The Scribble module regulates retromer-dependent endocytic trafficking during epithelial polarization

Abstract

Scribble (Scrib) module proteins are major regulators of cell polarity, but how they influence membrane traffic is not known. Endocytosis is also a key regulator of polarity through roles that remain unclear. Here we link Scrib to a specific arm of the endocytic trafficking system. Drosophila mutants that block AP-2-dependent endocytosis share many phenotypes with Scrib module mutants, but Scrib module mutants show intact internalization and endolysosomal transport. However, defective traffic of retromer pathway cargo is seen, and retromer components show strong genetic interactions with the Scrib module. The Scrib module is required for proper retromer localization to endosomes and promotes appropriate cargo sorting into the retromer pathway via both aPKC-dependent and -independent mechanisms. We propose that the Scrib module regulates epithelial polarity by influencing endocytic itineraries of Crumbs and other retromer-dependent cargo.

Keywords: Drosophila; Endocytosis; Epithelia; Polarity; Retromer.

Fig. 1.

Comparison of Scrib module and endocytic polarity phenotypes. Compared with WT (A), eye discs mutant for AP-2α (B), Chc (C) or shi (D) are overgrown, disorganized and multilayered [x-z sections; F-actin, red; nuclei, blue (DAPI)]. Par-6 (E) and Crb (G) are apical in WT. In dlg, both are mislocalized, but Par-6 remains at the plasma membrane (PM) (F) whereas Crb also shows a hazy subcortical distribution (H). Separate domains of apical aPKC (magenta) and basolateral Dlg (cyan) in WT follicle cells (I) are lost in AP-2 (J) and lgl (K). Separate domains of apical Crb (magenta) and basolateral Nrg (cyan) in WT (L) are lost in AP-2 (M) and lgl (N), but lgl displays a subcortical haze of Crb (arrowheads). Crb localization is shown in follicle cells knocked down for AP-2 (O), lgl (P) and for AP-2 and lgl (Q). (R) Quantitation of Crb cortical association; 1.0 reflects full association. n≥200 cells from at least five samples for each. ***P<0.001; n.s., not significant; individual P-values are given in supplementary material Table S1. Error bars indicate s.d. Scale bars: 100 μm in A; 25 μm in E; 10 μm in I.

Fig. 2.

Defective traffic of retromer cargo in Scrib module mutant discs. (A-J) Wing imaginal discs were stained with phalloidin (red). (A-D) Transgenic Wls and endogenous Crb are enriched subcortically in dlg but not WT. (E-H) Endogenous Ecad and transgenic CD8 localize to both WT and dlg PMs. (I,J) Activated aPKC is sufficient to induce Crb and Wls subcortical localization. (K) Quantitation of cortical association; 1.0 reflects full association. ***P<0.001; individual P-values are given in supplementary material Table S1. Error bars indicate s.d., n≥200 cells from at least five samples for each. (L-Q) In discs cultured with lysosomal inhibitors (LI), Ecad accumulation in dlg does not differ from WT, whereas the accumulation of Crb and Wls is increased. Scale bar: 10 μm.

Fig. 3.

Scrib module proteins control polarity via retromer. The WT wing (A) wrinkles upon mild lgl knockdown in the dorsal compartment (B). Heterozygosity for scrib (C) but not AP-2 (D) enhances this phenotype. Mild Vps35 (E) or Vps26 (F) knockdown has little effect alone, but strongly enhances mild lgl knockdown (G), including neoplastic transformation and pupal death (H). WT punctate localization of Rab9-YFP and Vps29-GFP (I,K) is dispersed in dlg discs (J,L). Quantitation (M) reveals that large Rab9-YFP and Vps29-GFP puncta are strongly depleted, whereas Rab5-YFP puncta are much less affected. Puncta numbers (N) do not change significantly. P-values are given in supplementary material Table S1. Error bars indicate s.d. n≥200 cells from at least five samples for each. PM levels of Wls-V5 in WT (O) are reduced upon Vps26 knockdown (P). Compared with dlg knockdown alone (Q), Vps26 dlg double knockdown shows reduced cortical and subcortical localization and enhanced punctate trapping (R) of Wls-V5. (I-L,O-R) Phalloidin staining is in red. Scale bars: 400 μm in A; 100 μm in H; 10 μm in I.

Fig. 4.

Scrib module trafficking regulation requires Par activity. (A-C′) Mutating baz in dlg mutant follicle cells largely prevents the inappropriate localization of Crb (magenta, with Nrg in cyan; A′-C′ show the Crb channel alone) and causes enhanced endosomal trapping. (D-F) Knocking down aPKC in dlg knockdown cells has a similar effect. Yellow lines mark WT cells. (G-I) Knocking down Cdc42 in dlg cells causes loss of most apical PM identity (as revealed by aPKC in white). Scale bar: 10 μm.