The brachyceran de novo gene PIP82, a phosphorylation target of aPKC, is essential for proper formation and maintenance of the rhabdomeric photoreceptor apical domain in Drosophila

Abstract

The Drosophila apical photoreceptor membrane is defined by the presence of two distinct morphological regions, the microvilli-based rhabdomere and the stalk membrane. The subdivision of the apical membrane contributes to the geometrical positioning and the stereotypical morphology of the rhabdomeres in compound eyes with open rhabdoms and neural superposition. Here we describe the characterization of the photoreceptor specific protein PIP82. We found that PIP82's subcellular localization demarcates the rhabdomeric portion of the apical membrane. We further demonstrate that PIP82 is a phosphorylation target of aPKC. PIP82 localization is modulated by phosphorylation, and in vivo, the loss of the aPKC/Crumbs complex results in an expansion of the PIP82 localization domain. The absence of PIP82 in photoreceptors leads to misshapped rhabdomeres as a result of misdirected cellular trafficking of rhabdomere proteins. Comparative analyses reveal that PIP82 originated de novo in the lineage leading to brachyceran Diptera, which is also characterized by the transition from fused to open rhabdoms. Taken together, these findings define a novel factor that delineates and maintains a specific apical membrane domain, and offers new insights into the functional organization and evolutionary history of the Drosophila retina.

In vivo identification of Drosophila rhodopsin interaction partners by biotin proximity labeling

“…6 ). For PIP82 1bp∆ the same result, that is no change in the rhodopsin content, has previously been described by Zelhof et al 48 . However, this mutant revealed partial misdirection of Rh1 and other rhabdomeral proteins to the basolateral membrane rather than to the base of the rhabdomere.…”

“…Rh1 is assumed to be incorporated into the rhabdomere via the apical stalk membrane, a part of the plasma membrane located between the rhabdomere and membrane adherence junctions, by a Crumbs and Myosin V-dependent transport 47 . Accordingly, identified proteins PIP82 and Stardust, which are located in this region, may interact with Rh1 during its incorporation into the apical membrane 48 , 49 .…”

“…In order to asses a possible functional role of selected putative Rh1 interaction partners for rhodopsin stability, we tested available mutants for the proteins PIP82, Rab32, Vap33 and Kazachoc for changes in the amount of Rh1 in Drosophila heads. The mutant PIP82 1bp∆ is a previously described null mutant 48 , Rab32, Vap33 and Kazachoc mutants were generated using tsCRISPR, by crossing an eyless-Gal4 driver line with the respective sgRNA lines. Using quantitative immunoblot analysis, we observed wild-type amounts of Rh1 in PIP82 and Rab32 mutants, but a reduced rhodopsin content in Vap33 (36% of control) and Kazachoc (38% of control) (Fig.…”

“…The following fly mutants were used: Rh1 null mutant: y w;; ninaE 17 ; Arr2 null mutant: w; arr2 3 ; NinaA null mutant: w; ninaA 2 ; PIP82 null mutant: PIP82 1bp∆ 48 ; sgRNA Vap33: Bloomington Drosophila Stock Center (BDSC) #81751; sgRNA Rab32: Vienna Drosophila Resource Center #342622; sgRNA Kcc: BDSC # 91934; ey driver line: P[GMR-w.IR]13D/FM7; ey > Gal4 UAS > uS-Cas9/CyO.…”

A TRiP RNAi screen to identify molecules necessary for Drosophila photoreceptor differentiation

“…Crumbs become localized to the stalk where it is essential for proper stalk membrane formation and maintenance ( Pellikka et al 2002 ) where the recruitment of aPKC limits the spread of rhabdomeric targeted proteins, e.g. PIP82 ( Zelhof et al 2020 ). The distinct photoreceptor membrane domains also house unique proteins thus requiring a sorting system necessary for both proper morphology and function of photoreceptors.…”