 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”.2In a third part, the authors address the role of PIP in 2 and show that the probe localizes mostly to the apical membrane. In the absence of Crumbs, PIP2 is no longer localized apically. Inhibiting Pten pharmacologically and genetically modulates the pool of PIP2 at the apical membrane. Importantly, Pten depletion suppress the formation of the PAMs that are induced by Crumbs depletion.the apical membrane. For this, they use a probe that specifically binds PIP2 apical levels are increased in the absence of Crumbs. This was stated along the first version of the manuscript and illustrated as well . However, to solidify our findings, we added the quantifications  of our microscopy data.Our results show that PIPThey 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.Drosophila salivary gland cells that Crb negatively regulates the phosphatase Pten required to maintain the apical PIP2/PIP3 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.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 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?2 and Phospho-Moesin that are never present in control salivary gland cells 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 PIP2 levels is such that when PIP2 levels are lowered in Crb-depleted SGs (by knocking down Pten or by using its inhibitor VO-OHpic), the formation of PAMS is suppressed  as well as the secretion defects .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 PIPTrue 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 PIP2 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.C1eyg/C53eygductless larvae, which highlights the importance of the SGs in larval survival.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  and food intake . While others have suggested that SGs might be dispensable for larval survival , 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 , but nevertheless they report a lethality of 72.8% in trans-heterozygous 2”) and included these points along our Discussion about their significance and their relation to the secretion defects.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  and the relevance of proper secretion for the pupation rate and food intake . We included an analysis of the microvilli density  as well as the size and frequency of PAMS PInstead of Prominin, we analyzed the localization of endogenous Cadherin99C, a TM protein regulating microvilli structure .2 levels by regulating Ocrl apical localization  and updated our Discussion about the connections between Crb, βHeavy-Spectrin and MyosinV with Pten2, and about the role of Crb in maintaining the localization of Pten at the apical membrane . We provide quantifications (and the corresponding source data) to most relevant microscopy data.We provide additional evidence that Crb regulates PIP1) 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 .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 . 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 . Using this tool, we confirmed the results obtained with the PNA line, and thus exchanged all figures with the ones obtained with SerpCBD-GFP .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?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.We have quantified the density of microvilli . Unfortunately, we cannot measure the 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  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 . Our results indicate that Cad99C is not properly localized to the apical aspect of Crb deficient cells  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 .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?2, 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.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 PIPDrosophila embryo . Further, it is well-known that Crb, and other polarity proteins, have tissue specific functions  some of which are mentioned in our Introduction and along our Discussion section.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 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?2” 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.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 PIP4.2) 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 . 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.”2 in Crb KD glands is likely due to an increase in Pten activity rather than a change in its localization.”“….suggests that accumulation of PIPTo state this, Pten activity should be measured directly, using extracts from WT and Crumbs mutant salivary glands with known substrates such as PIP3. A probe assessing PIP3 level should work if this cannot be done in vitro.2 and PIP3 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 PIP3 levels and the results are shown in Figure 5—figure supplement 2D-F. We quantified also the PIP2 levels by live imaging of glands expressing the probe for this phospholipid. Similarly, we quantified the distribution of Pten2-GFP and Ocrl-RFP . According to these measurements we made more conservative statements and updated our conclusions.Although there are commercially available kits to measure PIP6) 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?3 levels and activate AKT (i.e. GOF). Indeed, the PI3K gain of function increases both the salivary gland size  and the amount of phosphorylated AKT  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 PIP AKT see , lane 1.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 complexHeavy-Spectrin and MyosinV as interactors of Pten.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  and we have addressed this point in the Discussion section, and provided references that identified β7) Rabs:2/Pten section as the transition from Myo5 to Rabs is really abrupt and does not make a lot of sense.7.1) The reviewer suggests to put this part after the PIPThanks 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 PIP2 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 . Interestingly, activated Rab6, Rab11, and probably Rab30, are found to bind MyoV . 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 . 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 .2 is directly implicated in the regulation of exocytosis  and in all forms of endocytosis ”. However, as we also write in our Discussion section, we did not identify localization defects of Rab proteins involved in the endocytic route.The phosphoinositide composition of the plasma membrane is known to regulate every step of the membrane traffic machinery . As we write in our Introduction, “PIPRegarding 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 .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 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 PIP2 levels in cells: Skittles P2), Pten P3 to PIP2) and PI3K P2 to make PIP3). 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 PIP2 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 PIP2 is required for the formation of PAMs, but they do not make a very good case that Crb works through Pten to increase PIP2 levels. From the images provided, it is not at all clear that overall apical PIP2 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 PIP2 (which is known to bind and activate Moesin) does indeed appear to reduce PAM formation and increasing PIP2 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 PIP2 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 PIP2 levels are indeed higher with loss of crb.2 levels by measuring the fluorescence intensity using a well-established probe for PIP2 . Besides, by quantifying the distribution of an overexpressed Pten2 tagged with GFP we found that its distribution  decreases in the Crb-deficient salivary glands . Moreover, we extended our analyses to include the inositol polyphosphate 5-phosphatase Ocrl , that regulates PIP2 homeostasis by dephosphorylating PIP2. 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 PIP2 metabolism at different levels.We provide the quantification of PIP3) 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 . 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.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  using the RNAigfp 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.In fact, 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 . 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 .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 , 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.2 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 . 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.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 . Also, we added a quantification of the apical surface from confocal z-stacks and EM analyses P7) 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.2 levels”). Also see our answer to point 1 of reviewer #3.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 PReviewer #3:[…]1) All conclusions re: crb loss-of-function phenotypes  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.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 . Therefore, our Crb KD experiments are likely not affected by any miss-expression of fkh-Gal4.The 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  results in polarity defects. We used the deGradFP in combination with the GAL80ts 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 . In this experimental setup, we observed that Crb-GFPintra 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 . Thus, our study is based more on a sensitized Crb background rather than a DE-cad-mTomato . In this case we employed the deGradFPmediated knockout of Crb-GFP without the GAL80ts. In fact, the larvae raised at 29ºC expressing the nanobodies and Crb-GFPintra are smaller than their counterpart controls expressing the nanobodies with Crb-GFPextra. 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  for 4 or 7 hours at 29ºC. As shown in the Z-max projections in intra is not detectable at the apical membrane. Despite this, DE-cad-mTomato is still localized in a similar pattern as in the controls.To complement these observations and analyze possible polarity defects at earlier times, we used animals expressing 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.Taken together, the complete loss of Crb in differentiated cells is fatal and a result of many cellular processes affected. Our “milder” 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?2, as Pten knockdown suppresses the delay in pupation, while the Pi3K knockdown delays pupation even more than the Crb knockdown alone . 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 . Importantly, the Pten knockdown increases the food intake, even when Crb is silenced simultaneously .We analyzed whether the rescue of secretion by Pten knockdown also rescues the pupation as well as the feeding of the larvae . Our new results support the conclusion that defects in apical secretion in the salivary glands are mediated by an increase in PIPRegarding the localization of Crb during the transition to glue secretion, the protein remains apical during these stages see .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 . 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 . 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 .3) The authors should try to rule in/out increased endocytosis  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?tsShi), 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.We explored the role of endocytosis in three ways. By using the temperature sensitive allele of dynamin , 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: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 .Representative images of the results obtained are shown in 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 .The mechanism by which these inclusions form is unknown, but the reviewer is referred to a recent work  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.2 reporter or phospho-Moesin.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 PIP