Identification of Genes Required for Apical Protein Trafficking inDrosophilaPhotoreceptor Cells

Abstract

Drosophila melanogaster photoreceptor cells 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 the development of photoreceptor cells, normal vision, and photoreceptor cell 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 237 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.

Mutations in the splicing regulator Prp31 lead to retinal degeneration in Drosophila

“…Finally, we evaluated mRNA levels of three genes ( Table 2 ) that have been recently implicated in Rh1 trafficking ( Laffafian and Tepass, 2019 ). Of these, mRNA levels of only twinfilin ( twf ), which encodes an actin monomer-binding protein, is increased in Prp31 mutants (heterozygous and homozygous alleles) as compared to those of the respective genetic background.…”

“…Interestingly, the Prp31 mutants described here show increased mRNA levels of an evolutionary conserved actin monomer binding protein called twinfilin ( twf ), which inhibits actin polymerisation. Knockdown of twf results in excessive cytoplasmic Rh1 staining, suggesting defects in its trafficking ( Laffafian and Tepass, 2019 ). In Prp31 mutants, an increase in rhabdomeric Rh1 was observed as well as increased twf mRNA.…”

Hydroxylated sphingolipid biosynthesis regulates photoreceptor apical domain morphogenesis

“…Rhabdomere extension in the last quarter of metamorphosis coincides with a massive increase in the production of the apoprotein opsin, its maturation, and its trafficking as Rhodopsin through the secretory pathway to the rhabdomere via apically directed trafficking, mediated by the concerted action of Rab1, Syntaxin 5, Rab6, Rab11, Rip11, the exocyst complex, and MyoV ( Beronja et al, 2005 ; Iwanami et al, 2016 ; Karagiosis and Ready, 2004 ; Laffafian and Tepass, 2019 ; Li et al, 2007 ; Pocha et al, 2011 ; Satoh et al, 2005 ). To follow the conversion of opsin and its trafficking as Rh1 to the rhabdomeres, the BLICS (blue light–induced chromophore supply) assay was used ( Iwanami et al, 2016 ; Pocha et al, 2011 ; Satoh et al, 2005 ).…”

“…Biosynthesis of opsin (the protein moiety of Rhodopsin) is initiated in the last quarter of pupal development, followed by its apically directed transport toward the growing rhabdomere, and coincides with dramatic growth of the rhabdomere. Altered levels of Rhodopsin in the rhabdomere during this phase ( Kumar and Ready, 1995 ), as well as improper trafficking ( Beronja et al, 2005 ; Laffafian and Tepass, 2019 ; Satoh et al, 2005 , 2015 ), impairs rhabdomere morphogenesis. Apart from Rhodopsin 1 (Rh1) and actin regulators, Drosophila crb also plays a role in rhabdomere growth.…”

Application of Fluorescent Proteins for Functional Dissection of the Drosophila Visual System

“…In combination with Flp/FRT mosaic eyes, low Rh1::GFP fluorescence in rhabdomeres was also an indicator of degeneration in a screen that identified phosphoethanolamine cytidylyltransferase (PECT) activity as essential for phosphoethanolamine homeostasis, phototransduction and neuronal cell integrity [39]. As an alternative read-out, the waveguide properties of the rhabdomeres themselves have been used in combination with optical cornea neutralization to infer defects in rhabdomeral organization caused by knock-down of genes potentially involved in vesicle trafficking in an RNAi screen [40].…”