- Email: [email protected]
mTOR Signaling Regulates Megalin-Mediated Apical Endocytosis
Cell Reports
Report V-ATPase/mTOR Signaling Regulates Megalin-Mediated Apical Endocytosis Eva Maria Gleixner,1,2 Guillaume Canaud,5 Tobias Hermle,2 Maria Clara Guida,1,2 Oliver Kretz,1,3 Martin Helmsta¨dter,2 Tobias B. Huber,2,3 Stefan Eimer,1,3 Fabiola Terzi,5 and Matias Simons1,2,3,4,* 1Center
for Systems Biology (ZBSA), University of Freiburg, Habsburgerstrasse 49, 79104 Freiburg, Germany Division, University Hospital Freiburg, Hugstetter Strasse 55, 79106 Freiburg, Germany 3Center for Biological Signaling Studies (BIOSS), University of Freiburg, 79104 Freiburg, Germany 4Paris Descartes University–Sorbonne Paris Cite ´ , Imagine Institute, 24 Boulevard du Montparnasse, 75015 Paris, France 5INSERM U845, Centre de Recherche ‘‘Croissance et Signalisation,’’ Universite ´ Paris Descartes, Sorbonne Paris Cite´, Hoˆpital Necker–Enfants Malades, 75015 Paris, France *Correspondence: [email protected] http://dx.doi.org/10.1016/j.celrep.2014.05.035 This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/). 2Renal
SUMMARY
mTOR kinase is a master growth regulator that can be stimulated by multiple signals, including amino acids and the lysosomal small GTPase Rheb. Recent studies have proposed an important role for the V-ATPase in the sensing of amino acids in the lysosomal lumen. Using the Drosophila wing as a model epithelium, we show here that the V-ATPase is required for Rheb-dependent epithelial growth. We further uncover a positive feedback loop for the control of apical protein uptake that depends on V-ATPase/mTOR signaling. This feedback loop includes Rheb-dependent transcriptional regulation of the multiligand receptor Megalin, which itself is required for Rheb-induced endocytosis. In addition, we provide evidence that long-term mTOR inhibition with rapamycin in mice causes reduction of Megalin levels and proteinuria in the proximal tubular epithelium of the kidney. Thus, our findings unravel a homeostatic mechanism that allows epithelial cells to promote protein uptake under normal conditions and to prevent uptake in lysosomal stress conditions. INTRODUCTION The V-ATPase is a proton pump that acidifies intracellular compartments. Although acidification is important for a wide range of cell biological processes including endosomal ligand-receptor dissociation and lysosomal degradation, acidification-independent roles of the V-ATPase in secretion and membrane fusion have also been proposed (Wang and Hiesinger, 2013). The V-ATPase consists of multiple subunits that are organized in a V1 sector, which is important for ATP hydrolysis, and a V0 sector, that forms the proton pore. Our previous studies have focused on the functions of one particular subunit called VhaPRR in Drosophila and ATP6AP2 in mammals. In the initial description of this subunit, a small fragment of ATP6AP2 was found to co10 Cell Reports 8, 10–19, July 10, 2014 ª2014 The Authors
purifiy with the V0 sector (Ludwig et al., 1998). However, the cloning of full-length ATP6AP2 revealed a larger protein suggesting that the V-ATPase-associated fragment only contains the transmembrane domain and a short cytosolic tail but not the extracellular domain (Nguyen et al., 2002). Although the extracellular domain has been implicated in renin binding (Nguyen et al., 2002) and planar cell polarity signaling (Hermle et al., 2013), the transmembrane fragment contributes to V-ATPase functions such as endocytosis regulation, organellar acidification, and lysosomal degradation (Hermle et al., 2013; Kinouchi et al., 2010; Riediger et al., 2011). Sabatini and colleagues could recently demonstrate that the lysosomal V-ATPase plays an important role in mTOR signaling. Mammalian (or mechanistic) target of rapamycin (mTOR; Tor in Drosophila) is a kinase that integrates nutrient, energy, and growth factor signaling in higher eukaryotes. Several biological processes have been linked with active mTOR, most notably the promotion of anabolic and inhibition of catabolic pathways (Jewell et al., 2013). mTOR is found in a complex (called mTORC1) with Raptor, the pentameric Ragulator complex, and heterodimeric Rag GTPases (Kim et al., 2008; Sancak et al., 2008, 2010). In a current model for mTORC1 activation, the V-ATPase triggers Ragulator GEF activity for Rag GTPases in an amino-acid-dependent manner, followed by lysosomal recruitment of mTOR (Zoncu et al., 2011). At the lysosomal surface, the mTORC1 complex is brought in contact with the small GTPase Rheb, an important mTOR activator, which receives input from growth factor signaling. Rheb is repressed by the trimeric tuberous sclerosis complex (TSC) whose Tsc2 component harbors GTPase-activating protein activity toward Rheb (Bar-Peled et al., 2012; Sancak et al., 2010). The TSC complex is recruited to the lysosomal surface in response to amino acid starvation or the inhibition of growth factor signaling (Demetriades et al., 2014; Menon et al., 2014). Given the activation of the mTOR pathway at endolysosomal membranes, it is interesting that the pathway has also been associated with the regulation of endocytosis. Screening approaches in HeLa cells and Drosophila revealed that mTOR kinase modulates clathrin-dependent endocytosis (Hennig et al., 2006; Pelkmans et al., 2005). However, from these studies it
could not be concluded how this works on the mechanistic level. As the V-ATPase also plays a role in endocytosis (Hurtado-Lorenzo et al., 2006), it further remains to be determined whether the mTOR pathway and the V-ATPase have overlapping functions in the endocytic pathway. In this work, we show that Rheb-dependent tissue growth requires the V-ATPase in vivo. In addition, Rheb stimulates a Tor- and V-ATPase-dependent signaling pathway that causes apical endocytosis in epithelial cells. This pathway relies on the transcriptional control of the multiligand receptor Megalin, which itself is needed for Rheb-stimulated endocytosis. Thus, Tor kinase and V-ATPase act together to control endocytosis from the lysosomal surface. RESULTS V-ATPase Deficiency Impairs Tissue Size Maintenance and mTOR Signaling in the Drosophila Wing In addition to planar cell polarity phenotypes (Hermle et al., 2013), genetic manipulation of VhaPRR revealed severe tissue size defects. In the adult Drosophila wing, patched (ptc)-GAL4-mediated silencing of VhaPRR caused a reduction of the ptc compartment size, as reflected by the shortened distance between longitudinal L3 and L4 veins (Figures 1A, 1B, and 1G). Similar results were found when silencing Vha44 (V-ATPase C subunit; Figure 1G), indicating that compartment size reduction could be a V-ATPase-dependent phenotype. Reduced size of the ptc compartment was accompanied by an increase in hair density of adult wings (Figures 1I, 1J, and 1M), indicating that silencing of VhaPRR and Vha44 causes a reduction in cell size. Furthermore, when performing mosaic analyses with null alleles VhaPRR and Vha55 (B subunit) in pupal wings, we further noticed that mutant clones were smaller than their respective wild-type twin clones (Figure 1N; data not shown). We previously demonstrated that impairment of V-ATPase function causes JNK-dependent apoptosis, which could be blocked by coexpression of the negative regulator of JNK signaling Puckered (Puc) or the antiapoptotic baculoviral protein p35 (Hermle et al., 2013; Petzoldt et al., 2013). Because Puc was unable to restore tissue size (data not shown; Figure 2J for pupal wings), we excluded apoptosis as the main cause for the size phenotype. Instead, we turned to the mTOR pathway, which is a major regulator of cell and tissue size and has recently been shown to require the V-ATPase in Drosophila and mammalian cells (Zoncu et al., 2011). Similar to the V-ATPase, the expression of a dominant-negative version of Tor (Tor-DN; (Hennig and Neufeld, 2002) showed a decreased size of the ptc stripe as well as an increased hair density (Figures 1C, 1G, 1K, and 1M). When constitutively activating mTORC1, by overexpressing Rheb or hyperactivating endogenous Rheb through silencing of Tsc1, we observed a greatly increased tissue size (Figures 1D and 1H). In comparison, the overexpression of constitutively active (CA) and dominant-negative forms of RagA GTPase caused very modest alterations in tissue size (Figures S1A–S1D; Kim et al., 2008). Tissue enhancement by Rheb was blocked by Tor-DN (Figure 1H). A similar suppression was achieved with VhaPRR and Vha44 RNAi (Figures 1E, 1F, and 1H). Importantly, silencing of both VhaPRR and Tsc1 (or coexpressing VhaPRR RNAi and Rheb) caused a comparable ptc
domain reduction as silencing of VhaPRR alone (Figure 1H), suggesting that VhaPRR is epistatic to Rheb/Tsc1. We also found that mTOR signaling had a direct impact on V-ATPase expression. Although Tor-DN expression caused decreased mRNA levels of VhaPRR, Vha44, and Vha100-2, upregulation was detected upon Tsc1 knockdown (Figures S2A–S2G). This transcriptional regulation is in accordance with previous findings from cultured Tsc2 knockout cells (Pen˜a-Llopis et al., 2011; for more discussion see the legend of Figure S2). Further, we found increased protein levels of Vha55 in cells with activated mTOR signaling as well as decreased levels in cells with inactivated mTOR signaling (Figures 1O and 1P). Tsc1 knockdown cells were also probed for Vha13, Vha16, VhaSFD, and VhaPRR protein levels, which were all increased (Figures S2H–S2K). Taken together, these findings indicate that the V-ATPase is not only required for Rheb/Tsc1-dependent growth effects but is also under transcriptional control of Rheb/Tsc1. Apical Endocytosis Depends on V-ATPase/mTOR Signaling mTOR signaling has previously been shown to control bulk endocytosis in the Drosophila fat body (Hennig et al., 2006; Ibar et al., 2013). These studies made use of fluorescently labeled avidin as an endocytic tracer. Therefore, we tested whether V-ATPase and mTOR inhibition also affects avidin uptake in polarized epithelial cells of the wing. For this, we expressed several transgenes in the posterior engrailed (en) compartment or the ptc stripe of the wing, allowing for sideby-side comparison with the anterior wild-type compartment or the wild-type tissue flanking the ptc stripe, respectively (Figure 2A). Dissection was performed at the prepupal stage (5.5 hr after pupa formation [APF]), because at this stage there is no cuticle yet that could prevent uptake. Texas red (TR)-avidin was applied to the pupal wings for a 10 min pulse and internalized for another 10 min before fixation. For analysis, we focused on apical cell parts by confocal imaging. In wild-type cells, most of the applied TR-avidin readily entered the cell (Figures 2B and 2G). Within 10 min, internalized avidin had reached a Rab7-positive late endosomal compartment (Figure 2H’). V-ATPase deficiency, however, strongly compromised uptake. Both VhaPRR and Vha44 knockdown led to defective avidin uptake (Figures 2C and 2I), and a similar effect was seen in clones mutant for Vha55 and VhaPRR (Figures S3A–S3D). Uptake was already affected after a 10 min pulse, suggesting that early steps of endocytosis were compromised. Similarly, allowing for internalization for up to 60 min failed to improve uptake efficiency (Figures S3B–S3D). Thus, uptake was not just delayed but seemed to be defective at the level of the apical plasma membrane. As an alternative tracer, we also tested TR-dextran and observed a similar reduction in uptake (Figure S3E). Endocytosis defects were independent of apoptosis, because they could not be rescued by Puc (Figures 2I and 2J). Next, we assessed the role of mTOR signaling in avidin uptake in pupal wings. Similar to V-ATPase inactivation, the overexpression of Tor-DN caused reduced avidin uptake (Figure 2D). By contrast, the activation of mTOR signaling by Tsc1 knockdown or Rheb (but not Rag-CA) overexpression caused a strong increase in avidin uptake (Figures 2E, 2F, and S1E). Also, dextran Cell Reports 8, 10–19, July 10, 2014 ª2014 The Authors 11
Figure 1. Tsc1/Rheb-Dependent Growth in the Drosophila Wing Requires the V-ATPase in a Positive Feedback Loop (A–F) Indicated transgenes were expressed with ptc-GAL4 in the wing. The ptc domain is between L3 and L4 veins in adult wings. (G and H) Quantification is based on the ratio between the ptc stripe width (measured as indicated by the red line in A) and the length of the posterior cross vein (pcv; green line). Data are mean ± SD (*p < 0.001). Student’s test (two-tailed). (I–L) Hair densities within a defined area of the posterior wild-type side (upper panel) and the ptc stripe (lower panel) are depicted for control RNAi (I), VhaPRR RNAi (J), Tor-DN (K), and Tsc1 RNAi (L). For clarification, each wing cell usually develops only one wing hair.
(legend continued on next page)
12 Cell Reports 8, 10–19, July 10, 2014 ª2014 The Authors
uptake was enhanced by Rheb overexpression (Figure S3F). When coexpressing VhaPRR RNAi with ptc-GAL4, the uptake was, however, again similar to the expression of VhaPRR RNAi alone (Figures 2I and 2K). A comparable decrease in uptake was achieved by coexpressing Tor RNAi (data not shown). It can thus be concluded that apical endocytosis in pupal wings is controlled by V-ATPase/mTOR signaling and that Rheb-stimulated endocytosis requires the V-ATPase and Tor kinase. mTOR Signaling Controls Megalin Levels and Apical Surface Area Whereas dextrans are supposed to enter cells via fluid-phase uptake (Doherty and McMahon, 2009), it is not entirely clear what mode of endocytosis avidin is using for uptake. As avidin is a protein, we considered the involvement of the multiligand tandem receptors, Megalin and Cubilin. These two receptors have large ectodomains that account for apical epithelial uptake of a large number of proteins and hormones, most notably in proximal tubules of the kidney. Whereas Megalin has a cytosolic tail with endocytic sorting signals, Cubilin is a peripheral membrane protein that is endocytosed with the help of Megalin (Birn et al., 2000; Christensen and Birn, 2001). The Drosophila ortholog of Cubilin was recently shown to be required for endocytosis of the hemolymph protein ANF by pericardial nephrocytes (Zhang et al., 2013). We confirmed that Cubilin was also required for avidin uptake in nephrocytes (Figure S4B). Similarly, Megalin knockdown reduced avidin uptake in nephrocytes, but not to the same extent as Cubilin knockdown (Figure S4C). By contrast, Megalin but not Cubilin was required for uptake in pupal wings (Figures 3A, 3B, and S4E), consistent with a previous report on Megalin-dependent pigment protein endocytosis in pupal wings (Riedel et al., 2011). Interestingly, the increase in avidin uptake induced by Tsc1 RNAi was abolished by the coexpression of Megalin RNAi (Figure 3C). Therefore, we focused on Megalin for subsequent experiments. Immunostaining for Megalin revealed a high expression in pupal wings (Figures 3D–3I). The specificity of the antibody was confirmed by Megalin knockdown in the ptc stripe (Figure S4G). In accordance with mammalian epithelia, Megalin mainly localized to the apical membrane. However, in VhaPRR and Vha44 knockdown cells Megalin was strongly reduced (Figure 3E; data not shown). Similar results were shown for Tor-DN expression, albeit to a lesser extent (Figure 3F). By contrast, Megalin was significantly upregulated at the apical surface of cells expressing Tsc1 RNAi (Figure 3G). Comparable to the growth assay and avidin uptake, the combination of Rheb activation and V-ATPase or Tor inactivation suppressed Megalin increase. Instead, Megalin was downregulated to a similar degree as in cells with only inactivated V-ATPase or Tor kinase (Figures 3H and 3I; data not shown). Taken together, V-ATPase-deficient cells show the same phenotype with respect to Megalin-dependent endocytosis as Tor-deficient cells. Further, Rheb-induced endocytosis is V-ATPase- and Megalin-dependent.
As mTOR signaling can control protein levels via translation or transcription, we next asked whether apart from Megalin protein also Megalin mRNA is under the control of mTOR signaling. For this, we performed in situ hybridization on pupal wings. Whereas Tsc1 silencing in the posterior en compartment caused an increased signal for Megalin mRNA, the expression of Tor-DN showed less signal (Figures S4H and S4I). The specificity of the in situ probe for Megalin was confirmed by knocking down Megalin in the en compartment (Figure S4J). Interestingly, silencing of Megalin did not reduce tissue size or suppress Rheb-induced overgrowth (Figures 3C and S4D; data not shown). These results demonstrate that Megalin transcription is controlled by mTOR signaling. However, in contrast to the V-ATPase, Megalin is dispensable for the maintenance of tissue size. Because Megalin knockdown did not significantly affect dextran uptake (Figure S4F), we explored additional factors important for apical endocytosis. To determine the apical surface area, we assessed pupal wings by transmission electron microscopy. During wing disc eversion, the dorsoventral boundary of the larval wing disc pouch is protruding in such a manner that at pupal stages the dorsal epithelial layer will be positioned on top of the ventral layer. Whereas apical surfaces of both layers will be facing the outside, basal surfaces will be facing each other. Longitudinal sections of the pupal wing therefore provide a convenient way for comparing dorsal and ventral compartments (Figure 3J). Using this technique, we found that cells in the dorsal compartment expressing Rheb with apterous-GAL4 show a striking increase in apical surface area compared to the wild-type ventral side (Figures 3L and 3O). Microvilli were not only more numerous in relation to the apical surface length, but also more branched and longer. This effect was not observed when expressing a control transgene or Megalin RNAi in the dorsal compartment (Figures 3K and 3O; data not shown). By contrast, the apical surface was greatly reduced in posterior compartments expressing VhaPRR and Tor RNAi (Figures 3M–3O). Collectively, these findings strongly suggest that mTOR signaling controls apical endocytosis by two possibly unrelated mechanisms: first, the transcriptional regulation and surface expression of the multiligand receptor Megalin and second, the control of microvilli formation and apical surface area. Whereas avidin was mainly taken up in a Megalin-dependent manner, dextran uptake was independent of Megalin and presumably dependent on cell-surface area. Long-Term mTOR Inhibition Causes a Reduction of Megalin Levels in Proximal Tubules of the Kidney as well as Tubular Proteinuria Given the importance of Megalin-dependent apical endocytosis for proximal tubular (PT) cell function, we assessed the kidney phenotypes of long-term mTOR inhibition. Mice were treated with daily injection of either rapamycin or vehicle from postnatal day 14 onward. After 6 months of treatment, rapamycin led to an expected weight loss of 23% (Neff et al., 2013). Morphological
(M) Quantification of hair densities are based on the ratio between wild-type and ptc areas (*p < 0.001). Data are mean ± SD. Student’s t test (two-tailed). (N) Vha55 clones (marked by loss of GFP) in the pupal wing are smaller compared to the wild-type twin spot (marked by high levels (two copies) of GFP). (O and P) Vha55-GFP expression upon Tor-DN (O) and Tsc1 silencing (P) in the ptc stripe. Also, note the ptc compartment size difference. Scale bar, 20 mm. See also Figures S1 and S2.
Cell Reports 8, 10–19, July 10, 2014 ª2014 The Authors 13
Figure 2. Avidin Uptake Is Controlled by V-ATPase/mTOR Signaling (A) Anterior-posterior cross-sections of the pupal wing epithelium. Green areas indicate the expression domains of en-GAL4 and ptc-GAL4, respectively. TR-avidin (red dots) is taken up apically by prepupal wing cells (5.5 APF). Basal uptake is also possible because the medium can enter via the open hinge region. However, uptake was analyzed only in apical confocal planes depicted by black lines. (B–F) TR-avidin uptake upon control (PKD2) RNAi (B and B’), VhaPRR RNAi (C and C’), Tor-DN (D and D’), Tsc1 RNAi (E and E’), and Rheb (F and F’) expression in the en domain (marked by GFP). (G–I) Avidin uptake is normal in the ptc stripe expressing control RNAi (G) and reduced in Vha44 RNAi (H) and VhaPRR RNAi (I)-expressing cells. Coexpression of Rab7-YFP marks late endosomal compartments, which are positive for avidin (yellow dots) in wild-type but negative for avidin in RNAi-expressing tissue (green dots; H’). (J) Inhibition of apoptosis by Puc coexpression does not change uptake of cells expressing VhaPRR RNAi. (K) VhaPRR RNAi coexpression suppresses avidin uptake in Tsc1 RNAi-expressing cells. Scale bar, 20 mm. See also Figure S3.
examination of the kidneys revealed no significant difference in terms of glomerular lesions, tubular atrophy or fibrosis between the two groups of mice. However, a careful examination of the PTs showed that mice injected with rapamycin often displayed 14 Cell Reports 8, 10–19, July 10, 2014 ª2014 The Authors
large vacuoles occupying most of the apical cell portions (Figures 4A–4D). These vacuoles were positive for the lysosomal protein LAMP1 and the autophagosomal marker LC3B (Figures S5B and S5D), suggesting that they could represent autolysosomes.
Figure 3. mTOR Signaling Controls Megalin Levels and Apical Surface Area (A and B) Megalin (Mgl) knockdown reduces avidin uptake in the en (A and A’) and ptc (B) domain. (C) Megalin RNAi suppresses avidin uptake in cells expressing Tsc1 RNAi. Note that the size of the ptc domain is still enhanced. (D–F) Apical Megalin expression in ptc stripes (marked by mRFP) expressing control RNAi (D and D’), VhaPRR RNAi (E and E’), and Tor-DN expression (F and F’). Upper panel is an x-y and lower panel is an x-z projection with the apical membrane being the upper surface.
(legend continued on next page)
Cell Reports 8, 10–19, July 10, 2014 ª2014 The Authors 15
This was supported by the detection of increased levels of LC3B in western blot analysis (Figures S5E and S5F). Furthermore, decreased levels of P-S6RP and P-AKT (Figures 4E and 4F) were found, which is consistent with downregulation of mTORC1 and mTORC2 activity, respectively (Canaud et al., 2013). To validate the Drosophila results, we performed immunohistochemistry for Megalin (Figures 4C and 4D). Notably, we observed that the expression of Megalin was markedly reduced in mice treated with rapamycin. This reduction in expression was confirmed by western blot and quantitative PCR analysis (Figures 4E–4G). As Megalin is involved in PT cells transport of low-molecular-weight proteins, such as retinol-binding protein (Christensen and Birn, 2001), we next explored the functional consequences of Megalin decrease in rapamycin-treated mice by measuring the level of proteinuria. Coomassie staining and biochemical measurements revealed that rapamycin administration caused a high amount of low-molecular-weight proteinuria without albuminuria (Figures 4H–4J), consistent with impaired PT function. Taken together, these results suggest that longterm treatment with rapamycin decreases mTOR signaling and Megalin levels giving rise to low-molecular-weight proteinuria. DISCUSSION Here, we show that epithelial cells control apical endocytosis in a positive feedback loop that involves transcriptional regulation of Megalin and the V-ATPase as well as the regulation of apical surface area. This loop seems to be amplified at the lysosomal membrane, where the upstream TSC/Rheb module, the V-ATPase and Tor kinase come together. It is believed that the main pool of amino acids relevant for mTORC1 activation is either imported from the extracellular space or generated by metabolic processes in the cytoplasm before being sensed in the lysosomal lumen by the V-ATPase via unknown mechanisms (Jewell et al., 2013; Zoncu et al., 2011). By contrast, proteins entering the cell via endocytosis could, once they have been degraded in the lysosome, represent an important direct source of amino acids without the need for lysosomal import. In fact, it has been demonstrated that mTOR activation to trigger lysosome reformation that occurs after long starvation periods requires lysosomal protease and V-ATPase activity (Yu et al., 2010). Thus, the coupling of protein uptake, protein degradation, amino acid sensing, and transcriptional regulation by the lysosomal V-ATPase/mTORC1 complex could be a very efficient way of controlling anabolic cellular reactions. In renal proximal tubules, this regulatory system may be particularly important because here Megalin itself is needed for the uptake of lysosomal enzymes and, thus, for lysosomal degradation (Nielsen et al., 2013).
In addition to the amino acid input, growth factors such as Insulin that signal via the TSC/Rheb module represent a strong stimulus for cell and tissue growth (Saucedo et al., 2003). In cultured PT cells, it has been shown that Megalin transcription can be activated by Insulin (Hosojima et al., 2009), further emphasizing the intimate relationship between endocytosis and growth. As Megalin downregulation was not sufficient to reduce wing compartment size, it can be concluded that other endocytic pathways, including fluid-phase uptake, may act in a redundant manner with regard to mTOR-dependent cell and tissue growth. At the level of the lysosome, however, the compensatory mechanisms may be more limited. A decline in lysosomal function, e.g., due to V-ATPase deficiency, has devastating consequences for the cell, leading to accumulations of macromolecules and organelles in lysosomes and autolysosomes, respectively. Due to its strong endocytic activity, the PT epithelium of the kidney may be more vulnerable to lysosomal defects than other tissues. This notion is supported by several animal models and human genetic diseases: the constitutive knockout of the a4 V-ATPase subunit, for example, causes both lysosomal protein accumulations in PT cells and tubular proteinuria (Hennings et al., 2012), confirming the link between lysosomal function and apical endocytosis. Likewise, animal models for Dent’s disease and cystinosis, genetic disorders featured by a defective endolysosomal pathway, display tubular proteinuria and low levels of Megalin and Cubilin (Christensen et al., 2003; Piwon et al., 2000; Raggi et al., 2014). The findings in this paper suggest that an important cause for tubular proteinuria is the reduction of lysosomal mTOR signaling and subsequent alterations in the transcriptional profile of PT cells. It may even be speculated that proteinuria and loss of protein uptake is a cytoprotective response, because it prevents additional accumulation of protein and more damage in the endolysosomes. Thus, the control of apical endocytosis by switching mTOR signaling on and off at the lysosome is a simple but elegant mechanism to ensure proper protein uptake during tissue growth and to prevent uptake during stress conditions. An important next step will be to elucidate the downstream mechanisms by which Tor kinase regulates Megalin expression from the lysosome. EXPERIMENTAL PROCEDURES Fly Strains and Genetics Overexpression and transgenic RNAi studies were performed using the UAS/ GAL4 system. UAS-VhaPRR RNAi and UAS-Vha44 RNAi lines as well as VhaPRR and Vha55 alleles and the Vha55-eGFP gene trap line (Flytrap) have been characterized and validated previously (Hermle et al., 2010, 2013; Petzoldt et al., 2013). VhaPRR-GFP is a 3 kb genomic construct of the VhaPRR
(G) Megalin is increased in ptc stripes expressing Tsc1 RNAi. Confocal settings are optimized for the ptc stripe. Thus, expression outside the ptc stripe appears weaker. (H and I) VhaPRR RNAi suppresses Megalin expression in ptc stripes with activated mTOR signaling. Scale bar, 20 mm. (J) Cross-section in the proximal-distal axis of the prepupal wing (schematic in upper left). Dorsal is up and ventral is down. Black boxes depict the areas chosen for TEM pictures in (K) and (L). Transgenes are expressed on the dorsal side with apterous-GAL4. (K and L) Transmission electron microscopy shows a larger apical surface upon Rheb overexpression (K) compared to the wild-type ventral side (L). Black arrows show apical junctions. Area between the arrows represents the entire apical surface. Scale bar, 2 mm. (M and N) Tor (M) and VhaPRR (N) RNAi decrease apical surface area. (O) Dorsal apical surfaces (black bars) were quantified in relation to their ventral wild-type sides (white bar). Data are mean ± SEM (*p < 0.001). Student’s t test (two-tailed). AU, arbitrary unit. See also Figure S4.
16 Cell Reports 8, 10–19, July 10, 2014 ª2014 The Authors
Figure 4. mTOR Inhibition Results in Megalin Reduction and Proteinuria in Mice (A and B) Morphology of kidneys from mice injected either with the vehicle (A) and rapamycin (B) after 6 months. (C and D) Immunostaining of Megalin in vehicle (C) or rapamycin (D) injected mice after 6 months. Scale bars, 10 mm. Note that both the number of Megalinpositive tubuli and Megalin intensity are reduced. (E and F) Western blot (E) and quantification (F) of Megalin, P-Akt (Ser473), and P-S6RP in whole kidney lysate of vehicle- (n = 9) and rapamycin (n = 15)-injected mice after 6 months. (G) Megalin mRNA levels (normalized to RPL13) in vehicle- and rapamycin-treated mice. (H–J) Coomassie staining (H) and measurement of protein to creatinine ratio (I) as well as albumin to creatinine ratio (J) in urine from mice injected either with the vehicle or rapamycin at the age of 6 months, n = 9 and 15 for controls and rapamycin-injected mice, respectively. Bovine serum albumin (BSA) served as control for Coomassie staining. Data are means ± SEM (*p < 0.001). Mann-Whitney test: vehicle versus rapamycin-injected mice. See also Figure S5.
Cell Reports 8, 10–19, July 10, 2014 ª2014 The Authors 17
locus with a C-terminal GFP insertion. As drivers, ap-GAL4, ptc-GAL4 and en-GAL4 were used (all from Bloomington; en-GAL4 also contains UAS-Dicer2 and UAS–GFP). Crosses were grown on standard fly food at 25 C or 29 C. Clones were induced with the heat-shock Flippase (hsFlp). Other fly lines included UAS-PKD2 RNAi (ID:6941, Vienna Drosophila RNAi Center [VDRC]), UAS-Tsc1 RNAi (ID:31039), UAS-Tor RNAi (ID:34639; both from the Bloomington TRIP collection), UAS-Rheb, UAS-Tor-DN (both from Bloomington), Vha13eGFP (ID:CA07644), Vha16-eGFP (ID:ZCL0219), VhaSFD-eGFP (ID:G00259; all from Flytrap), GMR-GAL4;UAS-Rheb (from B. Edgar), tub-Rab7-YFP (from S. Eaton), and UAS-dRagAQ61L and UAS-dRagAT16N (from T. Neufeld). Immunostaining and Endocytic Tracer Studies Pupal wings (24 hr APF) were dissected, fixed in 8% paraformaldehyde for 60 min, and stained according to the standard procedure. The following antibodies were used: guinea pig anti-Megalin (from S. Eaton; Riedel et al., 2011) and rabbit anti-GFP (MBL International). For endocytic tracer uptake experiments, TR-conjugated avidin (Life Technologies) and TR-conjugated dextran (10 kDa, Life Technologies) were diluted with Schneider’s medium (PAN) to 0.2 and 0.5 mg/ml, respectively. Prepupal wings (5.5 hr APF) were dissected in Schneider’s medium and incubated in avidin or dextran for 10 min at room temperature followed by a 10 min chasing period with tracer-free Schneider’s medium. After, prepupal wings were fixed in 8% paraformaldehyde for 15 min. Imaging was performed on a Zeiss LSM 510 inverted confocal microscope.
Morphological Analysis and Immunohistochemistry Mouse kidneys were fixed in 4% paraformaldehyde and paraffin embedded, and 4 mm sections were stained with Periodic Acid Schiff (PAS) staining. For immunohistochemistry, 4 mm sections of paraffin-embedded kidneys were incubated with Megalin antibody (Abcam) followed by a sheep HRP-conjugated anti-rabbit antibody (Amersham). Staining was revealed by DAB. For immunofluorescence, 4 mm sections of paraffin-embedded kidneys were incubated with LAMP1 antibody (Sigma-Aldrich), anti-LC3B (Sigma-Aldrich), and Lotus Tetragonolobus Lectin (Vector Laboratories). The primary antibodies were revealed with the appropriate Alexa-488- or 555-conjugated secondary antibodies (Molecular Probes). Immunofluorescence staining was visualized using the Zeiss LSM 700 confocal microscope. Western Blot Western blots were performed as previously described (Canaud et al., 2013). In brief, protein extracts from kidneys were resolved by SDS-PAGE before being transferred onto the appropriate membrane and incubated with antiMegalin (Abcam), P-Akt Ser473 antibody (Cell Signaling Technology), P-S6 Ribosomal Protein antibody (Cell Signaling Technology), LC3B antibody, and anti-b-actin (both Sigma-Aldrich), followed by the appropriate peroxidaseconjugated secondary antibody. Chemiluminescence was acquired using a Fusion FX7 camera (Vilbert Lourmat) and densitometry was performed using Bio1D software (Certain Tech). SUPPLEMENTAL INFORMATION
Adult Wing Analysis For quantification of the ptc stripe width a minimum of 11 wings of each genotype were analyzed. The width of the ptc stripe was calculated by placing a line perpendicular to the L3 vein to the posterior cross vein. This distance was put into ratio to the length of the posterior cross vein. Quantification of hair densities was performed by counting the number of hairs in a minimum of 11 wings of each genotype in a defined area of the ptc stripe. The number of hairs was determined as a ratio to the hair number in an area of the same size in the adjacent posterior wild-type tissue. Electron Microscopy Drosophila prepupal wings (5.5 hr APF) were dissected and directly fixed with 4% paraformaldehyde and 1% glutaraldehyde in 0.1 M PB overnight at 4 C. After fixation, wings were washed in PB, treated with 2% OsO4, stained with uranyl acetate, dehydrated, and embedded in epoxy resin (Durcupan ACM, Fluka, and Sigma-Aldrich). Ultrathin cross-sections (40 nm) in the proximal-distal axis were cut using an ultramicrotome (Leica UC6) and analyzed with a Zeiss LEO 912 transmission electron microscope operated at 80 kV and equipped with a 2K CCD camera (Tro¨ndle). For quantitative analysis, randomly taken pictures of at least two independent wings with at least 20 cells per genotype were used. Quantification of apical membrane length as well as microvilli number, length, and branching was performed independently for cells in dorsal and ventral compartments, respectively. The values for the dorsal side were calculated in relation to the wild-type ventral value (= 1). Mice C57BL/6 male mice (Charles River Laboratories) were injected daily intraperitoneally with either saline + DMSO (vehicle) (n = 9) or rapamycin (LC Laboratories) 10 mg/kg (n = 15) from day 14 to day 180. We decided to start the treatment on day 14 to overcome any development effect. Animals were fed ad libitum and housed at constant ambient temperature in a 12-hr light cycle. Animal procedures were approved by the Departmental Director of ‘‘Services Ve´te´rinaires de la Pre´fecture de Police de Paris’’ and by the ethical committee of the Paris Descartes University. Urine and Plasma Analyses Urinary albumin, proteins, and creatinine concentrations were measured using an Olympus multiparametric analyzer (Instrumentation Laboratory). In addition, 20 ml of urine was run on Coomassie gels to visualize proteinuria.
18 Cell Reports 8, 10–19, July 10, 2014 ª2014 The Authors
Supplemental Information includes Supplemental Experimental Procedures and five figures and can be found with this article online at http://dx.doi.org/ 10.1016/j.celrep.2014.05.035. ACKNOWLEDGMENTS We thank Severine Kayser for excellent technical support. We thank Thomas Neufeld, Bruce Edgar, the Bloomington and Kyoto Stock Center, and the VDRC for fly strains. We thank Suzanne Eaton for the Megalin antibody. We are grateful to Mario Pende and Mila Roceri for help with the rapamycin experiment and to Sophie Berissi for mouse morphology. We thank Giorgos Pyrowolakis for critically reading the manuscript. The work has been supported by an Emmy-Noether grant (SI1303/2-1) by the Deutsche Forschungsgemeinschaft and a Gerontosys NephAge grant (0315896) by the German Ministry of Research and Education (BMBF). The TRiP RNAi resource at Harvard Medical School is supported by NIH grant R01GM084947. The mice experiments have been supported by INSERM, Universite´ Paris Descartes, Sorbonne Paris Cite´, and ANR. Received: January 16, 2014 Revised: April 16, 2014 Accepted: May 16, 2014 Published: June 19, 2014 REFERENCES Bar-Peled, L., Schweitzer, L.D., Zoncu, R., and Sabatini, D.M. (2012). Ragulator is a GEF for the rag GTPases that signal amino acid levels to mTORC1. Cell 150, 1196–1208. Birn, H., Fyfe, J.C., Jacobsen, C., Mounier, F., Verroust, P.J., Orskov, H., Willnow, T.E., Moestrup, S.K., and Christensen, E.I. (2000). Cubilin is an albumin binding protein important for renal tubular albumin reabsorption. J. Clin. Invest. 105, 1353–1361. Canaud, G., Bienaime´, F., Viau, A., Treins, C., Baron, W., Nguyen, C., Burtin, M., Berissi, S., Giannakakis, K., Muda, A.O., et al. (2013). AKT2 is essential to maintain podocyte viability and function during chronic kidney disease. Nat. Med. 19, 1288–1296. Christensen, E.I., and Birn, H. (2001). Megalin and cubilin: synergistic endocytic receptors in renal proximal tubule. Am. J. Physiol. Renal Physiol. 280, F562–F573.
Christensen, E.I., Devuyst, O., Dom, G., Nielsen, R., Van der Smissen, P., Verroust, P., Leruth, M., Guggino, W.B., and Courtoy, P.J. (2003). Loss of chloride channel ClC-5 impairs endocytosis by defective trafficking of megalin and cubilin in kidney proximal tubules. Proc. Natl. Acad. Sci. USA 100, 8472–8477.
Neff, F., Flores-Dominguez, D., Ryan, D.P., Horsch, M., Schro¨der, S., Adler, T., Afonso, L.C., Aguilar-Pimentel, J.A., Becker, L., Garrett, L., et al. (2013). Rapamycin extends murine lifespan but has limited effects on aging. J. Clin. Invest. 123, 3272–3291.
Demetriades, C., Doumpas, N., and Teleman, A.A. (2014). Regulation of TORC1 in response to amino acid starvation via lysosomal recruitment of TSC2. Cell 156, 786–799.
Nguyen, G., Delarue, F., Burckle´, C., Bouzhir, L., Giller, T., and Sraer, J.D. (2002). Pivotal role of the renin/prorenin receptor in angiotensin II production and cellular responses to renin. J. Clin. Invest. 109, 1417–1427.
Doherty, G.J., and McMahon, H.T. (2009). Mechanisms of endocytosis. Annu. Rev. Biochem. 78, 857–902. Hennig, K.M., and Neufeld, T.P. (2002). Inhibition of cellular growth and proliferation by dTOR overexpression in Drosophila. Genesis 34, 107–110.
Nielsen, R., Mollet, G., Esquivel, E.L., Weyer, K., Nielsen, P.K., Antignac, C., and Christensen, E.I. (2013). Increased lysosomal proteolysis counteracts protein accumulation in the proximal tubule during focal segmental glomerulosclerosis. Kidney Int. 84, 902–910.
Hennig, K.M., Colombani, J., and Neufeld, T.P. (2006). TOR coordinates bulk and targeted endocytosis in the Drosophila melanogaster fat body to regulate cell growth. J. Cell Biol. 173, 963–974.
Pelkmans, L., Fava, E., Grabner, H., Hannus, M., Habermann, B., Krausz, E., and Zerial, M. (2005). Genome-wide analysis of human kinases in clathrinand caveolae/raft-mediated endocytosis. Nature 436, 78–86.
Hennings, J.C., Picard, N., Huebner, A.K., Stauber, T., Maier, H., Brown, D., Jentsch, T.J., Vargas-Poussou, R., Eladari, D., and Hu¨bner, C.A. (2012). A mouse model for distal renal tubular acidosis reveals a previously unrecognized role of the V-ATPase a4 subunit in the proximal tubule. EMBO Mol. Med. 4, 1057–1071. Hermle, T., Saltukoglu, D., Grunewald, J., Walz, G., and Simons, M. (2010). Regulation of Frizzled-dependent planar polarity signaling by a V-ATPase subunit. Curr. Biol. 20, 1269–1276. Hermle, T., Guida, M.C., Beck, S., Helmsta¨dter, S., and Simons, M. (2013). Drosophila ATP6AP2/VhaPRR functions both as a novel planar cell polarity core protein and a regulator of endosomal trafficking. EMBO J. 32, 245–259. Hosojima, M., Sato, H., Yamamoto, K., Kaseda, R., Soma, T., Kobayashi, A., Suzuki, A., Kabasawa, H., Takeyama, A., Ikuyama, K., et al. (2009). Regulation of megalin expression in cultured proximal tubule cells by angiotensin II type 1A receptor- and insulin-mediated signaling cross talk. Endocrinology 150, 871–878. Hurtado-Lorenzo, A., Skinner, M., El Annan, J., Futai, M., Sun-Wada, G.H., Bourgoin, S., Casanova, J., Wildeman, A., Bechoua, S., Ausiello, D.A., et al. (2006). V-ATPase interacts with ARNO and Arf6 in early endosomes and regulates the protein degradative pathway. Nat. Cell Biol. 8, 124–136.
Pen˜a-Llopis, S., Vega-Rubin-de-Celis, S., Schwartz, J.C., Wolff, N.C., Tran, T.A., Zou, L., Xie, X.J., Corey, D.R., and Brugarolas, J. (2011). Regulation of TFEB and V-ATPases by mTORC1. EMBO J. 30, 3242–3258. Petzoldt, A.G., Gleixner, E.M., Fumagalli, A., Vaccari, T., and Simons, M. (2013). Elevated expression of the V-ATPase C subunit triggers JNK-dependent cell invasion and overgrowth in a Drosophila epithelium. Dis. Model. Mech. 6, 689–700. Piwon, N., Gu¨nther, W., Schwake, M., Bo¨sl, M.R., and Jentsch, T.J. (2000). ClC-5 Cl- -channel disruption impairs endocytosis in a mouse model for Dent’s disease. Nature 408, 369–373. Raggi, C., Luciani, A., Nevo, N., Antignac, C., Terryn, S., and Devuyst, O. (2014). Dedifferentiation and aberrations of the endolysosomal compartment characterize the early stage of nephropathic cystinosis. Hum. Mol. Genet. 23, 2266–2278. Riedel, F., Vorkel, D., and Eaton, S. (2011). Megalin-dependent yellow endocytosis restricts melanization in the Drosophila cuticle. Development 138, 149–158. Riediger, F., Quack, I., Qadri, F., Hartleben, B., Park, J.K., Potthoff, S.A., Sohn, D., Sihn, G., Rousselle, A., Fokuhl, V., et al. (2011). Prorenin receptor is essential for podocyte autophagy and survival. J. Am. Soc. Nephrol. 22, 2193–2202.
Ibar, C., Cataldo, V.F., Va´squez-Doorman, C., Olguı´n, P., and Glavic, A. (2013). Drosophila p53-related protein kinase is required for PI3K/TOR pathwaydependent growth. Development 140, 1282–1291.
Sancak, Y., Peterson, T.R., Shaul, Y.D., Lindquist, R.A., Thoreen, C.C., Bar-Peled, L., and Sabatini, D.M. (2008). The Rag GTPases bind raptor and mediate amino acid signaling to mTORC1. Science 320, 1496–1501.
Jewell, J.L., Russell, R.C., and Guan, K.L. (2013). Amino acid signalling upstream of mTOR. Nat. Rev. Mol. Cell Biol. 14, 133–139.
Sancak, Y., Bar-Peled, L., Zoncu, R., Markhard, A.L., Nada, S., and Sabatini, D.M. (2010). Ragulator-Rag complex targets mTORC1 to the lysosomal surface and is necessary for its activation by amino acids. Cell 141, 290–303.
Kim, E., Goraksha-Hicks, P., Li, L., Neufeld, T.P., and Guan, K.L. (2008). Regulation of TORC1 by Rag GTPases in nutrient response. Nat. Cell Biol. 10, 935–945. Kinouchi, K., Ichihara, A., Sano, M., Sun-Wada, G.H., Wada, Y., KurauchiMito, A., Bokuda, K., Narita, T., Oshima, Y., Sakoda, M., et al. (2010). The (pro)renin receptor/ATP6AP2 is essential for vacuolar H+-ATPase assembly in murine cardiomyocytes. Circ. Res. 107, 30–34. Ludwig, J., Kerscher, S., Brandt, U., Pfeiffer, K., Getlawi, F., Apps, D.K., and Scha¨gger, H. (1998). Identification and characterization of a novel 9.2-kDa membrane sector-associated protein of vacuolar proton-ATPase from chromaffin granules. J. Biol. Chem. 273, 10939–10947. Menon, S., Dibble, C.C., Talbott, G., Hoxhaj, G., Valvezan, A.J., Takahashi, H., Cantley, L.C., and Manning, B.D. (2014). Spatial control of the TSC complex integrates insulin and nutrient regulation of mTORC1 at the lysosome. Cell 156, 771–785.
Saucedo, L.J., Gao, X., Chiarelli, D.A., Li, L., Pan, D., and Edgar, B.A. (2003). Rheb promotes cell growth as a component of the insulin/TOR signalling network. Nat. Cell Biol. 5, 566–571. Wang, D., and Hiesinger, P.R. (2013). The vesicular ATPase: a missing link between acidification and exocytosis. J. Cell Biol. 203, 171–173. Yu, L., McPhee, C.K., Zheng, L., Mardones, G.A., Rong, Y., Peng, J., Mi, N., Zhao, Y., Liu, Z., Wan, F., et al. (2010). Termination of autophagy and reformation of lysosomes regulated by mTOR. Nature 465, 942–946. Zhang, F., Zhao, Y., Chao, Y., Muir, K., and Han, Z. (2013). Cubilin and amnionless mediate protein reabsorption in Drosophila nephrocytes. J. Am. Soc. Nephrol. 24, 209–216. Zoncu, R., Bar-Peled, L., Efeyan, A., Wang, S., Sancak, Y., and Sabatini, D.M. (2011). mTORC1 senses lysosomal amino acids through an inside-out mechanism that requires the vacuolar H(+)-ATPase. Science 334, 678–683.
Cell Reports 8, 10–19, July 10, 2014 ª2014 The Authors 19
Report V-ATPase/mTOR Signaling Regulates Megalin-Mediated Apical Endocytosis Eva Maria Gleixner,1,2 Guillaume Canaud,5 Tobias Hermle,2 Maria Clara Guida,1,2 Oliver Kretz,1,3 Martin Helmsta¨dter,2 Tobias B. Huber,2,3 Stefan Eimer,1,3 Fabiola Terzi,5 and Matias Simons1,2,3,4,* 1Center
for Systems Biology (ZBSA), University of Freiburg, Habsburgerstrasse 49, 79104 Freiburg, Germany Division, University Hospital Freiburg, Hugstetter Strasse 55, 79106 Freiburg, Germany 3Center for Biological Signaling Studies (BIOSS), University of Freiburg, 79104 Freiburg, Germany 4Paris Descartes University–Sorbonne Paris Cite ´ , Imagine Institute, 24 Boulevard du Montparnasse, 75015 Paris, France 5INSERM U845, Centre de Recherche ‘‘Croissance et Signalisation,’’ Universite ´ Paris Descartes, Sorbonne Paris Cite´, Hoˆpital Necker–Enfants Malades, 75015 Paris, France *Correspondence: [email protected] http://dx.doi.org/10.1016/j.celrep.2014.05.035 This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/). 2Renal
SUMMARY
mTOR kinase is a master growth regulator that can be stimulated by multiple signals, including amino acids and the lysosomal small GTPase Rheb. Recent studies have proposed an important role for the V-ATPase in the sensing of amino acids in the lysosomal lumen. Using the Drosophila wing as a model epithelium, we show here that the V-ATPase is required for Rheb-dependent epithelial growth. We further uncover a positive feedback loop for the control of apical protein uptake that depends on V-ATPase/mTOR signaling. This feedback loop includes Rheb-dependent transcriptional regulation of the multiligand receptor Megalin, which itself is required for Rheb-induced endocytosis. In addition, we provide evidence that long-term mTOR inhibition with rapamycin in mice causes reduction of Megalin levels and proteinuria in the proximal tubular epithelium of the kidney. Thus, our findings unravel a homeostatic mechanism that allows epithelial cells to promote protein uptake under normal conditions and to prevent uptake in lysosomal stress conditions. INTRODUCTION The V-ATPase is a proton pump that acidifies intracellular compartments. Although acidification is important for a wide range of cell biological processes including endosomal ligand-receptor dissociation and lysosomal degradation, acidification-independent roles of the V-ATPase in secretion and membrane fusion have also been proposed (Wang and Hiesinger, 2013). The V-ATPase consists of multiple subunits that are organized in a V1 sector, which is important for ATP hydrolysis, and a V0 sector, that forms the proton pore. Our previous studies have focused on the functions of one particular subunit called VhaPRR in Drosophila and ATP6AP2 in mammals. In the initial description of this subunit, a small fragment of ATP6AP2 was found to co10 Cell Reports 8, 10–19, July 10, 2014 ª2014 The Authors
purifiy with the V0 sector (Ludwig et al., 1998). However, the cloning of full-length ATP6AP2 revealed a larger protein suggesting that the V-ATPase-associated fragment only contains the transmembrane domain and a short cytosolic tail but not the extracellular domain (Nguyen et al., 2002). Although the extracellular domain has been implicated in renin binding (Nguyen et al., 2002) and planar cell polarity signaling (Hermle et al., 2013), the transmembrane fragment contributes to V-ATPase functions such as endocytosis regulation, organellar acidification, and lysosomal degradation (Hermle et al., 2013; Kinouchi et al., 2010; Riediger et al., 2011). Sabatini and colleagues could recently demonstrate that the lysosomal V-ATPase plays an important role in mTOR signaling. Mammalian (or mechanistic) target of rapamycin (mTOR; Tor in Drosophila) is a kinase that integrates nutrient, energy, and growth factor signaling in higher eukaryotes. Several biological processes have been linked with active mTOR, most notably the promotion of anabolic and inhibition of catabolic pathways (Jewell et al., 2013). mTOR is found in a complex (called mTORC1) with Raptor, the pentameric Ragulator complex, and heterodimeric Rag GTPases (Kim et al., 2008; Sancak et al., 2008, 2010). In a current model for mTORC1 activation, the V-ATPase triggers Ragulator GEF activity for Rag GTPases in an amino-acid-dependent manner, followed by lysosomal recruitment of mTOR (Zoncu et al., 2011). At the lysosomal surface, the mTORC1 complex is brought in contact with the small GTPase Rheb, an important mTOR activator, which receives input from growth factor signaling. Rheb is repressed by the trimeric tuberous sclerosis complex (TSC) whose Tsc2 component harbors GTPase-activating protein activity toward Rheb (Bar-Peled et al., 2012; Sancak et al., 2010). The TSC complex is recruited to the lysosomal surface in response to amino acid starvation or the inhibition of growth factor signaling (Demetriades et al., 2014; Menon et al., 2014). Given the activation of the mTOR pathway at endolysosomal membranes, it is interesting that the pathway has also been associated with the regulation of endocytosis. Screening approaches in HeLa cells and Drosophila revealed that mTOR kinase modulates clathrin-dependent endocytosis (Hennig et al., 2006; Pelkmans et al., 2005). However, from these studies it
could not be concluded how this works on the mechanistic level. As the V-ATPase also plays a role in endocytosis (Hurtado-Lorenzo et al., 2006), it further remains to be determined whether the mTOR pathway and the V-ATPase have overlapping functions in the endocytic pathway. In this work, we show that Rheb-dependent tissue growth requires the V-ATPase in vivo. In addition, Rheb stimulates a Tor- and V-ATPase-dependent signaling pathway that causes apical endocytosis in epithelial cells. This pathway relies on the transcriptional control of the multiligand receptor Megalin, which itself is needed for Rheb-stimulated endocytosis. Thus, Tor kinase and V-ATPase act together to control endocytosis from the lysosomal surface. RESULTS V-ATPase Deficiency Impairs Tissue Size Maintenance and mTOR Signaling in the Drosophila Wing In addition to planar cell polarity phenotypes (Hermle et al., 2013), genetic manipulation of VhaPRR revealed severe tissue size defects. In the adult Drosophila wing, patched (ptc)-GAL4-mediated silencing of VhaPRR caused a reduction of the ptc compartment size, as reflected by the shortened distance between longitudinal L3 and L4 veins (Figures 1A, 1B, and 1G). Similar results were found when silencing Vha44 (V-ATPase C subunit; Figure 1G), indicating that compartment size reduction could be a V-ATPase-dependent phenotype. Reduced size of the ptc compartment was accompanied by an increase in hair density of adult wings (Figures 1I, 1J, and 1M), indicating that silencing of VhaPRR and Vha44 causes a reduction in cell size. Furthermore, when performing mosaic analyses with null alleles VhaPRR and Vha55 (B subunit) in pupal wings, we further noticed that mutant clones were smaller than their respective wild-type twin clones (Figure 1N; data not shown). We previously demonstrated that impairment of V-ATPase function causes JNK-dependent apoptosis, which could be blocked by coexpression of the negative regulator of JNK signaling Puckered (Puc) or the antiapoptotic baculoviral protein p35 (Hermle et al., 2013; Petzoldt et al., 2013). Because Puc was unable to restore tissue size (data not shown; Figure 2J for pupal wings), we excluded apoptosis as the main cause for the size phenotype. Instead, we turned to the mTOR pathway, which is a major regulator of cell and tissue size and has recently been shown to require the V-ATPase in Drosophila and mammalian cells (Zoncu et al., 2011). Similar to the V-ATPase, the expression of a dominant-negative version of Tor (Tor-DN; (Hennig and Neufeld, 2002) showed a decreased size of the ptc stripe as well as an increased hair density (Figures 1C, 1G, 1K, and 1M). When constitutively activating mTORC1, by overexpressing Rheb or hyperactivating endogenous Rheb through silencing of Tsc1, we observed a greatly increased tissue size (Figures 1D and 1H). In comparison, the overexpression of constitutively active (CA) and dominant-negative forms of RagA GTPase caused very modest alterations in tissue size (Figures S1A–S1D; Kim et al., 2008). Tissue enhancement by Rheb was blocked by Tor-DN (Figure 1H). A similar suppression was achieved with VhaPRR and Vha44 RNAi (Figures 1E, 1F, and 1H). Importantly, silencing of both VhaPRR and Tsc1 (or coexpressing VhaPRR RNAi and Rheb) caused a comparable ptc
domain reduction as silencing of VhaPRR alone (Figure 1H), suggesting that VhaPRR is epistatic to Rheb/Tsc1. We also found that mTOR signaling had a direct impact on V-ATPase expression. Although Tor-DN expression caused decreased mRNA levels of VhaPRR, Vha44, and Vha100-2, upregulation was detected upon Tsc1 knockdown (Figures S2A–S2G). This transcriptional regulation is in accordance with previous findings from cultured Tsc2 knockout cells (Pen˜a-Llopis et al., 2011; for more discussion see the legend of Figure S2). Further, we found increased protein levels of Vha55 in cells with activated mTOR signaling as well as decreased levels in cells with inactivated mTOR signaling (Figures 1O and 1P). Tsc1 knockdown cells were also probed for Vha13, Vha16, VhaSFD, and VhaPRR protein levels, which were all increased (Figures S2H–S2K). Taken together, these findings indicate that the V-ATPase is not only required for Rheb/Tsc1-dependent growth effects but is also under transcriptional control of Rheb/Tsc1. Apical Endocytosis Depends on V-ATPase/mTOR Signaling mTOR signaling has previously been shown to control bulk endocytosis in the Drosophila fat body (Hennig et al., 2006; Ibar et al., 2013). These studies made use of fluorescently labeled avidin as an endocytic tracer. Therefore, we tested whether V-ATPase and mTOR inhibition also affects avidin uptake in polarized epithelial cells of the wing. For this, we expressed several transgenes in the posterior engrailed (en) compartment or the ptc stripe of the wing, allowing for sideby-side comparison with the anterior wild-type compartment or the wild-type tissue flanking the ptc stripe, respectively (Figure 2A). Dissection was performed at the prepupal stage (5.5 hr after pupa formation [APF]), because at this stage there is no cuticle yet that could prevent uptake. Texas red (TR)-avidin was applied to the pupal wings for a 10 min pulse and internalized for another 10 min before fixation. For analysis, we focused on apical cell parts by confocal imaging. In wild-type cells, most of the applied TR-avidin readily entered the cell (Figures 2B and 2G). Within 10 min, internalized avidin had reached a Rab7-positive late endosomal compartment (Figure 2H’). V-ATPase deficiency, however, strongly compromised uptake. Both VhaPRR and Vha44 knockdown led to defective avidin uptake (Figures 2C and 2I), and a similar effect was seen in clones mutant for Vha55 and VhaPRR (Figures S3A–S3D). Uptake was already affected after a 10 min pulse, suggesting that early steps of endocytosis were compromised. Similarly, allowing for internalization for up to 60 min failed to improve uptake efficiency (Figures S3B–S3D). Thus, uptake was not just delayed but seemed to be defective at the level of the apical plasma membrane. As an alternative tracer, we also tested TR-dextran and observed a similar reduction in uptake (Figure S3E). Endocytosis defects were independent of apoptosis, because they could not be rescued by Puc (Figures 2I and 2J). Next, we assessed the role of mTOR signaling in avidin uptake in pupal wings. Similar to V-ATPase inactivation, the overexpression of Tor-DN caused reduced avidin uptake (Figure 2D). By contrast, the activation of mTOR signaling by Tsc1 knockdown or Rheb (but not Rag-CA) overexpression caused a strong increase in avidin uptake (Figures 2E, 2F, and S1E). Also, dextran Cell Reports 8, 10–19, July 10, 2014 ª2014 The Authors 11
Figure 1. Tsc1/Rheb-Dependent Growth in the Drosophila Wing Requires the V-ATPase in a Positive Feedback Loop (A–F) Indicated transgenes were expressed with ptc-GAL4 in the wing. The ptc domain is between L3 and L4 veins in adult wings. (G and H) Quantification is based on the ratio between the ptc stripe width (measured as indicated by the red line in A) and the length of the posterior cross vein (pcv; green line). Data are mean ± SD (*p < 0.001). Student’s test (two-tailed). (I–L) Hair densities within a defined area of the posterior wild-type side (upper panel) and the ptc stripe (lower panel) are depicted for control RNAi (I), VhaPRR RNAi (J), Tor-DN (K), and Tsc1 RNAi (L). For clarification, each wing cell usually develops only one wing hair.
(legend continued on next page)
12 Cell Reports 8, 10–19, July 10, 2014 ª2014 The Authors
uptake was enhanced by Rheb overexpression (Figure S3F). When coexpressing VhaPRR RNAi with ptc-GAL4, the uptake was, however, again similar to the expression of VhaPRR RNAi alone (Figures 2I and 2K). A comparable decrease in uptake was achieved by coexpressing Tor RNAi (data not shown). It can thus be concluded that apical endocytosis in pupal wings is controlled by V-ATPase/mTOR signaling and that Rheb-stimulated endocytosis requires the V-ATPase and Tor kinase. mTOR Signaling Controls Megalin Levels and Apical Surface Area Whereas dextrans are supposed to enter cells via fluid-phase uptake (Doherty and McMahon, 2009), it is not entirely clear what mode of endocytosis avidin is using for uptake. As avidin is a protein, we considered the involvement of the multiligand tandem receptors, Megalin and Cubilin. These two receptors have large ectodomains that account for apical epithelial uptake of a large number of proteins and hormones, most notably in proximal tubules of the kidney. Whereas Megalin has a cytosolic tail with endocytic sorting signals, Cubilin is a peripheral membrane protein that is endocytosed with the help of Megalin (Birn et al., 2000; Christensen and Birn, 2001). The Drosophila ortholog of Cubilin was recently shown to be required for endocytosis of the hemolymph protein ANF by pericardial nephrocytes (Zhang et al., 2013). We confirmed that Cubilin was also required for avidin uptake in nephrocytes (Figure S4B). Similarly, Megalin knockdown reduced avidin uptake in nephrocytes, but not to the same extent as Cubilin knockdown (Figure S4C). By contrast, Megalin but not Cubilin was required for uptake in pupal wings (Figures 3A, 3B, and S4E), consistent with a previous report on Megalin-dependent pigment protein endocytosis in pupal wings (Riedel et al., 2011). Interestingly, the increase in avidin uptake induced by Tsc1 RNAi was abolished by the coexpression of Megalin RNAi (Figure 3C). Therefore, we focused on Megalin for subsequent experiments. Immunostaining for Megalin revealed a high expression in pupal wings (Figures 3D–3I). The specificity of the antibody was confirmed by Megalin knockdown in the ptc stripe (Figure S4G). In accordance with mammalian epithelia, Megalin mainly localized to the apical membrane. However, in VhaPRR and Vha44 knockdown cells Megalin was strongly reduced (Figure 3E; data not shown). Similar results were shown for Tor-DN expression, albeit to a lesser extent (Figure 3F). By contrast, Megalin was significantly upregulated at the apical surface of cells expressing Tsc1 RNAi (Figure 3G). Comparable to the growth assay and avidin uptake, the combination of Rheb activation and V-ATPase or Tor inactivation suppressed Megalin increase. Instead, Megalin was downregulated to a similar degree as in cells with only inactivated V-ATPase or Tor kinase (Figures 3H and 3I; data not shown). Taken together, V-ATPase-deficient cells show the same phenotype with respect to Megalin-dependent endocytosis as Tor-deficient cells. Further, Rheb-induced endocytosis is V-ATPase- and Megalin-dependent.
As mTOR signaling can control protein levels via translation or transcription, we next asked whether apart from Megalin protein also Megalin mRNA is under the control of mTOR signaling. For this, we performed in situ hybridization on pupal wings. Whereas Tsc1 silencing in the posterior en compartment caused an increased signal for Megalin mRNA, the expression of Tor-DN showed less signal (Figures S4H and S4I). The specificity of the in situ probe for Megalin was confirmed by knocking down Megalin in the en compartment (Figure S4J). Interestingly, silencing of Megalin did not reduce tissue size or suppress Rheb-induced overgrowth (Figures 3C and S4D; data not shown). These results demonstrate that Megalin transcription is controlled by mTOR signaling. However, in contrast to the V-ATPase, Megalin is dispensable for the maintenance of tissue size. Because Megalin knockdown did not significantly affect dextran uptake (Figure S4F), we explored additional factors important for apical endocytosis. To determine the apical surface area, we assessed pupal wings by transmission electron microscopy. During wing disc eversion, the dorsoventral boundary of the larval wing disc pouch is protruding in such a manner that at pupal stages the dorsal epithelial layer will be positioned on top of the ventral layer. Whereas apical surfaces of both layers will be facing the outside, basal surfaces will be facing each other. Longitudinal sections of the pupal wing therefore provide a convenient way for comparing dorsal and ventral compartments (Figure 3J). Using this technique, we found that cells in the dorsal compartment expressing Rheb with apterous-GAL4 show a striking increase in apical surface area compared to the wild-type ventral side (Figures 3L and 3O). Microvilli were not only more numerous in relation to the apical surface length, but also more branched and longer. This effect was not observed when expressing a control transgene or Megalin RNAi in the dorsal compartment (Figures 3K and 3O; data not shown). By contrast, the apical surface was greatly reduced in posterior compartments expressing VhaPRR and Tor RNAi (Figures 3M–3O). Collectively, these findings strongly suggest that mTOR signaling controls apical endocytosis by two possibly unrelated mechanisms: first, the transcriptional regulation and surface expression of the multiligand receptor Megalin and second, the control of microvilli formation and apical surface area. Whereas avidin was mainly taken up in a Megalin-dependent manner, dextran uptake was independent of Megalin and presumably dependent on cell-surface area. Long-Term mTOR Inhibition Causes a Reduction of Megalin Levels in Proximal Tubules of the Kidney as well as Tubular Proteinuria Given the importance of Megalin-dependent apical endocytosis for proximal tubular (PT) cell function, we assessed the kidney phenotypes of long-term mTOR inhibition. Mice were treated with daily injection of either rapamycin or vehicle from postnatal day 14 onward. After 6 months of treatment, rapamycin led to an expected weight loss of 23% (Neff et al., 2013). Morphological
(M) Quantification of hair densities are based on the ratio between wild-type and ptc areas (*p < 0.001). Data are mean ± SD. Student’s t test (two-tailed). (N) Vha55 clones (marked by loss of GFP) in the pupal wing are smaller compared to the wild-type twin spot (marked by high levels (two copies) of GFP). (O and P) Vha55-GFP expression upon Tor-DN (O) and Tsc1 silencing (P) in the ptc stripe. Also, note the ptc compartment size difference. Scale bar, 20 mm. See also Figures S1 and S2.
Cell Reports 8, 10–19, July 10, 2014 ª2014 The Authors 13
Figure 2. Avidin Uptake Is Controlled by V-ATPase/mTOR Signaling (A) Anterior-posterior cross-sections of the pupal wing epithelium. Green areas indicate the expression domains of en-GAL4 and ptc-GAL4, respectively. TR-avidin (red dots) is taken up apically by prepupal wing cells (5.5 APF). Basal uptake is also possible because the medium can enter via the open hinge region. However, uptake was analyzed only in apical confocal planes depicted by black lines. (B–F) TR-avidin uptake upon control (PKD2) RNAi (B and B’), VhaPRR RNAi (C and C’), Tor-DN (D and D’), Tsc1 RNAi (E and E’), and Rheb (F and F’) expression in the en domain (marked by GFP). (G–I) Avidin uptake is normal in the ptc stripe expressing control RNAi (G) and reduced in Vha44 RNAi (H) and VhaPRR RNAi (I)-expressing cells. Coexpression of Rab7-YFP marks late endosomal compartments, which are positive for avidin (yellow dots) in wild-type but negative for avidin in RNAi-expressing tissue (green dots; H’). (J) Inhibition of apoptosis by Puc coexpression does not change uptake of cells expressing VhaPRR RNAi. (K) VhaPRR RNAi coexpression suppresses avidin uptake in Tsc1 RNAi-expressing cells. Scale bar, 20 mm. See also Figure S3.
examination of the kidneys revealed no significant difference in terms of glomerular lesions, tubular atrophy or fibrosis between the two groups of mice. However, a careful examination of the PTs showed that mice injected with rapamycin often displayed 14 Cell Reports 8, 10–19, July 10, 2014 ª2014 The Authors
large vacuoles occupying most of the apical cell portions (Figures 4A–4D). These vacuoles were positive for the lysosomal protein LAMP1 and the autophagosomal marker LC3B (Figures S5B and S5D), suggesting that they could represent autolysosomes.
Figure 3. mTOR Signaling Controls Megalin Levels and Apical Surface Area (A and B) Megalin (Mgl) knockdown reduces avidin uptake in the en (A and A’) and ptc (B) domain. (C) Megalin RNAi suppresses avidin uptake in cells expressing Tsc1 RNAi. Note that the size of the ptc domain is still enhanced. (D–F) Apical Megalin expression in ptc stripes (marked by mRFP) expressing control RNAi (D and D’), VhaPRR RNAi (E and E’), and Tor-DN expression (F and F’). Upper panel is an x-y and lower panel is an x-z projection with the apical membrane being the upper surface.
(legend continued on next page)
Cell Reports 8, 10–19, July 10, 2014 ª2014 The Authors 15
This was supported by the detection of increased levels of LC3B in western blot analysis (Figures S5E and S5F). Furthermore, decreased levels of P-S6RP and P-AKT (Figures 4E and 4F) were found, which is consistent with downregulation of mTORC1 and mTORC2 activity, respectively (Canaud et al., 2013). To validate the Drosophila results, we performed immunohistochemistry for Megalin (Figures 4C and 4D). Notably, we observed that the expression of Megalin was markedly reduced in mice treated with rapamycin. This reduction in expression was confirmed by western blot and quantitative PCR analysis (Figures 4E–4G). As Megalin is involved in PT cells transport of low-molecular-weight proteins, such as retinol-binding protein (Christensen and Birn, 2001), we next explored the functional consequences of Megalin decrease in rapamycin-treated mice by measuring the level of proteinuria. Coomassie staining and biochemical measurements revealed that rapamycin administration caused a high amount of low-molecular-weight proteinuria without albuminuria (Figures 4H–4J), consistent with impaired PT function. Taken together, these results suggest that longterm treatment with rapamycin decreases mTOR signaling and Megalin levels giving rise to low-molecular-weight proteinuria. DISCUSSION Here, we show that epithelial cells control apical endocytosis in a positive feedback loop that involves transcriptional regulation of Megalin and the V-ATPase as well as the regulation of apical surface area. This loop seems to be amplified at the lysosomal membrane, where the upstream TSC/Rheb module, the V-ATPase and Tor kinase come together. It is believed that the main pool of amino acids relevant for mTORC1 activation is either imported from the extracellular space or generated by metabolic processes in the cytoplasm before being sensed in the lysosomal lumen by the V-ATPase via unknown mechanisms (Jewell et al., 2013; Zoncu et al., 2011). By contrast, proteins entering the cell via endocytosis could, once they have been degraded in the lysosome, represent an important direct source of amino acids without the need for lysosomal import. In fact, it has been demonstrated that mTOR activation to trigger lysosome reformation that occurs after long starvation periods requires lysosomal protease and V-ATPase activity (Yu et al., 2010). Thus, the coupling of protein uptake, protein degradation, amino acid sensing, and transcriptional regulation by the lysosomal V-ATPase/mTORC1 complex could be a very efficient way of controlling anabolic cellular reactions. In renal proximal tubules, this regulatory system may be particularly important because here Megalin itself is needed for the uptake of lysosomal enzymes and, thus, for lysosomal degradation (Nielsen et al., 2013).
In addition to the amino acid input, growth factors such as Insulin that signal via the TSC/Rheb module represent a strong stimulus for cell and tissue growth (Saucedo et al., 2003). In cultured PT cells, it has been shown that Megalin transcription can be activated by Insulin (Hosojima et al., 2009), further emphasizing the intimate relationship between endocytosis and growth. As Megalin downregulation was not sufficient to reduce wing compartment size, it can be concluded that other endocytic pathways, including fluid-phase uptake, may act in a redundant manner with regard to mTOR-dependent cell and tissue growth. At the level of the lysosome, however, the compensatory mechanisms may be more limited. A decline in lysosomal function, e.g., due to V-ATPase deficiency, has devastating consequences for the cell, leading to accumulations of macromolecules and organelles in lysosomes and autolysosomes, respectively. Due to its strong endocytic activity, the PT epithelium of the kidney may be more vulnerable to lysosomal defects than other tissues. This notion is supported by several animal models and human genetic diseases: the constitutive knockout of the a4 V-ATPase subunit, for example, causes both lysosomal protein accumulations in PT cells and tubular proteinuria (Hennings et al., 2012), confirming the link between lysosomal function and apical endocytosis. Likewise, animal models for Dent’s disease and cystinosis, genetic disorders featured by a defective endolysosomal pathway, display tubular proteinuria and low levels of Megalin and Cubilin (Christensen et al., 2003; Piwon et al., 2000; Raggi et al., 2014). The findings in this paper suggest that an important cause for tubular proteinuria is the reduction of lysosomal mTOR signaling and subsequent alterations in the transcriptional profile of PT cells. It may even be speculated that proteinuria and loss of protein uptake is a cytoprotective response, because it prevents additional accumulation of protein and more damage in the endolysosomes. Thus, the control of apical endocytosis by switching mTOR signaling on and off at the lysosome is a simple but elegant mechanism to ensure proper protein uptake during tissue growth and to prevent uptake during stress conditions. An important next step will be to elucidate the downstream mechanisms by which Tor kinase regulates Megalin expression from the lysosome. EXPERIMENTAL PROCEDURES Fly Strains and Genetics Overexpression and transgenic RNAi studies were performed using the UAS/ GAL4 system. UAS-VhaPRR RNAi and UAS-Vha44 RNAi lines as well as VhaPRR and Vha55 alleles and the Vha55-eGFP gene trap line (Flytrap) have been characterized and validated previously (Hermle et al., 2010, 2013; Petzoldt et al., 2013). VhaPRR-GFP is a 3 kb genomic construct of the VhaPRR
(G) Megalin is increased in ptc stripes expressing Tsc1 RNAi. Confocal settings are optimized for the ptc stripe. Thus, expression outside the ptc stripe appears weaker. (H and I) VhaPRR RNAi suppresses Megalin expression in ptc stripes with activated mTOR signaling. Scale bar, 20 mm. (J) Cross-section in the proximal-distal axis of the prepupal wing (schematic in upper left). Dorsal is up and ventral is down. Black boxes depict the areas chosen for TEM pictures in (K) and (L). Transgenes are expressed on the dorsal side with apterous-GAL4. (K and L) Transmission electron microscopy shows a larger apical surface upon Rheb overexpression (K) compared to the wild-type ventral side (L). Black arrows show apical junctions. Area between the arrows represents the entire apical surface. Scale bar, 2 mm. (M and N) Tor (M) and VhaPRR (N) RNAi decrease apical surface area. (O) Dorsal apical surfaces (black bars) were quantified in relation to their ventral wild-type sides (white bar). Data are mean ± SEM (*p < 0.001). Student’s t test (two-tailed). AU, arbitrary unit. See also Figure S4.
16 Cell Reports 8, 10–19, July 10, 2014 ª2014 The Authors
Figure 4. mTOR Inhibition Results in Megalin Reduction and Proteinuria in Mice (A and B) Morphology of kidneys from mice injected either with the vehicle (A) and rapamycin (B) after 6 months. (C and D) Immunostaining of Megalin in vehicle (C) or rapamycin (D) injected mice after 6 months. Scale bars, 10 mm. Note that both the number of Megalinpositive tubuli and Megalin intensity are reduced. (E and F) Western blot (E) and quantification (F) of Megalin, P-Akt (Ser473), and P-S6RP in whole kidney lysate of vehicle- (n = 9) and rapamycin (n = 15)-injected mice after 6 months. (G) Megalin mRNA levels (normalized to RPL13) in vehicle- and rapamycin-treated mice. (H–J) Coomassie staining (H) and measurement of protein to creatinine ratio (I) as well as albumin to creatinine ratio (J) in urine from mice injected either with the vehicle or rapamycin at the age of 6 months, n = 9 and 15 for controls and rapamycin-injected mice, respectively. Bovine serum albumin (BSA) served as control for Coomassie staining. Data are means ± SEM (*p < 0.001). Mann-Whitney test: vehicle versus rapamycin-injected mice. See also Figure S5.
Cell Reports 8, 10–19, July 10, 2014 ª2014 The Authors 17
locus with a C-terminal GFP insertion. As drivers, ap-GAL4, ptc-GAL4 and en-GAL4 were used (all from Bloomington; en-GAL4 also contains UAS-Dicer2 and UAS–GFP). Crosses were grown on standard fly food at 25 C or 29 C. Clones were induced with the heat-shock Flippase (hsFlp). Other fly lines included UAS-PKD2 RNAi (ID:6941, Vienna Drosophila RNAi Center [VDRC]), UAS-Tsc1 RNAi (ID:31039), UAS-Tor RNAi (ID:34639; both from the Bloomington TRIP collection), UAS-Rheb, UAS-Tor-DN (both from Bloomington), Vha13eGFP (ID:CA07644), Vha16-eGFP (ID:ZCL0219), VhaSFD-eGFP (ID:G00259; all from Flytrap), GMR-GAL4;UAS-Rheb (from B. Edgar), tub-Rab7-YFP (from S. Eaton), and UAS-dRagAQ61L and UAS-dRagAT16N (from T. Neufeld). Immunostaining and Endocytic Tracer Studies Pupal wings (24 hr APF) were dissected, fixed in 8% paraformaldehyde for 60 min, and stained according to the standard procedure. The following antibodies were used: guinea pig anti-Megalin (from S. Eaton; Riedel et al., 2011) and rabbit anti-GFP (MBL International). For endocytic tracer uptake experiments, TR-conjugated avidin (Life Technologies) and TR-conjugated dextran (10 kDa, Life Technologies) were diluted with Schneider’s medium (PAN) to 0.2 and 0.5 mg/ml, respectively. Prepupal wings (5.5 hr APF) were dissected in Schneider’s medium and incubated in avidin or dextran for 10 min at room temperature followed by a 10 min chasing period with tracer-free Schneider’s medium. After, prepupal wings were fixed in 8% paraformaldehyde for 15 min. Imaging was performed on a Zeiss LSM 510 inverted confocal microscope.
Morphological Analysis and Immunohistochemistry Mouse kidneys were fixed in 4% paraformaldehyde and paraffin embedded, and 4 mm sections were stained with Periodic Acid Schiff (PAS) staining. For immunohistochemistry, 4 mm sections of paraffin-embedded kidneys were incubated with Megalin antibody (Abcam) followed by a sheep HRP-conjugated anti-rabbit antibody (Amersham). Staining was revealed by DAB. For immunofluorescence, 4 mm sections of paraffin-embedded kidneys were incubated with LAMP1 antibody (Sigma-Aldrich), anti-LC3B (Sigma-Aldrich), and Lotus Tetragonolobus Lectin (Vector Laboratories). The primary antibodies were revealed with the appropriate Alexa-488- or 555-conjugated secondary antibodies (Molecular Probes). Immunofluorescence staining was visualized using the Zeiss LSM 700 confocal microscope. Western Blot Western blots were performed as previously described (Canaud et al., 2013). In brief, protein extracts from kidneys were resolved by SDS-PAGE before being transferred onto the appropriate membrane and incubated with antiMegalin (Abcam), P-Akt Ser473 antibody (Cell Signaling Technology), P-S6 Ribosomal Protein antibody (Cell Signaling Technology), LC3B antibody, and anti-b-actin (both Sigma-Aldrich), followed by the appropriate peroxidaseconjugated secondary antibody. Chemiluminescence was acquired using a Fusion FX7 camera (Vilbert Lourmat) and densitometry was performed using Bio1D software (Certain Tech). SUPPLEMENTAL INFORMATION
Adult Wing Analysis For quantification of the ptc stripe width a minimum of 11 wings of each genotype were analyzed. The width of the ptc stripe was calculated by placing a line perpendicular to the L3 vein to the posterior cross vein. This distance was put into ratio to the length of the posterior cross vein. Quantification of hair densities was performed by counting the number of hairs in a minimum of 11 wings of each genotype in a defined area of the ptc stripe. The number of hairs was determined as a ratio to the hair number in an area of the same size in the adjacent posterior wild-type tissue. Electron Microscopy Drosophila prepupal wings (5.5 hr APF) were dissected and directly fixed with 4% paraformaldehyde and 1% glutaraldehyde in 0.1 M PB overnight at 4 C. After fixation, wings were washed in PB, treated with 2% OsO4, stained with uranyl acetate, dehydrated, and embedded in epoxy resin (Durcupan ACM, Fluka, and Sigma-Aldrich). Ultrathin cross-sections (40 nm) in the proximal-distal axis were cut using an ultramicrotome (Leica UC6) and analyzed with a Zeiss LEO 912 transmission electron microscope operated at 80 kV and equipped with a 2K CCD camera (Tro¨ndle). For quantitative analysis, randomly taken pictures of at least two independent wings with at least 20 cells per genotype were used. Quantification of apical membrane length as well as microvilli number, length, and branching was performed independently for cells in dorsal and ventral compartments, respectively. The values for the dorsal side were calculated in relation to the wild-type ventral value (= 1). Mice C57BL/6 male mice (Charles River Laboratories) were injected daily intraperitoneally with either saline + DMSO (vehicle) (n = 9) or rapamycin (LC Laboratories) 10 mg/kg (n = 15) from day 14 to day 180. We decided to start the treatment on day 14 to overcome any development effect. Animals were fed ad libitum and housed at constant ambient temperature in a 12-hr light cycle. Animal procedures were approved by the Departmental Director of ‘‘Services Ve´te´rinaires de la Pre´fecture de Police de Paris’’ and by the ethical committee of the Paris Descartes University. Urine and Plasma Analyses Urinary albumin, proteins, and creatinine concentrations were measured using an Olympus multiparametric analyzer (Instrumentation Laboratory). In addition, 20 ml of urine was run on Coomassie gels to visualize proteinuria.
18 Cell Reports 8, 10–19, July 10, 2014 ª2014 The Authors
Supplemental Information includes Supplemental Experimental Procedures and five figures and can be found with this article online at http://dx.doi.org/ 10.1016/j.celrep.2014.05.035. ACKNOWLEDGMENTS We thank Severine Kayser for excellent technical support. We thank Thomas Neufeld, Bruce Edgar, the Bloomington and Kyoto Stock Center, and the VDRC for fly strains. We thank Suzanne Eaton for the Megalin antibody. We are grateful to Mario Pende and Mila Roceri for help with the rapamycin experiment and to Sophie Berissi for mouse morphology. We thank Giorgos Pyrowolakis for critically reading the manuscript. The work has been supported by an Emmy-Noether grant (SI1303/2-1) by the Deutsche Forschungsgemeinschaft and a Gerontosys NephAge grant (0315896) by the German Ministry of Research and Education (BMBF). The TRiP RNAi resource at Harvard Medical School is supported by NIH grant R01GM084947. The mice experiments have been supported by INSERM, Universite´ Paris Descartes, Sorbonne Paris Cite´, and ANR. Received: January 16, 2014 Revised: April 16, 2014 Accepted: May 16, 2014 Published: June 19, 2014 REFERENCES Bar-Peled, L., Schweitzer, L.D., Zoncu, R., and Sabatini, D.M. (2012). Ragulator is a GEF for the rag GTPases that signal amino acid levels to mTORC1. Cell 150, 1196–1208. Birn, H., Fyfe, J.C., Jacobsen, C., Mounier, F., Verroust, P.J., Orskov, H., Willnow, T.E., Moestrup, S.K., and Christensen, E.I. (2000). Cubilin is an albumin binding protein important for renal tubular albumin reabsorption. J. Clin. Invest. 105, 1353–1361. Canaud, G., Bienaime´, F., Viau, A., Treins, C., Baron, W., Nguyen, C., Burtin, M., Berissi, S., Giannakakis, K., Muda, A.O., et al. (2013). AKT2 is essential to maintain podocyte viability and function during chronic kidney disease. Nat. Med. 19, 1288–1296. Christensen, E.I., and Birn, H. (2001). Megalin and cubilin: synergistic endocytic receptors in renal proximal tubule. Am. J. Physiol. Renal Physiol. 280, F562–F573.
Christensen, E.I., Devuyst, O., Dom, G., Nielsen, R., Van der Smissen, P., Verroust, P., Leruth, M., Guggino, W.B., and Courtoy, P.J. (2003). Loss of chloride channel ClC-5 impairs endocytosis by defective trafficking of megalin and cubilin in kidney proximal tubules. Proc. Natl. Acad. Sci. USA 100, 8472–8477.
Neff, F., Flores-Dominguez, D., Ryan, D.P., Horsch, M., Schro¨der, S., Adler, T., Afonso, L.C., Aguilar-Pimentel, J.A., Becker, L., Garrett, L., et al. (2013). Rapamycin extends murine lifespan but has limited effects on aging. J. Clin. Invest. 123, 3272–3291.
Demetriades, C., Doumpas, N., and Teleman, A.A. (2014). Regulation of TORC1 in response to amino acid starvation via lysosomal recruitment of TSC2. Cell 156, 786–799.
Nguyen, G., Delarue, F., Burckle´, C., Bouzhir, L., Giller, T., and Sraer, J.D. (2002). Pivotal role of the renin/prorenin receptor in angiotensin II production and cellular responses to renin. J. Clin. Invest. 109, 1417–1427.
Doherty, G.J., and McMahon, H.T. (2009). Mechanisms of endocytosis. Annu. Rev. Biochem. 78, 857–902. Hennig, K.M., and Neufeld, T.P. (2002). Inhibition of cellular growth and proliferation by dTOR overexpression in Drosophila. Genesis 34, 107–110.
Nielsen, R., Mollet, G., Esquivel, E.L., Weyer, K., Nielsen, P.K., Antignac, C., and Christensen, E.I. (2013). Increased lysosomal proteolysis counteracts protein accumulation in the proximal tubule during focal segmental glomerulosclerosis. Kidney Int. 84, 902–910.
Hennig, K.M., Colombani, J., and Neufeld, T.P. (2006). TOR coordinates bulk and targeted endocytosis in the Drosophila melanogaster fat body to regulate cell growth. J. Cell Biol. 173, 963–974.
Pelkmans, L., Fava, E., Grabner, H., Hannus, M., Habermann, B., Krausz, E., and Zerial, M. (2005). Genome-wide analysis of human kinases in clathrinand caveolae/raft-mediated endocytosis. Nature 436, 78–86.
Hennings, J.C., Picard, N., Huebner, A.K., Stauber, T., Maier, H., Brown, D., Jentsch, T.J., Vargas-Poussou, R., Eladari, D., and Hu¨bner, C.A. (2012). A mouse model for distal renal tubular acidosis reveals a previously unrecognized role of the V-ATPase a4 subunit in the proximal tubule. EMBO Mol. Med. 4, 1057–1071. Hermle, T., Saltukoglu, D., Grunewald, J., Walz, G., and Simons, M. (2010). Regulation of Frizzled-dependent planar polarity signaling by a V-ATPase subunit. Curr. Biol. 20, 1269–1276. Hermle, T., Guida, M.C., Beck, S., Helmsta¨dter, S., and Simons, M. (2013). Drosophila ATP6AP2/VhaPRR functions both as a novel planar cell polarity core protein and a regulator of endosomal trafficking. EMBO J. 32, 245–259. Hosojima, M., Sato, H., Yamamoto, K., Kaseda, R., Soma, T., Kobayashi, A., Suzuki, A., Kabasawa, H., Takeyama, A., Ikuyama, K., et al. (2009). Regulation of megalin expression in cultured proximal tubule cells by angiotensin II type 1A receptor- and insulin-mediated signaling cross talk. Endocrinology 150, 871–878. Hurtado-Lorenzo, A., Skinner, M., El Annan, J., Futai, M., Sun-Wada, G.H., Bourgoin, S., Casanova, J., Wildeman, A., Bechoua, S., Ausiello, D.A., et al. (2006). V-ATPase interacts with ARNO and Arf6 in early endosomes and regulates the protein degradative pathway. Nat. Cell Biol. 8, 124–136.
Pen˜a-Llopis, S., Vega-Rubin-de-Celis, S., Schwartz, J.C., Wolff, N.C., Tran, T.A., Zou, L., Xie, X.J., Corey, D.R., and Brugarolas, J. (2011). Regulation of TFEB and V-ATPases by mTORC1. EMBO J. 30, 3242–3258. Petzoldt, A.G., Gleixner, E.M., Fumagalli, A., Vaccari, T., and Simons, M. (2013). Elevated expression of the V-ATPase C subunit triggers JNK-dependent cell invasion and overgrowth in a Drosophila epithelium. Dis. Model. Mech. 6, 689–700. Piwon, N., Gu¨nther, W., Schwake, M., Bo¨sl, M.R., and Jentsch, T.J. (2000). ClC-5 Cl- -channel disruption impairs endocytosis in a mouse model for Dent’s disease. Nature 408, 369–373. Raggi, C., Luciani, A., Nevo, N., Antignac, C., Terryn, S., and Devuyst, O. (2014). Dedifferentiation and aberrations of the endolysosomal compartment characterize the early stage of nephropathic cystinosis. Hum. Mol. Genet. 23, 2266–2278. Riedel, F., Vorkel, D., and Eaton, S. (2011). Megalin-dependent yellow endocytosis restricts melanization in the Drosophila cuticle. Development 138, 149–158. Riediger, F., Quack, I., Qadri, F., Hartleben, B., Park, J.K., Potthoff, S.A., Sohn, D., Sihn, G., Rousselle, A., Fokuhl, V., et al. (2011). Prorenin receptor is essential for podocyte autophagy and survival. J. Am. Soc. Nephrol. 22, 2193–2202.
Ibar, C., Cataldo, V.F., Va´squez-Doorman, C., Olguı´n, P., and Glavic, A. (2013). Drosophila p53-related protein kinase is required for PI3K/TOR pathwaydependent growth. Development 140, 1282–1291.
Sancak, Y., Peterson, T.R., Shaul, Y.D., Lindquist, R.A., Thoreen, C.C., Bar-Peled, L., and Sabatini, D.M. (2008). The Rag GTPases bind raptor and mediate amino acid signaling to mTORC1. Science 320, 1496–1501.
Jewell, J.L., Russell, R.C., and Guan, K.L. (2013). Amino acid signalling upstream of mTOR. Nat. Rev. Mol. Cell Biol. 14, 133–139.
Sancak, Y., Bar-Peled, L., Zoncu, R., Markhard, A.L., Nada, S., and Sabatini, D.M. (2010). Ragulator-Rag complex targets mTORC1 to the lysosomal surface and is necessary for its activation by amino acids. Cell 141, 290–303.
Kim, E., Goraksha-Hicks, P., Li, L., Neufeld, T.P., and Guan, K.L. (2008). Regulation of TORC1 by Rag GTPases in nutrient response. Nat. Cell Biol. 10, 935–945. Kinouchi, K., Ichihara, A., Sano, M., Sun-Wada, G.H., Wada, Y., KurauchiMito, A., Bokuda, K., Narita, T., Oshima, Y., Sakoda, M., et al. (2010). The (pro)renin receptor/ATP6AP2 is essential for vacuolar H+-ATPase assembly in murine cardiomyocytes. Circ. Res. 107, 30–34. Ludwig, J., Kerscher, S., Brandt, U., Pfeiffer, K., Getlawi, F., Apps, D.K., and Scha¨gger, H. (1998). Identification and characterization of a novel 9.2-kDa membrane sector-associated protein of vacuolar proton-ATPase from chromaffin granules. J. Biol. Chem. 273, 10939–10947. Menon, S., Dibble, C.C., Talbott, G., Hoxhaj, G., Valvezan, A.J., Takahashi, H., Cantley, L.C., and Manning, B.D. (2014). Spatial control of the TSC complex integrates insulin and nutrient regulation of mTORC1 at the lysosome. Cell 156, 771–785.
Saucedo, L.J., Gao, X., Chiarelli, D.A., Li, L., Pan, D., and Edgar, B.A. (2003). Rheb promotes cell growth as a component of the insulin/TOR signalling network. Nat. Cell Biol. 5, 566–571. Wang, D., and Hiesinger, P.R. (2013). The vesicular ATPase: a missing link between acidification and exocytosis. J. Cell Biol. 203, 171–173. Yu, L., McPhee, C.K., Zheng, L., Mardones, G.A., Rong, Y., Peng, J., Mi, N., Zhao, Y., Liu, Z., Wan, F., et al. (2010). Termination of autophagy and reformation of lysosomes regulated by mTOR. Nature 465, 942–946. Zhang, F., Zhao, Y., Chao, Y., Muir, K., and Han, Z. (2013). Cubilin and amnionless mediate protein reabsorption in Drosophila nephrocytes. J. Am. Soc. Nephrol. 24, 209–216. Zoncu, R., Bar-Peled, L., Efeyan, A., Wang, S., Sancak, Y., and Sabatini, D.M. (2011). mTORC1 senses lysosomal amino acids through an inside-out mechanism that requires the vacuolar H(+)-ATPase. Science 334, 678–683.
Cell Reports 8, 10–19, July 10, 2014 ª2014 The Authors 19