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. 1A-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. 1B). In contrast, PH(Akt)-GFP is only weakly associated with the plasma membrane but also shows a cytoplasmic and vesicular-associated distribution (Fig. 1C). Nonetheless, surface views show a strand-like pattern too, but these strands do not co-localize with Sns (Fig. 1D) 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. 2B 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. 2A-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. 2E), 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. 2D 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. 2D). 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. 2G). In contrast, it displayed a diffuse localization with some perinuclear accumulation in Sktl-RNAi expressing nephrocytes (Fig. 2H). Moreover, downregulation of the exocyst complex components Exo70 and Sec3 resulted in similar loss of slit diaphragms as Sktl-RNAi (Fig. 2I 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. 2F). 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. 3F). 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. 3A-D, quantified in 3F). Notably, PI3K-CA-expressing nephrocytes are larger than control nephrocytes (Fig. 3G). 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. 3H).
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. 3E-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. 3F, 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. 3F,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. 4A-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. 4D-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.