PI(4,5)P2 Controls Slit Diaphragm Formation and Endocytosis in Drosophila Nephrocytes

PI(4,5)P2 Controls Slit Diaphragm Formation and Endocytosis in Drosophila Nephrocytes | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article PI(4,5)P2 Controls Slit Diaphragm Formation and Endocytosis in Drosophila Nephrocytes Maximilian Gass, Sarah Borkowsky, Marie-Luise Lotz, Rita Schröter, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-739266/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 18 Apr, 2022 Read the published version in Cellular and Molecular Life Sciences → Version 1 posted 5 You are reading this latest preprint version Abstract Drosophila nephrocytes are an emerging model system for mammalian podocytes and podocyte-associated diseases. Like podocytes, nephrocytes exhibit characteristics of epithelial cells, but the role of phospholipids in polarization of these cells is yet unclear. In epithelia phosphatidylinositol(4,5)bisphosphate (PI(4,5)P2) and phosphatidylinositol(3,4,5)-trisphosphate (PI(3,4,5)P3) are asymmetrically distributed in the plasma membrane and determine apical-basal polarity. Here we demonstrate that both phospholipids are present in the plasma membrane of nephrocytes, but only PI(4,5)P2 accumulates at slit diaphragms. Knockdown of Skittles, a phosphatidylinositol(4)phosphate 5-kinase, which produces PI(4,5)P2, abolished slit diaphragm formation and led to strongly reduced endocytosis. Notably, reduction in PI(3,4,5)P3 by overexpression of PTEN or expression of a dominant-negative phosphatidylinositol-3-Kinase did not affect nephrocyte function, whereas enhanced formation of PI(3,4,5)P3 by constitutively active phosphatidylinositol-3-Kinase resulted in strong slit diaphragm and endocytosis defects by ectopic activation of the Akt/mTOR pathway. Thus, PI(4,5)P2 but not PI(3,4,5)P3 is essential for slit diaphragm formation and nephrocyte function. However, PI(3,4,5)P3 has to be tightly controlled to ensure nephrocyte development. Cellular & Molecular Neuroscience Nephrocyte podocyte slit diaphragm Phosphoinositides PI3-Kinase Phospholipids PTEN Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction In Drosophila , pericardial nephrocytes located along the heart tube and garland nephrocytes surrounding the proventriculus filtrate the hemolymph and endocytose proteins and toxins to store the latter permanently in order to inactivate them [ 1 ]. Nephrocytes were shown to share several key features with podocytes in vertebrates, qualifying them as a model system to study mammalian podocyte function and podocyte-associated diseases [ 2 – 4 ]. Like in podocytes, homologues of Nephrin- and Neph1 (Sticks and stones (Sns)/Hybris and Kind of irre (Kirre)/Dumbfounded) form the slit diaphragm, thereby separating the lacunae from the body cavity with hemolymph. These lacunae are formed by invaginations of the plasma membrane and form channel-like structures with both ends connected to the extra cellular space [ 5 ]. Due to the high endocytosis capacity in these lacunae and the expression of endocytosis receptors like Cubilin, Megalin and Amnionless, nephrocytes are used as a model system for proximal tubules of the kidney, too [ 6 ]. Apart from the core components of the Nephrin/Neph1 family, the slit diaphragm is stabilized by adapter proteins, e.g. the Podocin homologue Mec2 [ 7 , 1 ] and the ZO-1 homologue Polychaetoid [ 1 , 8 ]. Furthermore, we recently showed, that regulators of classical apical-basal polarity in epithelia are partly localized to slit diaphragm complexes [ 9 ]. Knockdown studies revealed that apical polarity regulators, such as Crumbs/Stardust and the PAR/aPKC complex as well as the basolateral polarity determinants Scribble/Lethal (2) giant larvae and PAR-1 are essential for slit diaphragm formation and - at least some of them - for endocytosis [ 10 , 11 , 9 ]. In classical epithelia, these polarity regulators are targeted to either the apical (Crumbs- and PAR/aPKC-complex) or the basolateral (Scribble/Dlg/Lgl-complex, PAR-1/LKB1) plasma membrane and are essential for the establishment and maintenance of apical-basal polarity and cell-cell contacts [ 12 ]. However, not only proteins are involved in this process, but also distinct phospholipids are enriched either in the apical or the basolateral plasma membrane: In particular, phosphatidylinositol(4,5)bisphosphate (PI(4,5)P2) accumulates in the apical membrane, whereas phosphatidylinositol(3,4,5)trisphosphate (PI(3,4,5)P3) is preferentially found in the basolateral membrane domain [ 13 , 14 ]. Notably, PTEN, which dephosphorylates PI(3,4,5)P3 to generate PI(4,5)P2, is recruited to the plasma membrane by PAR-3, the core scaffolding protein of the PAR/aPKC-complex [ 15 – 17 ]. Thereby, junctionally localized PAR-3/PTEN establishes a segregation point for PI(3,4,5)P3 and PI(4,5)P2 [ 13 ]. In turn, PAR-3 directly binds to PI(4,5)P2 and PI(3,4,5)P3, which contributes to its targeting to the plasma membrane [ 18 , 19 ]. During epithelial polarization, phosphatidylinositol-3-kinase (PI3K), which phosphorylates PI(4,5)P2 to PI(3,4,5)P3, seems to function as one of the first cues to determine the basolateral, PI(3,4,5)P3-enriched plasma membrane domain [ 20 ]. Moreover, disruption of the PI(4,5)P2/PI(3,4,5)P3 balance results in severe polarity defects, suggesting a role of phospholipids as regulators of apical-basal cell polarity [ 13 , 14 ]. Although podocytes and nephrocytes share key features with classical epithelial cells, like cell-cell junctions and apical-basal polarity, little is known about the distribution and function of PI(4,5)P2 and PI(3,4,5)P3 in these cell types. Moreover, several studies suggest different functions of PI3K and PTEN in cultured podocytes [ 21 – 24 ], but the role of these key enzymes in vivo is still unclear. Therefore, the aim of this study was to investigate the subcellular accumulation of these two phospholipids as well as their function in slit diaphragm assembly and nephrocyte development. Materials And Methods Drosophila stocks and genetics Fly stocks were cultured on standard cornmeal agar food and maintained at 25°C. For downregulation or overexpression of specific genes for immunostainings and electron microscopy, sns::GAL4 [ 25 ], was crossed with the following lines: UAS::Akt-RNAi (#103703), UAS::Exo70-RNAi (#103717), UAS::Or83b-RNAi (negative control, #100825), UAS::PTEN-RNAi (#01475), UAS::Sec3-RNAi (#108085), UAS::Sktl-RNAi (#101624) (provided by Vienna Drosophila Resource Center, Austria), UAS::PI3K92E-CAAX (PI3K-CA, #8294), UAS::PI3K92E.A2860C (PI3K-DN, #8289), UAS:Sktl (#39675), UAS::PH(PLCδ)-mCherry (#51658), tubP::GAL80ts (65406), UAS::dTOR-RNAi (#34639) (all obtained from Bloomington stock center). UAS::Myr::Akt was provided by Hugo Stocker [ 26 ] and UAS::Myc-Sktl was obtained from Sandra Claret [ 27 ]. UASt::PTEN was established by PhiC31-Integrase Insertion using attP86F. UAS::PH(Akt)-GFP was constructed by fusing the PH domain of mammalian Akt1 to the N-terminus of GFP in the pUASt-vector. Transgenic flies were generated by P-element-mediated germ line transformation. An insertion on second chromosome was used in this study. For all RNAi and overexpression experiments, crosses were kept for 3 days at 25°C and larvae subsequently shifted to 29°C, in order to obtain maximum expression. PH(PLCδ)-mCherry was expressed at 25°C, PH(Akt)-GFP was analyzed at 18°C, 21°C and 25°C, with best results at 18°C, because at higher temperature, the expression of the chimeric protein was too strong and found overall the cell, likely due to the limited amount of PI(3,4,5)P3 to bind to. Endocytosis assays For the ANP-2xGFP accumulation assay, garland nephrocytes from wandering third instar larvae were dissected in HL3.1 saline [ 28 ], fixed in 4% PFA in PBS for 10min, stained with DAPI for 20min, washed with PBS, and mounted in Mowiol. ANP-2xGFP accumulation per nephrocyte area (CTCF = Corrected Total Cell Fluorescence) was analyzed and quantified with ImageJ after subtracting the autofluorescent background of dissected larvae. For each genotype, at least 100 nephrocytes of 15 independent larvae were quantified. Immunohistochemistry Garland nephrocytes were dissected as described above and heat-fixed for 20 seconds in boiling heat fix saline (0.03% Triton-X100). Subsequently, nephrocytes were washed three times in PBS + 0.2% Triton X-100 and blocked with 1% BSA for 1h, incubated over night with primary antibodies in PBS + 0.2% Triton X-100 + 1% BSA, washed three times and incubated for 2h with secondary antibodies. After three washing steps and DAPI-staining, nephrocytes were mounted with Mowiol. Primary antibodies used were as follows: anti Baz [1:250, 29], rabbit anti Exo70 [1:500, 30], goat anti GFP (1:500, #600-101-215, Rockland), mouse anti Myc (1:100, 9E10, Developmental Studies Hybridoma Bank (DSHB)), chicken anti Sns [1:1000, 10], mouse anti Talin (1:20, E16B, DSHB). Secondary antibodies conjugated with Alexa 488, Alexa 568 and Alexa 647 (Life technologies) were used at 1:400. Images were taken on a Leica SP8 confocal microscope using lightning program and processed using ImageJ. Transmission electron microscopy Garland nephrocytes of third instar larvae were dissected in HL3.1 saline, high pressure frozen (EM-PACT2, Leica, Wetzlar, Germany), freeze-substituted in acetone / 1% OsO4 / 5% H 2 O / 0.25% uranyl acetate (AFS2, Leica, Wetzlar, Germany) and embedded in Epon. For transmission electron microscopy, 70nm thick sections were cut using an ultramicrotome (Leica UC7, Wetzlar, Germany). All samples were imaged with a transmission electron microscope (ZEISS, Libra 120, Germany). Results PI(4,5)P2 but not PI(3,4,5)P3 is enriched at slit diaphragms In classical epithelia, PI(4,5)P2 is enriched in the apical plasma membrane, whereas PI(3,4,5)P3 accumulates in the basolateral plasma membrane [ 13 ]. In contrast, nothing is known about the distribution of specific phospholipids in mammalian podocytes or Drosophila nephrocytes. Therefore, we first investigated the distribution of PI(4,5)P2 and PI(3,4,5)P3 in Drosophila garland nephrocytes by expressing fusion proteins consisting of a fluorescent protein and a Pleckstrin homology (PH) domain, which preferentially bind to PI(4,5)P2 (PH domain of PLCδ [ 31 ]) or to PI(3,4,5)P3 (PH domain of Akt1, this study). mCherry-PH(PLCδ) is substantially associated with the plasma membrane (Fig. 1 A-B) but it is also found in intracellular pools, partly associated with vesicular structures. Surface views reveal that its cortical association form strand-like structures, which to some extent co-stain with endogenous Sns, a marker for slit diaphragms (Fig. 1 B). In contrast, PH(Akt)-GFP is only weakly associated with the plasma membrane but also shows a cytoplasmic and vesicular-associated distribution (Fig. 1 C). Nonetheless, surface views show a strand-like pattern too, but these strands do not co-localize with Sns (Fig. 1 D) but are rather found between the Sns-strands. These findings suggest, that PI(4,5)P2 in the plasma membrane accumulates at slit diaphragms, whereas PI(3,4,5)P3 is enriched in the free plasma membrane between slit diaphragms. Impaired PI(4,5)P2 production results in strong developmental and slit diaphragm defects In order to test whether PI(4,5)P2 is essential for nephrocyte development and function, in particular regarding slit diaphragm assembly and maintenance, we used RNA-interference (RNAi) to knockdown the ubiquitously expressed PI(4)P5-Kinase Skittles (Sktl), which is responsible for converting PI(4)P to PI(4,5)P2 using the nephrocyte-specific driver line sns::GAL4. In Drosophila , Sktl has been described to regulate apical-basal polarity by targeting PAR-3 to the apical junctions in follicular epithelial cells [ 32 ] and to the anterior cortex in the oocyte [ 33 ]. In tracheal tubes, Sktl-produced PI(4,5)P2 was proposed to recruit the formin Diaphanous to the apical membrane [ 34 ]. In nephrocytes, Sktl partly colocalizes with Sns at slit diaphragms (Fig. S1A), opening the possibility of a local accumulation of PI(4,5)P2 in microdomains of the plasma membrane at slit diaphragms. Indeed, impaired expression of Sktl resulted in dramatic morphological changes with fused nephrocytes (Fig. 2 B compared to control RNAi in 2A). Furthermore, the typical strand-like structures of Sns-labelled slit diaphragm observed at the surface of control nephrocytes was completely abolished in Sktl-RNAi expressing nephrocytes, resulting in a dispersion of Sns to intracellular puncta (Fig. 2 A-D). Besides Sns, the basal polarity determinant Talin and the apical polarity regulator PAR-3 (Bazooka (Baz) in Drosophila) are lost from the cortex, too. In contrast to impaired PI(4,5)P2 levels, overexpression of Sktl in order to increase PI(4,5)P2 did not affect nephrocyte morphology or slit diaphragm assembly (Fig. S1B, quantified in Fig. 2 E), although the amount of PI(4,5)P2 seemed to be significantly increased, as demonstrated by enhanced accumulation of mCherry-PH(PLCδ) at the plasma membrane (Fig. S1C). Skittles is essential for slit diaphragm assembly by regulating exocytosis Analysis of Sktl-RNAi expressing nephrocytes by electron microscopy confirmed an almost complete absence of slit diaphragms (Fig. 2 D compared to control in C). Notably, these nephrocytes do not form regular lacunae but accumulate large electron-light vesicles below the plasma membrane (marked with asterisks in Fig. 2 D). This phenotype suggests severe defects in exocytosis, which is essential for the delivery of transmembrane proteins of the slit diaphragm complex (Sns, Kirre and Crb). During exocytosis, clustering of PI(4,5)P2 facilitates the docking of the exocyst complex to the plasma membrane by direct binding of its components Exo70 and Sec3 in yeast and in mammalian cells [ 35 – 38 ]. In a second step, PI(4,5)P2 is also essential for vesicle fusion and several proteins involved in regulation of fusion directly interact with PI(4,5)P2 [reviewed by 39]. In order to test whether Sktl-produced PI(4,5)P2 recruits Exocyst complex components in nephrocytes, we stained for endogenous Exo70. In control nephrocytes, apart from intracellular giant vesicles, a substantial pool of Exo70 was found at the plasma membrane, co-localizing with Sns (Fig. 2 G). In contrast, it displayed a diffuse localization with some perinuclear accumulation in Sktl-RNAi expressing nephrocytes (Fig. 2 H). Moreover, downregulation of the exocyst complex components Exo70 and Sec3 resulted in similar loss of slit diaphragms as Sktl-RNAi (Fig. 2 I and Fig. S1D), which is in line with a recent study reporting a crucial role of the exocyst complex in slit diaphragm formation/maintenance [ 33 ]. Decreased PI(4,5)P2 levels impair endocytosis in nephrocytes Apart from exocytosis, PI(4,5)P2 also regulates clathrin-dependent and -independent endocytosis by recruiting several proteins involved in early steps of endocytosis to the plasma membrane and by inducing actin remodeling during micropinocytosis [reviewed by 39]. In nephrocytes, endocytosis is essential for the uptake of filtrated proteins, toxins and metabolites, which are then stored and inactivated. Disturbance of slit diaphragm formation as well as of endocytic receptors and proteins involved in the endocytosis machinery have been reported to reduce endocytosis [ 25 , 1 , 10 , 11 , 6 , 40 – 43 ]. In order to test, whether PI(4,5)P2 is essential for endocytosis in nephrocytes, we quantified the accumulation of secreted ANP-2xGFP [ 9 ] which is secreted into the hemolymph, filtrated by nephrocytes and taken up by endocytosis. Indeed, downregulation of Sktl in nephrocytes, reducing PI(4,5)P2 levels, resulted in a strong decrease of ANP-2xGFP accumulation in nephrocytes, consistent with impaired endocytosis (Fig. 2 F). This is in line with reports from the Drosophila oocyte, where Sktl is essential for Rab5-mediated endocytosis of yolk protein [ 44 ]. PI(3,4,5)P3 is not essential for nephrocyte function but ectopic production results in dominant negative effects In contrast to PI(4,5)P2, reducing PI(3,4,5)P3 by overexpression of PTEN or expression of a dominant negative version of PI3K (PI3K-DN) did not affect nephrocyte morphology or slit diaphragm formation (Fig. S2A-B and Fig. 3 F). However, overexpression of a constitutively active PI3K (PI3K-CA), which is targeted to the plasma membrane by attachment of a prenylation anchor (CAAX-motif), in nephrocytes resulted in a strong fusion phenotype and a disturbed pattern of slit diaphragms (Fig. 3 A-D, quantified in 3F). Notably, PI3K-CA-expressing nephrocytes are larger than control nephrocytes (Fig. 3 G). In addition to slit diaphragm defects, overexpression of PI3K-CA resulted in a drastic decrease in ANP-2xGFP uptake, suggesting a defect in endocytosis (Fig. 3 H). Like PI3K-CA, enhanced accumulation of PI(3,4,5)P3 by knockdown of PTEN resulted in similar but milder phenotypes regarding slit diaphragms, whereas cell size was not increased (Fig. 3 E-G). This is likely due to the limited abundance of PI(3,4,5)P3 within the plasma membrane. Ectopic production of PI(3,4,5)P3 from PI(4,5)P2 by PI3K-CA likely produces higher levels of PI(3,4,5)P3 in the plasma membrane due to the larger pool of PI(4,5)P2 [ 45 ], whereas inhibition of dephosphorylation of PI(3,4,5)P3 to PI(4,5)P2 only moderately increases PI(3,4,5)P3 levels in the plasma membrane. These data suggest that slit diaphragm assembly might be more sensitive to enhanced PI(3,4,5)P3 level than cell size regulation. Phenotypes of increased PI(3,4,5)P3 are induced by the Akt/mTOR pathway Increased PI(3,4,5)P3 in the plasma membrane leads to activation of the Akt/mTOR signaling cascade, which, among various other functions, results in cell survival and increased cell size and proliferation [reviewed by 46]. In order to test whether the phenotypes observed in nephrocytes expressing PI3K-CA are caused by ectopic Akt/mTOR activation, we introduced a constitutively active variant of Akt (Myr-Akt), which is recruited to the plasma membrane and activated independently of PI(3,4,5)P3 due to the fusion of a myristoylation-signal [ 26 ]. Indeed, these nephrocytes mimicked the PI3K-CA overexpression phenotype with disrupted slit diaphragms, increased size and fusion phenotypes (Fig. 3 F, G, I). However, cell size of Myr-Akt expressing nephrocytes was not as strongly increased as in PI3K-CA expressing ones (albeit higher than in case of PTEN-RNAi), whereas slit diaphragm assembly is severely disturbed and comparable with Pi3K-CA and PTEN-RNAi-expressing nephrocytes. Thus, these data provide additional support to the notion that slit diaphragm assembly and size regulation show different susceptibility to levels of PI(3,4,5)P3. To further substantiate our hypothesis that the defects observed in PI3K-CA expressing nephrocytes are due to ectopic activation of Akt/mTOR signaling upon increased levels of PI(3,4,5)P3, we knocked down Akt or Drosophila Tor (dTOR) in PI3K-CA expressing nephrocytes. As depicted in Fig. 3 F,G,J and Fig. S2C, downregulation of Akt or dTOR rescued to a large extent the slit diaphragm defects as well as size differences in PI3K-CA expressing nephrocytes, confirming that the dominant negative function of PI(3,4,5)P3 is mediated by the Akt/mTOR-pathway. Changes in PI(4,5)P2 and PI(3,4,5)P3 levels cause rapid defects In order to elucidate whether slit diaphragm defects are established early in development during formation of nephrocytes or whether PI(4,5)P2 and PI(3,4,5)P3 levels are also essential for the turnover and maintenance of slit diaphragms, we used a temperature-sensitive GAL80 (GAL80ts), which suppresses GAL4 activity at the permissive temperature at 18°C. After molting to L3, larvae were shifted to 29°C for 24h prior to dissection, inactivating the GAL80 and thus releasing GAL4, which induces the UAS-transgene. In Sktl-RNAi expressing nephrocytes dissected from animals raised under these conditions, we observed similar defects in morphology as well as impaired Sns strands (Fig. 4 A-C), indicating that PI(4,5)P2 is essential for the turnover/maintenance after the initial establishment of slit diaphragms during the development of nephrocytes. In contrast, short-term induction of Pi3K-CA did not produce phenotypes comparable to continuous expression of this transgene (Fig. 4 D-F), indicating that the Akt/mTOR-mediated effect of ectopic PI(3,4,5)P3 production is either critical during nephrocyte development or it takes longer time to get established, presumably due to the delay upon transcriptional reprogramming of the cell as a consequence of mTOR target activation. Discussion Our findings demonstrate that PI(4,5)P2, but not PI(3,4,5)P3 is essential for nephrocyte function and slit diaphragm formation. Of note, PI(4,5)P2 is not evenly distributed in the entire plasma membrane but displays a strand-like pattern, partly colocalizing with Sns as a maker for slit diaphragms. Although PI(4,5)P2 has been found in other cell types at the entire plasma membrane – or, in epithelial cells, enriched in the apical plasma membrane domain - there is increasing evidence that this phospholipid is concentrated in distinct microdomains of the plasma membrane [discussed by 47,48]: In cultured fibroblasts, freeze-fracture membrane preparation and subsequent electron microscopy revealed three distinct pools of PH(PLCδ) at the rim of caveolae, in coated pits and at the free plasma membrane [ 49 ]. Notably, these three pools exhibited different kinetics upon regulatory stimuli, suggesting different types of regulation. PI(4,5)P2 was also reported to accumulate in lipid rafts of distinct (phospho)lipid and cholesterol composition within the plasma membrane, promoting local actin remodeling or receptor clustering [ 50 – 52 ]. Sarmento et al. observed a Ca(2+)-dependent PI(4,5)P2 clustering in liposomes in vitro under physiological Ca(2+) and PI(4,5)P2 concentrations [ 53 ]. Thus, PI(4,5)P2 may accumulate in distinct microdomains of the plasma membrane adjacent to slit diaphragms in order to regulate vesicle trafficking – to the plasma membrane by inducing fusion of vesicles and from the plasma membrane by regulating endocytosis. The dramatic phenotypes observed in Sktl-RNAi expressing nephrocytes underline the critical role of PI(4,5)P2 as an important regulator in these processes. Notably, the human homologue of Sktl, PIP5Kα, was described to be recruited by the Chloride Intracellular Channel 5 (CLIC5A) to cortical Ezrin, inducing clusters of PI(4,5)P2 in the plasma membrane of COS-7 cells [ 54 ]. In podocytes, Ezrin is part of the Ezrin-NHERF2-Podocalyxin complex, an essential component of the glycocalyx. Furthermore, in glomeruli of CLIC5A-deficient mice, cortical Ezrin/NHERF2 as well as glomerular Podocalyxin are reduced [ 54 ]. Another hint to an important role of PI(4,5)P2 in regulating podocyte morphology comes from a study reporting that the PI5P-Phosphatase Ship2 can be recruited and activated by Nephrin via Nck-Pak1-Filamin in cultured human podocytes [ 55 ]. Ship2 dephosphorylates PI(3,4,5)P3 to PI(3,4)P2, thus its activation by Nephrin in this systems results in an increase of PI(3,4)P2, which activates Lamellipodin, a regulator of Ena/Vasp proteins, resulting in the formation of lamellipodia. Finally, the Nephrin/Ship2 interaction was increased in a podocyte injury model in vivo , suggesting that lamellipodia formation upon Nephrin-mediated Ship2-activation contributes to foot process effacement observed upon podocyte damage. However, it remains unclear how the Ship2-regulated balance between PI(3,4,5)P3 and PI(3,4)P2 at the Nephrin-complex contributes to slit diaphragm assembly/maintenance and podocyte function under physiological conditions. PI(4,5)P2 as well as PI(3,4,5)P3 are capable of regulating the actin cytoskeleton by recruiting and activating the small GTPases Rac1 and Cdc42 as well as proteins of the WASP family [ 56 – 58 ]. Notably, a coordinated actin cytoskeleton remodeling is essential for cortical Nephrin localization and slit diaphragm assembly in Drosophila nephrocytes [ 59 , 60 ] as well as in mammalian podocytes [ 61 ]. Vice versa , activated Nephrin recruits PI3K resulting in Rac1 activation, actin branching and lamellipodia formation in cultured rat podocytes [ 21 ]. Notably, PTEN is downregulated in podocytes of patients suffering from diabetic nephropathy and inhibition or podocyte-specific knockout of PTEN in mice results in cytoskeleton rearrangements, foot process effacement and proteinuria [ 62 ]. Apart from their impact on the actin cytoskeleton, PI(4,5)P2 and PI(3,4,5)P3-activated Rac1/Cdc42 and actin regulators are essential for remodeling and stability of tight junctions as well as adherens junctions in classical epithelia [reviewed by 63]. Increasing evidence suggests that the slit diaphragms connecting the foot processes of neighboring podocytes emerge from transformation of the tight junctions of the epithelial podocyte progenitor cells [ 64 ]. Indeed, several proteins of the adherens- and tight junctions can also be found to be components of the slit diaphragm, e.g. ZO-1, Crumbs, PAR/aPKC-complex [ 10 , 8 , 1 , 65 – 71 , 11 ]. Thus, it is likely that changes in PI(4,5)P2 and PI(3,4,5)P3 affect slit diaphragm formation and maintenance/stability like they affect adherens junctions/tight junctions in classical epithelia. Declarations Acknowledgements We thank E. Chan, S. Claret, the Bloomington Drosophila stock center at the University of Indiana (USA), the Vienna Drosophila Resource Center (Austria) and the Developmental Studies Hybridoma Bank at the University of Iowa (USA) for providing reagents. We also thank Kerstin Seiling for technical assistance with electron microscopic work. This work was supported by grants of the German research foundation (DFG) to M. P. K. (CRC1348-A05), M.M. (CRC1348-A03) and S.L. (CRC1348-B10). Author contributions M.G., S.B. M.-L.L. performed the experiments and analyzed the data, S.L. established the UAS::Akt-GFP line and revised the manuscript, A.R., R.S. and M.M. performed electron microscopy analysis and revised the paper, P.N. and M.P.K. supervised the experiments and wrote the manuscript. Competing interests The authors declare no competing interests. Data availability All data are available in main und supplemental figures. 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(C) Overexpression of Sktl results in increased accumulation of PH(PLCδ)-mCherry at the plasma membrane. Scale bars are 5µm in A, A’’, B’, B’’, C, C’, 50µm in B and 2,5µm in insets in A’. Figure S2. Decrease of PI(3,4,5)P3 does not affect slit diaphragms Related to Fig. 3. (A-B) Nephrocytes overexpressing a dominant negative Pi3K (Pi3K-DN, A) or PTEN (B) were stained with the indicated antibodies. (D) RNAi targeting dTOR was expressed in nephrocytes with expression of Pi3K-CA. Scale bars are 25µm in A, B and C and 5µm in A’, A’’, B’, B’’, C’ and C’’. Cite Share Download PDF Status: Published Journal Publication published 18 Apr, 2022 Read the published version in Cellular and Molecular Life Sciences → Version 1 posted Editorial decision: Major Revision 27 Aug, 2021 Reviews received at journal 03 Aug, 2021 Reviewers invited by journal 03 Aug, 2021 Editor assigned by journal 22 Jul, 2021 First submitted to journal 20 Jul, 2021 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-739266","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":43686589,"identity":"de93a368-c6a5-4409-8201-44f452b8b2e0","order_by":0,"name":"Maximilian Gass","email":"","orcid":"","institution":"University of Münster: Westfalische Wilhelms-Universitat Munster","correspondingAuthor":false,"prefix":"","firstName":"Maximilian","middleName":"","lastName":"Gass","suffix":""},{"id":43686590,"identity":"94fa4fe3-6275-4655-80cf-e0babd700cb6","order_by":1,"name":"Sarah Borkowsky","email":"","orcid":"","institution":"University of Münster: Westfalische Wilhelms-Universitat Munster","correspondingAuthor":false,"prefix":"","firstName":"Sarah","middleName":"","lastName":"Borkowsky","suffix":""},{"id":43686591,"identity":"981b97ff-bf1c-4bc9-bbf2-44eed5bb7780","order_by":2,"name":"Marie-Luise Lotz","email":"","orcid":"","institution":"University of Münster: Westfalische Wilhelms-Universitat Munster","correspondingAuthor":false,"prefix":"","firstName":"Marie-Luise","middleName":"","lastName":"Lotz","suffix":""},{"id":43686592,"identity":"88e745df-cb5e-48e0-85e5-c8190cb3a8da","order_by":3,"name":"Rita Schröter","email":"","orcid":"","institution":"University of Münster: Westfalische Wilhelms-Universitat Munster","correspondingAuthor":false,"prefix":"","firstName":"Rita","middleName":"","lastName":"Schröter","suffix":""},{"id":43686593,"identity":"d7fd10db-b7f2-4e2f-b06e-00017e2aa1b1","order_by":4,"name":"Pavel Nedvetsky","email":"","orcid":"","institution":"University of Münster: Westfalische Wilhelms-Universitat Munster","correspondingAuthor":false,"prefix":"","firstName":"Pavel","middleName":"","lastName":"Nedvetsky","suffix":""},{"id":43686594,"identity":"e03a8988-d1ff-4078-be15-5a456ca929bb","order_by":5,"name":"Stefan Luschnig","email":"","orcid":"","institution":"University of Münster: Westfalische Wilhelms-Universitat Munster","correspondingAuthor":false,"prefix":"","firstName":"Stefan","middleName":"","lastName":"Luschnig","suffix":""},{"id":43686595,"identity":"03a86056-8b24-4726-b999-b817983a0193","order_by":6,"name":"Astrid Rohlmann","email":"","orcid":"","institution":"University of Münster: Westfalische Wilhelms-Universitat Munster","correspondingAuthor":false,"prefix":"","firstName":"Astrid","middleName":"","lastName":"Rohlmann","suffix":""},{"id":43686596,"identity":"7eb7383f-1a09-41eb-803e-e9582a9ec681","order_by":7,"name":"Markus Missler","email":"","orcid":"","institution":"University of Münster: Westfalische Wilhelms-Universitat Munster","correspondingAuthor":false,"prefix":"","firstName":"Markus","middleName":"","lastName":"Missler","suffix":""},{"id":43686597,"identity":"1e3f8d61-3d6f-48da-94fe-15aef866c2fe","order_by":8,"name":"Michael P. 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A and C are sections through the equatorial region of the nephrocyte and B/D are onviews onto the surface of these nephrocytes. Scale bars are 5µm and 1µm in insets. ","description":"","filename":"fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-739266/v1/d8f929f1cf4abacfca162af6.jpg"},{"id":12182333,"identity":"be9c33a2-9cc7-462f-891c-91195ecde728","added_by":"auto","created_at":"2021-08-06 14:34:07","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":148864,"visible":true,"origin":"","legend":"PI(4,5)P2 produced by Skittles is essential for slit diaphragm formation and endocytosis\n(A-B) Garland nephrocytes from 3rd instar larvae expressing either control RNAi (A) or Sktl-RNAi (B) were stained with the indicated antibodies. (C-D) Transmission electron microscopy of garland nephrocytes of control third instar larvae (C) and Sktl-RNAi-expressing larvae (D). Some slit diaphragms were labeled with arrows in control nephrocytes. Slit diaphragms were absent in Sktl-RNAi expressing nephrocytes. Arrow heads mark the basement membrane. Asterisks indicate electron-bright vesicles, which resembles lacunae in control nephrocytes. (E) Slit diaphragms of nephrocytes expressing Sktl or control were quantified from surface views. For this, a 5µm line perpendicular to the Sns-strands was drawn and the number of strands quantified. 5 lines/nephrocyte and at least 5 nephrocytes were quantified per genotype. Sktl-RNAi expressing nephrocytes were not characterized as they did not display detectable Sns strands at the surface but exhibited a rather diffuse Sns staining. (F) Endocytosis of a secreted ANP-2xGFP by garland nephrocytes expressing the indicated RNAi’s was quantified as described in the methods section. At least 100 nephrocytes from at least 15 different larvae were evaluated. (G-H) Nephrocytes expressing control RNAi (G) or Sktl RNAi (H) were co-stained with Exo70 and Sns. (I) Immunostainings of nephrocytes expressing Exo70-RNAi. Scales bars are 15µm in A and B, 5µm in A, A’’, B’, B’’ and G-I, 2.5µm in insets in G and H and 1µm in C-D. Error bars are standard error of the means. Significance was determined by Mann-Whitney test: *** p\u003c0.001, ** p\u003c0.01,* p\u003c0.05. n.s. not significant.","description":"","filename":"fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-739266/v1/b63a5f52ba035676902d545c.jpg"},{"id":12182339,"identity":"451d610e-cb0c-4655-90a4-faaba6436c79","added_by":"auto","created_at":"2021-08-06 14:34:08","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":117329,"visible":true,"origin":"","legend":"PI(3,4,5)P3 is not essential for slit diaphragm assembly and maintenance. \n(A-B) Garland nephrocytes from 3rd instar larvae either of controls (sns::GAL4 crossed with the empty attP40 line, A) or of animals expressing a constitutively activated Pi3K (Pi3K-CA, B) in nephrocytes were stained with the indicated antibodies. (C-D) Transmission electron microscopy of garland nephrocytes of control third instar larvae (C) and PI3K-CA expressing larvae (D). Slit diaphragms were labeled with arrows and arrow heads mark the basement membrane. (E) Immunostainings of nephrocytes expressing RNAi against PTEN. (F) Slit diaphragms of nephrocytes expressing the indicated transgenes were quantified from surface views. For this, a 5µm line perpendicular to the Sns-strands was drawn and the number of strands quantified. 5 lines/nephrocyte and at least 5 nephrocytes were quantified per genotype. (G) The size of nephrocytes expressing the indicated transgenes was quantified by measuring the cell area of equatorial sections. At least 120 nephrocytes from at least 10 larvae were quantified. (H) Endocytosis of a secreted ANP-2xGFP by garland nephrocytes expressing the indicated controls was quantified as described in the methods section. At least 100 nephrocytes from at least 15 different larvae were evaluated. (I) Myr-Akt expressing nephrocytes were stained with the indicated antibodies. (J) Immunostainings of nephrocytes expressing Pi3K-CA together with RNAi targeting Akt. Scale bars are 50µm in A, B, E, I and J, 5µm in A’, A’’, B’, B’’, E’, E’’, I’, I’’, J’ and J’’, 2.5µm in insets in A’’, B’’, E’’, I’’, J’’ and 1µm in C and D. Error bars are standard error of the means. Significance was determined by Mann-Whitney test: *** p\u003c0.001, ** p\u003c0.01,* p\u003c0.05. n.s. not significant.","description":"","filename":"fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-739266/v1/1df347c78a49c5ce9e705324.jpg"},{"id":12182247,"identity":"35978941-9f16-4d36-ba0b-68c7a3095441","added_by":"auto","created_at":"2021-08-06 14:33:58","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":103256,"visible":true,"origin":"","legend":"Rapid effects of PI(4,5)P2 reduction and PI(3,4,5)P3 accumulation. \n(A-F) Immunostainings of nephrocytes from 3rd instar larvae expressing GAL80ts together with sns::GAL4 and Sktl-RNAi (A-C) or Pi3K-CA (D-F), which were first raised at 18°C in order to suppress expression of the transgenes. In L3, larvae were shifted to 29°C for 24h prior to dissection in order to induce expression of the transgenes. Scale bars are 50µm in A and D, 5µm in B,C, E and F and 2.5µm in insets in C and F.","description":"","filename":"fig4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-739266/v1/5a5cdb32b19a2046b81f811c.jpg"},{"id":20523634,"identity":"1f280a05-6556-4852-a557-9b0f7c5244f4","added_by":"auto","created_at":"2022-04-19 21:18:51","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":896017,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-739266/v1/49451698-9adf-4699-acff-f7c80ec0aae5.pdf"},{"id":12182264,"identity":"02b91981-5447-4e8d-88a8-fec708395186","added_by":"auto","created_at":"2021-08-06 14:34:04","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2894222,"visible":true,"origin":"","legend":"Figure S1. Overexpression of Sktl does not affect slit diaphragm assembly. \nRelated to Fig. 2. \n(A) Immunostaining of Skittles-GFP, Sns and Baz in nephrocytes. (B) Nephrocytes overexpressing Sktl were stained with the indicated antibodies. (C) Overexpression of Sktl results in increased accumulation of PH(PLCδ)-mCherry at the plasma membrane. Scale bars are 5µm in A, A’’, B’, B’’, C, C’, 50µm in B and 2,5µm in insets in A’.\n\nFigure S2. Decrease of PI(3,4,5)P3 does not affect slit diaphragms\nRelated to Fig. 3. \n(A-B) Nephrocytes overexpressing a dominant negative Pi3K (Pi3K-DN, A) or PTEN (B) were stained with the indicated antibodies. (D) RNAi targeting dTOR was expressed in nephrocytes with expression of Pi3K-CA. Scale bars are 25µm in A, B and C and 5µm in A’, A’’, B’, B’’, C’ and C’’.","description":"","filename":"FigureS1S2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-739266/v1/8aa9738a96349041146abf40.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003ePI(4,5)P2 Controls Slit Diaphragm Formation and Endocytosis in \u003cem\u003eDrosophila \u003c/em\u003eNephrocytes\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIn \u003cem\u003eDrosophila\u003c/em\u003e, pericardial nephrocytes located along the heart tube and garland nephrocytes surrounding the proventriculus filtrate the hemolymph and endocytose proteins and toxins to store the latter permanently in order to inactivate them [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Nephrocytes were shown to share several key features with podocytes in vertebrates, qualifying them as a model system to study mammalian podocyte function and podocyte-associated diseases [\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Like in podocytes, homologues of Nephrin- and Neph1 (Sticks and stones (Sns)/Hybris and Kind of irre (Kirre)/Dumbfounded) form the slit diaphragm, thereby separating the lacunae from the body cavity with hemolymph. These lacunae are formed by invaginations of the plasma membrane and form channel-like structures with both ends connected to the extra cellular space [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Due to the high endocytosis capacity in these lacunae and the expression of endocytosis receptors like Cubilin, Megalin and Amnionless, nephrocytes are used as a model system for proximal tubules of the kidney, too [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eApart from the core components of the Nephrin/Neph1 family, the slit diaphragm is stabilized by adapter proteins, e.g. the Podocin homologue Mec2 [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e] and the ZO-1 homologue Polychaetoid [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Furthermore, we recently showed, that regulators of classical apical-basal polarity in epithelia are partly localized to slit diaphragm complexes [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Knockdown studies revealed that apical polarity regulators, such as Crumbs/Stardust and the PAR/aPKC complex as well as the basolateral polarity determinants Scribble/Lethal (2) giant larvae and PAR-1 are essential for slit diaphragm formation and - at least some of them - for endocytosis [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn classical epithelia, these polarity regulators are targeted to either the apical (Crumbs- and PAR/aPKC-complex) or the basolateral (Scribble/Dlg/Lgl-complex, PAR-1/LKB1) plasma membrane and are essential for the establishment and maintenance of apical-basal polarity and cell-cell contacts [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. However, not only proteins are involved in this process, but also distinct phospholipids are enriched either in the apical or the basolateral plasma membrane: In particular, phosphatidylinositol(4,5)bisphosphate (PI(4,5)P2) accumulates in the apical membrane, whereas phosphatidylinositol(3,4,5)trisphosphate (PI(3,4,5)P3) is preferentially found in the basolateral membrane domain [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Notably, PTEN, which dephosphorylates PI(3,4,5)P3 to generate PI(4,5)P2, is recruited to the plasma membrane by PAR-3, the core scaffolding protein of the PAR/aPKC-complex [\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Thereby, junctionally localized PAR-3/PTEN establishes a segregation point for PI(3,4,5)P3 and PI(4,5)P2 [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In turn, PAR-3 directly binds to PI(4,5)P2 and PI(3,4,5)P3, which contributes to its targeting to the plasma membrane [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. During epithelial polarization, phosphatidylinositol-3-kinase (PI3K), which phosphorylates PI(4,5)P2 to PI(3,4,5)P3, seems to function as one of the first cues to determine the basolateral, PI(3,4,5)P3-enriched plasma membrane domain [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Moreover, disruption of the PI(4,5)P2/PI(3,4,5)P3 balance results in severe polarity defects, suggesting a role of phospholipids as regulators of apical-basal cell polarity [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAlthough podocytes and nephrocytes share key features with classical epithelial cells, like cell-cell junctions and apical-basal polarity, little is known about the distribution and function of PI(4,5)P2 and PI(3,4,5)P3 in these cell types. Moreover, several studies suggest different functions of PI3K and PTEN in cultured podocytes [\u003cspan additionalcitationids=\"CR22 CR23\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], but the role of these key enzymes \u003cem\u003ein vivo\u003c/em\u003e is still unclear. Therefore, the aim of this study was to investigate the subcellular accumulation of these two phospholipids as well as their function in slit diaphragm assembly and nephrocyte development.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cp\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eDrosophila\u003c/span\u003e \u003cb\u003estocks and genetics\u003c/b\u003e\u003c/p\u003e \u003cp\u003eFly stocks were cultured on standard cornmeal agar food and maintained at 25\u0026deg;C. For downregulation or overexpression of specific genes for immunostainings and electron microscopy, \u003cem\u003esns::GAL4\u003c/em\u003e [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], was crossed with the following lines: UAS::Akt-RNAi (#103703), UAS::Exo70-RNAi (#103717), UAS::Or83b-RNAi (negative control, #100825), UAS::PTEN-RNAi (#01475), UAS::Sec3-RNAi (#108085), UAS::Sktl-RNAi (#101624) (provided by Vienna \u003cem\u003eDrosophila\u003c/em\u003e Resource Center, Austria), UAS::PI3K92E-CAAX (PI3K-CA, #8294), UAS::PI3K92E.A2860C (PI3K-DN, #8289), UAS:Sktl (#39675), UAS::PH(PLCδ)-mCherry (#51658), tubP::GAL80ts (65406), UAS::dTOR-RNAi (#34639) (all obtained from Bloomington stock center). UAS::Myr::Akt was provided by Hugo Stocker [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] and UAS::Myc-Sktl was obtained from Sandra Claret [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. UASt::PTEN was established by PhiC31-Integrase Insertion using attP86F. UAS::PH(Akt)-GFP was constructed by fusing the PH domain of mammalian Akt1 to the N-terminus of GFP in the pUASt-vector. Transgenic flies were generated by P-element-mediated germ line transformation. An insertion on second chromosome was used in this study. For all RNAi and overexpression experiments, crosses were kept for 3 days at 25\u0026deg;C and larvae subsequently shifted to 29\u0026deg;C, in order to obtain maximum expression. PH(PLCδ)-mCherry was expressed at 25\u0026deg;C, PH(Akt)-GFP was analyzed at 18\u0026deg;C, 21\u0026deg;C and 25\u0026deg;C, with best results at 18\u0026deg;C, because at higher temperature, the expression of the chimeric protein was too strong and found overall the cell, likely due to the limited amount of PI(3,4,5)P3 to bind to.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eEndocytosis assays\u003c/h2\u003e \u003cp\u003eFor the ANP-2xGFP accumulation assay, garland nephrocytes from wandering third instar larvae were dissected in HL3.1 saline [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], fixed in 4% PFA in PBS for 10min, stained with DAPI for 20min, washed with PBS, and mounted in Mowiol. ANP-2xGFP accumulation per nephrocyte area (CTCF\u0026thinsp;=\u0026thinsp;Corrected Total Cell Fluorescence) was analyzed and quantified with ImageJ after subtracting the autofluorescent background of dissected larvae. For each genotype, at least 100 nephrocytes of 15 independent larvae were quantified.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemistry\u003c/h2\u003e \u003cp\u003eGarland nephrocytes were dissected as described above and heat-fixed for 20 seconds in boiling heat fix saline (0.03% Triton-X100). Subsequently, nephrocytes were washed three times in PBS\u0026thinsp;+\u0026thinsp;0.2% Triton X-100 and blocked with 1% BSA for 1h, incubated over night with primary antibodies in PBS\u0026thinsp;+\u0026thinsp;0.2% Triton X-100\u0026thinsp;+\u0026thinsp;1% BSA, washed three times and incubated for 2h with secondary antibodies. After three washing steps and DAPI-staining, nephrocytes were mounted with Mowiol. Primary antibodies used were as follows: anti Baz [1:250, 29], rabbit anti Exo70 [1:500, 30], goat anti GFP (1:500, #600-101-215, Rockland), mouse anti Myc (1:100, 9E10, Developmental Studies Hybridoma Bank (DSHB)), chicken anti Sns [1:1000, 10], mouse anti Talin (1:20, E16B, DSHB). Secondary antibodies conjugated with Alexa 488, Alexa 568 and Alexa 647 (Life technologies) were used at 1:400. Images were taken on a Leica SP8 confocal microscope using lightning program and processed using ImageJ.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eTransmission electron microscopy\u003c/h2\u003e \u003cp\u003eGarland nephrocytes of third instar larvae were dissected in HL3.1 saline, high pressure frozen (EM-PACT2, Leica, Wetzlar, Germany), freeze-substituted in acetone / 1% OsO4 / 5% H\u003csub\u003e2\u003c/sub\u003eO / 0.25% uranyl acetate (AFS2, Leica, Wetzlar, Germany) and embedded in Epon. For transmission electron microscopy, 70nm thick sections were cut using an ultramicrotome (Leica UC7, Wetzlar, Germany). All samples were imaged with a transmission electron microscope (ZEISS, Libra 120, Germany).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv class=\"Section2\" id=\"Sec7\"\u003e\n \u003ch2\u003ePI(4,5)P2 but not PI(3,4,5)P3 is enriched at slit diaphragms\u003c/h2\u003e\n \u003cp\u003eIn classical epithelia, PI(4,5)P2 is enriched in the apical plasma membrane, whereas PI(3,4,5)P3 accumulates in the basolateral plasma membrane [\u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e]. In contrast, nothing is known about the distribution of specific phospholipids in mammalian podocytes or \u003cem\u003eDrosophila\u003c/em\u003e nephrocytes. Therefore, we first investigated the distribution of PI(4,5)P2 and PI(3,4,5)P3 in \u003cem\u003eDrosophila\u003c/em\u003e garland nephrocytes by expressing fusion proteins consisting of a fluorescent protein and a Pleckstrin homology (PH) domain, which preferentially bind to PI(4,5)P2 (PH domain of PLC\u0026delta; [\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e]) or to PI(3,4,5)P3 (PH domain of Akt1, this study).\u003c/p\u003e\n \u003cp\u003emCherry-PH(PLC\u0026delta;) is substantially associated with the plasma membrane (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA-B) but it is also found in intracellular pools, partly associated with vesicular structures. Surface views reveal that its cortical association form strand-like structures, which to some extent co-stain with endogenous Sns, a marker for slit diaphragms (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB). In contrast, PH(Akt)-GFP is only weakly associated with the plasma membrane but also shows a cytoplasmic and vesicular-associated distribution (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eC). Nonetheless, surface views show a strand-like pattern too, but these strands do not co-localize with Sns (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eD) but are rather found between the Sns-strands. These findings suggest, that PI(4,5)P2 in the plasma membrane accumulates at slit diaphragms, whereas PI(3,4,5)P3 is enriched in the free plasma membrane between slit diaphragms.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec8\"\u003e\n \u003ch2\u003eImpaired PI(4,5)P2 production results in strong developmental and slit diaphragm defects\u003c/h2\u003e\n \u003cp\u003eIn order to test whether PI(4,5)P2 is essential for nephrocyte development and function, in particular regarding slit diaphragm assembly and maintenance, we used RNA-interference (RNAi) to knockdown the ubiquitously expressed PI(4)P5-Kinase Skittles (Sktl), which is responsible for converting PI(4)P to PI(4,5)P2 using the nephrocyte-specific driver line sns::GAL4. In \u003cem\u003eDrosophila\u003c/em\u003e, Sktl has been described to regulate apical-basal polarity by targeting PAR-3 to the apical junctions in follicular epithelial cells [\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e] and to the anterior cortex in the oocyte [\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e]. In tracheal tubes, Sktl-produced PI(4,5)P2 was proposed to recruit the formin Diaphanous to the apical membrane [\u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e]. In nephrocytes, Sktl partly colocalizes with Sns at slit diaphragms (Fig. S1A), opening the possibility of a local accumulation of PI(4,5)P2 in microdomains of the plasma membrane at slit diaphragms. Indeed, impaired expression of Sktl resulted in dramatic morphological changes with fused nephrocytes (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB compared to control RNAi in 2A). Furthermore, the typical strand-like structures of Sns-labelled slit diaphragm observed at the surface of control nephrocytes was completely abolished in Sktl-RNAi expressing nephrocytes, resulting in a dispersion of Sns to intracellular puncta (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA-D). Besides Sns, the basal polarity determinant Talin and the apical polarity regulator PAR-3 (Bazooka (Baz) in Drosophila) are lost from the cortex, too. In contrast to impaired PI(4,5)P2 levels, overexpression of Sktl in order to increase PI(4,5)P2 did not affect nephrocyte morphology or slit diaphragm assembly (Fig. S1B, quantified in Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eE), although the amount of PI(4,5)P2 seemed to be significantly increased, as demonstrated by enhanced accumulation of mCherry-PH(PLC\u0026delta;) at the plasma membrane (Fig. S1C).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec9\"\u003e\n \u003ch2\u003eSkittles is essential for slit diaphragm assembly by regulating exocytosis\u003c/h2\u003e\n \u003cp\u003eAnalysis of Sktl-RNAi expressing nephrocytes by electron microscopy confirmed an almost complete absence of slit diaphragms (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eD compared to control in C). Notably, these nephrocytes do not form regular lacunae but accumulate large electron-light vesicles below the plasma membrane (marked with asterisks in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eD). This phenotype suggests severe defects in exocytosis, which is essential for the delivery of transmembrane proteins of the slit diaphragm complex (Sns, Kirre and Crb). During exocytosis, clustering of PI(4,5)P2 facilitates the docking of the exocyst complex to the plasma membrane by direct binding of its components Exo70 and Sec3 in yeast and in mammalian cells [\u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e38\u003c/span\u003e]. In a second step, PI(4,5)P2 is also essential for vesicle fusion and several proteins involved in regulation of fusion directly interact with PI(4,5)P2 [reviewed by 39]. In order to test whether Sktl-produced PI(4,5)P2 recruits Exocyst complex components in nephrocytes, we stained for endogenous Exo70. In control nephrocytes, apart from intracellular giant vesicles, a substantial pool of Exo70 was found at the plasma membrane, co-localizing with Sns (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eG). In contrast, it displayed a diffuse localization with some perinuclear accumulation in Sktl-RNAi expressing nephrocytes (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eH). Moreover, downregulation of the exocyst complex components Exo70 and Sec3 resulted in similar loss of slit diaphragms as Sktl-RNAi (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eI and Fig. S1D), which is in line with a recent study reporting a crucial role of the exocyst complex in slit diaphragm formation/maintenance [\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec10\"\u003e\n \u003ch2\u003eDecreased PI(4,5)P2 levels impair endocytosis in nephrocytes\u003c/h2\u003e\n \u003cp\u003eApart from exocytosis, PI(4,5)P2 also regulates clathrin-dependent and -independent endocytosis by recruiting several proteins involved in early steps of endocytosis to the plasma membrane and by inducing actin remodeling during micropinocytosis [reviewed by 39]. In nephrocytes, endocytosis is essential for the uptake of filtrated proteins, toxins and metabolites, which are then stored and inactivated. Disturbance of slit diaphragm formation as well as of endocytic receptors and proteins involved in the endocytosis machinery have been reported to reduce endocytosis [\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e43\u003c/span\u003e]. In order to test, whether PI(4,5)P2 is essential for endocytosis in nephrocytes, we quantified the accumulation of secreted ANP-2xGFP [\u003cspan class=\"CitationRef\"\u003e9\u003c/span\u003e] which is secreted into the hemolymph, filtrated by nephrocytes and taken up by endocytosis. Indeed, downregulation of Sktl in nephrocytes, reducing PI(4,5)P2 levels, resulted in a strong decrease of ANP-2xGFP accumulation in nephrocytes, consistent with impaired endocytosis (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eF). This is in line with reports from the \u003cem\u003eDrosophila\u003c/em\u003e oocyte, where Sktl is essential for Rab5-mediated endocytosis of yolk protein [\u003cspan class=\"CitationRef\"\u003e44\u003c/span\u003e].\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec11\"\u003e\n \u003ch2\u003ePI(3,4,5)P3 is not essential for nephrocyte function but ectopic production results in dominant negative effects\u003c/h2\u003e\n \u003cp\u003eIn contrast to PI(4,5)P2, reducing PI(3,4,5)P3 by overexpression of PTEN or expression of a dominant negative version of PI3K (PI3K-DN) did not affect nephrocyte morphology or slit diaphragm formation (Fig. S2A-B and Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eF). However, overexpression of a constitutively active PI3K (PI3K-CA), which is targeted to the plasma membrane by attachment of a prenylation anchor (CAAX-motif), in nephrocytes resulted in a strong fusion phenotype and a disturbed pattern of slit diaphragms (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA-D, quantified in 3F). Notably, PI3K-CA-expressing nephrocytes are larger than control nephrocytes (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eG). In addition to slit diaphragm defects, overexpression of PI3K-CA resulted in a drastic decrease in ANP-2xGFP uptake, suggesting a defect in endocytosis (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eH).\u003c/p\u003e\n \u003cp\u003eLike PI3K-CA, enhanced accumulation of PI(3,4,5)P3 by knockdown of PTEN resulted in similar but milder phenotypes regarding slit diaphragms, whereas cell size was not increased (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eE-G). This is likely due to the limited abundance of PI(3,4,5)P3 within the plasma membrane. Ectopic production of PI(3,4,5)P3 from PI(4,5)P2 by PI3K-CA likely produces higher levels of PI(3,4,5)P3 in the plasma membrane due to the larger pool of PI(4,5)P2 [\u003cspan class=\"CitationRef\"\u003e45\u003c/span\u003e], whereas inhibition of dephosphorylation of PI(3,4,5)P3 to PI(4,5)P2 only moderately increases PI(3,4,5)P3 levels in the plasma membrane. These data suggest that slit diaphragm assembly might be more sensitive to enhanced PI(3,4,5)P3 level than cell size regulation.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec12\"\u003e\n \u003ch2\u003ePhenotypes of increased PI(3,4,5)P3 are induced by the Akt/mTOR pathway\u003c/h2\u003e\n \u003cp\u003eIncreased PI(3,4,5)P3 in the plasma membrane leads to activation of the Akt/mTOR signaling cascade, which, among various other functions, results in cell survival and increased cell size and proliferation [reviewed by 46]. In order to test whether the phenotypes observed in nephrocytes expressing PI3K-CA are caused by ectopic Akt/mTOR activation, we introduced a constitutively active variant of Akt (Myr-Akt), which is recruited to the plasma membrane and activated independently of PI(3,4,5)P3 due to the fusion of a myristoylation-signal [\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e]. Indeed, these nephrocytes mimicked the PI3K-CA overexpression phenotype with disrupted slit diaphragms, increased size and fusion phenotypes (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eF, G, I). However, cell size of Myr-Akt expressing nephrocytes was not as strongly increased as in PI3K-CA expressing ones (albeit higher than in case of PTEN-RNAi), whereas slit diaphragm assembly is severely disturbed and comparable with Pi3K-CA and PTEN-RNAi-expressing nephrocytes. Thus, these data provide additional support to the notion that slit diaphragm assembly and size regulation show different susceptibility to levels of PI(3,4,5)P3.\u003c/p\u003e\n \u003cp\u003eTo further substantiate our hypothesis that the defects observed in PI3K-CA expressing nephrocytes are due to ectopic activation of Akt/mTOR signaling upon increased levels of PI(3,4,5)P3, we knocked down Akt or \u003cem\u003eDrosophila\u003c/em\u003e Tor (dTOR) in PI3K-CA expressing nephrocytes. As depicted in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eF,G,J and Fig. S2C, downregulation of Akt or dTOR rescued to a large extent the slit diaphragm defects as well as size differences in PI3K-CA expressing nephrocytes, confirming that the dominant negative function of PI(3,4,5)P3 is mediated by the Akt/mTOR-pathway.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec13\"\u003e\n \u003ch2\u003eChanges in PI(4,5)P2 and PI(3,4,5)P3 levels cause rapid defects\u003c/h2\u003e\n \u003cp\u003eIn order to elucidate whether slit diaphragm defects are established early in development during formation of nephrocytes or whether PI(4,5)P2 and PI(3,4,5)P3 levels are also essential for the turnover and maintenance of slit diaphragms, we used a temperature-sensitive GAL80 (GAL80ts), which suppresses GAL4 activity at the permissive temperature at 18\u0026deg;C. After molting to L3, larvae were shifted to 29\u0026deg;C for 24h prior to dissection, inactivating the GAL80 and thus releasing GAL4, which induces the UAS-transgene. In Sktl-RNAi expressing nephrocytes dissected from animals raised under these conditions, we observed similar defects in morphology as well as impaired Sns strands (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA-C), indicating that PI(4,5)P2 is essential for the turnover/maintenance after the initial establishment of slit diaphragms during the development of nephrocytes. In contrast, short-term induction of Pi3K-CA did not produce phenotypes comparable to continuous expression of this transgene (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eD-F), indicating that the Akt/mTOR-mediated effect of ectopic PI(3,4,5)P3 production is either critical during nephrocyte development or it takes longer time to get established, presumably due to the delay upon transcriptional reprogramming of the cell as a consequence of mTOR target activation.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eOur findings demonstrate that PI(4,5)P2, but not PI(3,4,5)P3 is essential for nephrocyte function and slit diaphragm formation. Of note, PI(4,5)P2 is not evenly distributed in the entire plasma membrane but displays a strand-like pattern, partly colocalizing with Sns as a maker for slit diaphragms. Although PI(4,5)P2 has been found in other cell types at the entire plasma membrane \u0026ndash; or, in epithelial cells, enriched in the apical plasma membrane domain - there is increasing evidence that this phospholipid is concentrated in distinct microdomains of the plasma membrane [discussed by 47,48]: In cultured fibroblasts, freeze-fracture membrane preparation and subsequent electron microscopy revealed three distinct pools of PH(PLCδ) at the rim of caveolae, in coated pits and at the free plasma membrane [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Notably, these three pools exhibited different kinetics upon regulatory stimuli, suggesting different types of regulation. PI(4,5)P2 was also reported to accumulate in lipid rafts of distinct (phospho)lipid and cholesterol composition within the plasma membrane, promoting local actin remodeling or receptor clustering [\u003cspan additionalcitationids=\"CR51\" citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Sarmento et al. observed a Ca(2+)-dependent PI(4,5)P2 clustering in liposomes \u003cem\u003ein vitro\u003c/em\u003e under physiological Ca(2+) and PI(4,5)P2 concentrations [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Thus, PI(4,5)P2 may accumulate in distinct microdomains of the plasma membrane adjacent to slit diaphragms in order to regulate vesicle trafficking \u0026ndash; to the plasma membrane by inducing fusion of vesicles and from the plasma membrane by regulating endocytosis. The dramatic phenotypes observed in Sktl-RNAi expressing nephrocytes underline the critical role of PI(4,5)P2 as an important regulator in these processes. Notably, the human homologue of Sktl, PIP5Kα, was described to be recruited by the Chloride Intracellular Channel 5 (CLIC5A) to cortical Ezrin, inducing clusters of PI(4,5)P2 in the plasma membrane of COS-7 cells [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. In podocytes, Ezrin is part of the Ezrin-NHERF2-Podocalyxin complex, an essential component of the glycocalyx. Furthermore, in glomeruli of CLIC5A-deficient mice, cortical Ezrin/NHERF2 as well as glomerular Podocalyxin are reduced [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Another hint to an important role of PI(4,5)P2 in regulating podocyte morphology comes from a study reporting that the PI5P-Phosphatase Ship2 can be recruited and activated by Nephrin via Nck-Pak1-Filamin in cultured human podocytes [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Ship2 dephosphorylates PI(3,4,5)P3 to PI(3,4)P2, thus its activation by Nephrin in this systems results in an increase of PI(3,4)P2, which activates Lamellipodin, a regulator of Ena/Vasp proteins, resulting in the formation of lamellipodia. Finally, the Nephrin/Ship2 interaction was increased in a podocyte injury model \u003cem\u003ein vivo\u003c/em\u003e, suggesting that lamellipodia formation upon Nephrin-mediated Ship2-activation contributes to foot process effacement observed upon podocyte damage. However, it remains unclear how the Ship2-regulated balance between PI(3,4,5)P3 and PI(3,4)P2 at the Nephrin-complex contributes to slit diaphragm assembly/maintenance and podocyte function under physiological conditions.\u003c/p\u003e \u003cp\u003ePI(4,5)P2 as well as PI(3,4,5)P3 are capable of regulating the actin cytoskeleton by recruiting and activating the small GTPases Rac1 and Cdc42 as well as proteins of the WASP family [\u003cspan additionalcitationids=\"CR57\" citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. Notably, a coordinated actin cytoskeleton remodeling is essential for cortical Nephrin localization and slit diaphragm assembly in \u003cem\u003eDrosophila\u003c/em\u003e nephrocytes [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e] as well as in mammalian podocytes [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. \u003cem\u003eVice versa\u003c/em\u003e, activated Nephrin recruits PI3K resulting in Rac1 activation, actin branching and lamellipodia formation in cultured rat podocytes [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Notably, PTEN is downregulated in podocytes of patients suffering from diabetic nephropathy and inhibition or podocyte-specific knockout of PTEN in mice results in cytoskeleton rearrangements, foot process effacement and proteinuria [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eApart from their impact on the actin cytoskeleton, PI(4,5)P2 and PI(3,4,5)P3-activated Rac1/Cdc42 and actin regulators are essential for remodeling and stability of tight junctions as well as adherens junctions in classical epithelia [reviewed by 63]. Increasing evidence suggests that the slit diaphragms connecting the foot processes of neighboring podocytes emerge from transformation of the tight junctions of the epithelial podocyte progenitor cells [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. Indeed, several proteins of the adherens- and tight junctions can also be found to be components of the slit diaphragm, e.g. ZO-1, Crumbs, PAR/aPKC-complex [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan additionalcitationids=\"CR66 CR67 CR68 CR69 CR70\" citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Thus, it is likely that changes in PI(4,5)P2 and PI(3,4,5)P3 affect slit diaphragm formation and maintenance/stability like they affect adherens junctions/tight junctions in classical epithelia.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank E. Chan, S. Claret, the Bloomington \u003cem\u003eDrosophila\u003c/em\u003e stock center at the University of Indiana (USA), the Vienna Drosophila Resource Center (Austria) and the Developmental Studies Hybridoma Bank at the University of Iowa (USA) for providing reagents. We also thank Kerstin Seiling for technical assistance with electron microscopic work. This work was supported by grants of the German research foundation (DFG) to M. P. K. (CRC1348-A05), M.M. (CRC1348-A03) and S.L. (CRC1348-B10).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eM.G., S.B. M.-L.L. performed the experiments and analyzed the data, S.L. established the UAS::Akt-GFP line and revised the manuscript, A.R., R.S. and M.M. performed electron microscopy analysis and revised the paper, P.N. and M.P.K. supervised the experiments and wrote the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data are available in main und supplemental figures.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eWeavers H, Prieto-Sanchez S, Grawe F, Garcia-Lopez A, Artero R, Wilsch-Brauninger M, Ruiz-Gomez M, Skaer H, Denholm B (2009) The insect nephrocyte is a podocyte-like cell with a filtration slit diaphragm. 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Journal of the American Society of Nephrology: JASN 32(5):1053\u0026ndash;1070. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1681/ASN.2020040501\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cellular-and-molecular-life-sciences","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"life","sideBox":"Learn more about [Cellular and Molecular Life Sciences](https://link.springer.com/journal/18)","snPcode":"18","submissionUrl":"https://www.editorialmanager.com/life/default2.aspx","title":"Cellular and Molecular Life Sciences","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Open","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Nephrocyte, podocyte, slit diaphragm, Phosphoinositides, PI3-Kinase, Phospholipids, PTEN","lastPublishedDoi":"10.21203/rs.3.rs-739266/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-739266/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Drosophila nephrocytes are an emerging model system for mammalian podocytes and podocyte-associated diseases. Like podocytes, nephrocytes exhibit characteristics of epithelial cells, but the role of phospholipids in polarization of these cells is yet unclear. In epithelia phosphatidylinositol(4,5)bisphosphate (PI(4,5)P2) and phosphatidylinositol(3,4,5)-trisphosphate (PI(3,4,5)P3) are asymmetrically distributed in the plasma membrane and determine apical-basal polarity. Here we demonstrate that both phospholipids are present in the plasma membrane of nephrocytes, but only PI(4,5)P2 accumulates at slit diaphragms. Knockdown of Skittles, a phosphatidylinositol(4)phosphate 5-kinase, which produces PI(4,5)P2, abolished slit diaphragm formation and led to strongly reduced endocytosis. Notably, reduction in PI(3,4,5)P3 by overexpression of PTEN or expression of a dominant-negative phosphatidylinositol-3-Kinase did not affect nephrocyte function, whereas enhanced formation of PI(3,4,5)P3 by constitutively active phosphatidylinositol-3-Kinase resulted in strong slit diaphragm and endocytosis defects by ectopic activation of the Akt/mTOR pathway. Thus, PI(4,5)P2 but not PI(3,4,5)P3 is essential for slit diaphragm formation and nephrocyte function. However, PI(3,4,5)P3 has to be tightly controlled to ensure nephrocyte development.","manuscriptTitle":"PI(4,5)P2 Controls Slit Diaphragm Formation and Endocytosis in Drosophila Nephrocytes","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-08-06 14:31:02","doi":"10.21203/rs.3.rs-739266/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major Revision","date":"2021-08-27T10:24:31+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2021-08-03T13:30:43+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2021-08-03T07:34:25+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2021-07-22T16:23:13+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cellular and Molecular Life Sciences","date":"2021-07-21T02:54:37+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cellular-and-molecular-life-sciences","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"life","sideBox":"Learn more about [Cellular and Molecular Life Sciences](https://link.springer.com/journal/18)","snPcode":"18","submissionUrl":"https://www.editorialmanager.com/life/default2.aspx","title":"Cellular and Molecular Life Sciences","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Open","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"62639377-c9b8-4313-9bcf-ff4ba924294e","owner":[],"postedDate":"August 6th, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":6259272,"name":"Cellular \u0026 Molecular Neuroscience"}],"tags":[],"updatedAt":"2022-04-19T21:18:43+00:00","versionOfRecord":{"articleIdentity":"rs-739266","link":"https://doi.org/10.1007/s00018-022-04273-7","journal":{"identity":"cellular-and-molecular-life-sciences","isVorOnly":false,"title":"Cellular and Molecular Life Sciences"},"publishedOn":"2022-04-18 21:18:43","publishedOnDateReadable":"April 18th, 2022"},"versionCreatedAt":"2021-08-06 14:31:02","video":"","vorDoi":"10.1007/s00018-022-04273-7","vorDoiUrl":"https://doi.org/10.1007/s00018-022-04273-7","workflowStages":[]},"version":"v1","identity":"rs-739266","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-739266","identity":"rs-739266","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}