Hydroxylated sphingolipid biosynthesis regulates photoreceptor apical domain morphogenesis

Apical domains of epithelial cells often undergo dramatic changes during morphogenesis to form specialized structures, such as microvilli. Here, we addressed the role of lipids during morphogenesis of the rhabdomere, the microvilli-based photosensitive organelle of Drosophila photoreceptor cells. Shotgun lipidomics analysis performed on mutant alleles of the polarity regulator crumbs, exhibiting varying rhabdomeric growth defects, revealed a correlation between increased abundance of hydroxylated sphingolipids and abnormal rhabdomeric growth. This could be attributed to an up-regulation of fatty acid hydroxylase transcription. Indeed, direct genetic perturbation of the hydroxylated sphingolipid metabolism modulated rhabdomere growth in a crumbs mutant background. One of the pathways targeted by sphingolipid metabolism turned out to be the secretory route of newly synthesized Rhodopsin, a major rhabdomeric protein. In particular, altered biosynthesis of hydroxylated sphingolipids impaired apical trafficking via Rab11, and thus apical membrane growth. The intersection of lipid metabolic pathways with apical domain growth provides a new facet to our understanding of apical growth during morphogenesis.

Superoxide increases surface NKCC2 in the rat thick ascending limbs via PKC

The apical Na+-K+-2Cl− cotransporter (NKCC2) mediates NaCl reabsorption by the thick ascending limb (TAL). The free radical superoxide ([Formula: see text]) stimulates TAL NaCl absorption by enhancing NKCC2 activity. In contrast, nitric oxide (NO) scavenges [Formula: see text] and inhibits NKCC2. NKCC2 activity depends on the number of NKCC2 transporters in the TAL apical membrane and its phosphorylation. We hypothesized that [Formula: see text] stimulates NKCC2 activity by enhancing apical surface NKCC2 expression. We measured surface NKCC2 expression in rat TALs by surface biotinylation and Western blot analysis. Treatment of TALs with [Formula: see text] produced by exogenous xanthine oxidase (1 mU/ml) and hypoxanthine (500 µM) stimulated surface NKCC2 expression by ~18 ± 5% ( P < 0.05). [Formula: see text]-stimulated surface NKCC2 expression was blocked by the [Formula: see text] scavenger tempol (50 µM). Scavenging H2O2 with 100 U/ml catalase did not block the stimulatory effect of xanthine oxidase-hypoxanthine (22 ± 8% increase from control, P < 0.05). Inhibition of endogenous NO production with Nω-nitro-l-arginine methyl ester enhanced surface NKCC2 expression by 21 ± 6% and, when added together with xanthine oxidase-hypoxanthine, increased surface NKCC2 by 41 ± 10% ( P < 0.05). Scavenging [Formula: see text] with superoxide dismutase (300 U/ml) decreased this stimulatory effect by 60% (39 ± 4% to 15 ± 10%, P < 0.05). Protein kinase C inhibition with Gö-6976 (100 nM) blocked [Formula: see text]-stimulated surface NKCC2 expression ( P < 0.05). [Formula: see text] did not affect NKCC2 phosphorylation at Thr96/101 or its upstream kinases STE20/SPS1-related proline/alanine-rich kinase-oxidative stress-responsive kinase 1. We conclude that [Formula: see text] increases surface NKCC2 expression by stimulating protein kinase C and that this effect is blunted by endogenous NO. [Formula: see text]-stimulated apical trafficking of NKCC2 may be involved in the enhanced surface NKCC2 expression observed in Dahl salt-sensitive rats.

Identification of genes required for apical protein trafficking in Drosophila photoreceptor cells

Drosophila photoreceptor cells (PRCs) are highly polarized epithelial cells. Their apical membrane is further subdivided into the stalk membrane and the light-sensing rhabdomere. The photo-pigment Rhodopsin1 (Rh1) localizes to the rhabdomere, whereas the apical determinant Crumbs (Crb) is enriched at the stalk membrane. The proteoglycan Eyes shut (Eys) is secreted through the apical membrane into an inter-rhabdomeral space. Rh1, Crb, and Eys are essential for PRC development, normal vision, and PRC survival. Human orthologs of all three proteins have been linked to retinal degenerative diseases. Here, we describe an RNAi-based screen examining the importance of approximately 240 trafficking-related genes in apical trafficking of Eys, Rh1, and Crb. We found 28 genes that have an effect on the localization and/or levels of these apical proteins and analyzed several factors in more detail. We show that the Arf GEF protein Sec71 is required for biosynthetic traffic of both apical and basolateral proteins, that the exocyst complex and the microtubule-based motor proteins dynein and kinesin promote the secretion of Eys and Rh1, and that Syntaxin 7/Avalanche controls the endocytosis of Rh1, Eys, and Crb.Article summeryPhotoreceptor cells (PRCs) rely on polarized vesicle trafficking to deliver key secreted and transmembrane proteins to their correct locations. Failure to do so causes defects in PRC development, function, and survival leading to retinal disease. Using the fruit fly Drosophila as a model we have identified 28 genes that are required for the trafficking of the three apical proteins Rhodopsin 1, Crumbs, and Eyes Shut. Human homologs of all three genes are associated with retinal degeneration. We characterized several genes to reveal novel mechanisms of vesicle trafficking in photoreceptor cells at different points in the biosynthetic or endocytotic pathways.

A V0-ATPase-dependent apical trafficking pathway maintains the polarity of the intestinal absorptive membrane

Intestine function relies on the strong polarity of intestinal epithelial cells and the array of microvilli forming a brush border at their luminal pole. Combining genetic RNAi screen and in vivo super-resolution imaging in the C. elegans intestine, we uncovered that the V0 sector of the V-ATPase (V0-ATPase) controls a late apical trafficking step, involving RAB-11 endosomes and the SNARE SNAP-29, necessary to maintain the polarized localization of both apical polarity modules and brush border proteins. We show that the V0-ATPase pathway also genetically interacts with glycosphingolipids in enterocyte polarity maintenance. Finally, we demonstrate that depletion of the V0-ATPase fully recapitulates the severe structural, polarity and trafficking defects observed in enterocytes from patients with Microvillus inclusion disease (MVID) and used this new in vivo MVID model to follow the dynamics of microvillus inclusions. Hence, we describe a new function for the V0-ATPase in apical trafficking and epithelial polarity maintenance and the promising use of C. elegans intestine as an in vivo model to better understand the molecular mechanisms of rare genetic enteropathies.Summary statementV0-ATPase controls a late apical trafficking step involved in the maintenance of the apical absorptive intestinal membrane and its depletion phenocopies the trafficking and structural defects of MVID in C. elegans.