Apical-basal polarity regulators are essential for slit diaphragm assembly and endocytosis in Drosophila nephrocytes

Abstract

Apical-basal polarity is a key feature of most epithelial cells and it is regulated by highly conserved protein complexes. In mammalian podocytes, which emerge from columnar epithelial cells, this polarity is preserved and the tight junctions are converted to the slit diaphragms, establishing the filtration barrier. In Drosophila, nephrocytes show several structural and functional similarities with mammalian podocytes and proximal tubular cells. However, in contrast to podocytes, little is known about the role of apical-basal polarity regulators in these cells. In this study, we used expansion microscopy and found the apical polarity determinants of the PAR/aPKC and Crb-complexes to be predominantly targeted to the cell cortex in proximity to the nephrocyte diaphragm, whereas basolateral regulators also accumulate intracellularly. Knockdown of PAR-complex proteins results in severe endocytosis and nephrocyte diaphragm defects, which is due to impaired aPKC recruitment to the plasma membrane. Similar, downregulation of most basolateral polarity regulators disrupts Nephrin localization but had surprisingly divergent effects on endocytosis. Our findings suggest that morphology and slit diaphragm assembly/maintenance of nephrocytes is regulated by classical apical-basal polarity regulators, which have distinct functions in endocytosis.

Using Drosophila Nephrocytes to Understand the Formation and Maintenance of the Podocyte Slit Diaphragm

“…The apical and basal plasma membrane domains are defined by the junctional zone and SD/lacunar channel diaphragms in mammalian podocytes and fly nephrocytes, respectively. Moreover, apical-basal polarity in both cell types is governed by the same classical polarity proteins, which have been found to play key roles in SD assembly, maintenance and endocytosis ( Michgehl et al, 2017 ; Heiden et al, 2021 ) ( Figure 2 and Table 1 ).…”

“…This transformation requires (re-)localization of many apical junction proteins to the basal side of the podocyte, where they are gradually replaced by the SD ( Ichimura et al, 2017 ). Even though nephrocytes are not derived from epithelial progenitor cells, they do show the distinct apical-basal polarity ( Heiden et al, 2021 ). The apical and basal plasma membrane domains are defined by the junctional zone and SD/lacunar channel diaphragms in mammalian podocytes and fly nephrocytes, respectively.…”

Podocyte Endocytosis in Regulating the Glomerular Filtration Barrier

“…These results suggest that apical polarity determinants are important for endocytosis and slit diaphragm homeostasis. Basolateral polarity regulators are also critical for mammalian nephrin homolog Sns localization in Drosophila ( 116 ). Basolateral polarity proteins- Discs large, Scribble, Lethal giant larvae, and Par-1 are important for normal endocytic trafficking of slit diaphragm proteins including zonula occludens-1 (ZO-1), which was confirmed by colocalization with early endosome Rab5, recycling endosome Rab7, and late endosome Rab11, respectively.…”

PI(4,5)P2 controls slit diaphragm formation and endocytosis in Drosophila nephrocytes

“…Disturbance of slit diaphragm formation as well as of endocytic receptors and proteins involved in the endocytosis machinery have been reported to reduce endocytosis (Soukup et al, 2009;Weavers et al, 2009;Zhuang et al, 2009;Zhang et al, 2013a;Hochapfel et al, 2017;Weide et al, 2017;Hermle et al, 2018;Kampf et al, 2019;Troha et al, 2019). In order to test, whether PI(4,5)P2 is essential for endocytosis in nephrocytes, we quantified the accumulation of secreted ANP-2xGFP (Heiden et al, 2021) 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.…”

“…Furthermore, we recently showed, that regulators of classical apical-basal polarity in epithelia are partly localized to slit diaphragm complexes (Heiden et al, 2021). 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 (Hochapfel et al, 2017;Weide et al, 2017;Heiden et al, 2021).…”