Loss of class 3 PI3K complex leads to retention of Wg at the apical membrane in polarized tissue

ABSTRACT Wnt/Wingless (Wg) signalling is a key regulator of tissue patterning and morphogenesis in Drosophila, coordinating cell fate decisions and long-range morphogen signalling. In wing imaginal discs, Wg proteins rely on specialized trafficking machinery, including the cargo receptor Evi/Wls, and are secreted via multiple routes, comprising a glypican-dependent Wg pool and an endocytosis-dependent Wg pool. However, the cellular mechanisms controlling Wg secretion and post-endocytic trafficking in Drosophila remain incompletely understood. We performed an in vivo kinome- and phosphatome-wide CRISPR-Cas9 screen in Drosophila wing imaginal discs using endogenous fluorescently tagged Wg as a readout. Genetic perturbations were combined with super-resolution microscopy and ex vivo pharmacological treatments to resolve Wg and Evi/Wls secretion dynamics. We identified Vps15, a core subunit of the class III phosphatidylinositol 3-kinase (PI3K (III)) complex, as a critical factor of Wg secretion. Loss of Vps15 caused apical accumulation of Wg in Wg-secreting cells, selectively impairing an endocytosis-dependent Wg pool while leaving a glypican-mediated Wg pool intact. In contrast to Wg, Evi/Wls did not accumulate in PI3K (III) mutant cells, but instead was subject to proteasome-dependent degradation. Super-resolution imaging further revealed frequent spatial separation of Wg and Evi/Wls in Wg-secreting cells prior to the uptake of the endocytosis-dependent Wg pool. Our study establishes PI3K (III) perturbation as a powerful approach to dissect distinct Wg secretion routes in Drosophila wing imaginal discs. We uncovered divergent post-endocytic fates of Wg and Evi/Wls upon perturbation and provided new mechanistic insight into Wg-Evi/Wls dynamics. Competing Interest Statement The authors have declared no competing interest. Footnotes The new version of the manuscript contains additional experiments and revised text. LIST OF ABBREVIATIONS - A - Anterior - Act - Actin - AFI - Average Fluorescence intensity - Cas9 - Caspase 9 - CRISPR - Clustered Regularly Interspaced Short Palindromic Repeats - D - Dorsal - Dll - Distalless - Dlp - Dally-like protein - DMSO - Dimethyl sulfoxide - dTom - dTomato - DV - Dorso-ventral (boundary) - ECM - Extracellular matrix - ER - Endoplasmic Reticulum - Evi/Wls - Evenness-interrupted/Wntless - EviIn - Internalized Evi/Wls - GFP - Green Fluorescent protein - HD_CFD - Heidelberg CRISPR Fly Design (library) - hh - Hedgehog - Mut - Mutated - P - Posterior - Pdm2 - POU domain protein 2 - PI - Phosphoinositol - PI3K (III) - Phosphoinositol 3 Kinase class III - PI3P - Phosphoinositol-3-phosphate - RNAi - RNA interference - ROI - Region of interest - sgRNA - small guide RNA - Shi - Shibire - UAS - Upstream activation sequence - V - Ventral - Wg - Wingless - WgEx - Extracellular Wg - WgIn - Internalized Wg - Wt - Wildtype