Genetic identification of intracellular trafficking regulators involved in Notch-dependent binary cell fate acquisition following asymmetric cell division.

Notch signalling is involved in numerous cellular processes during development and throughout adult life. Although ligands and receptors are largely expressed in the whole organism, activation of Notch receptors only takes place in a subset of cells and/or tissues and is accurately regulated in time and space. Previous studies have demonstrated that endocytosis and recycling of both ligands and/or receptors are essential for this regulation. However, the precise endocytic routes, compartments and regulators involved in the spatiotemporal regulation are largely unknown. In order to identify intracellular trafficking regulators of Notch signalling, we have undertaken a tissue-specific dsRNA genetic screen of candidates potentially involved in endocytosis and recycling within the endolysosomal pathway. dsRNA against 418 genes was induced in the Drosophila melanogaster sensory organ lineage in which Notch signalling regulates binary cell fate acquisition. Gain or loss of Notch signalling phenotypes were observed in adult sensory organs for 113 of them. Furthermore, 26 genes were found to regulate the steady state localisation of Notch, Sanpodo, a Notch co-factor, and/or Delta in the pupal lineage. In particular, we identified 20 genes with previously unknown function in D. melanogaster intracellular trafficking. Among them, we identified CG2747 and we show that it regulates the localisation of clathrin adaptor AP-1 complex, a negative regulator of Notch signalling. Together, our results further demonstrate the essential function of intracellular trafficking in regulating Notch-signalling-dependent binary cell fate acquisition and constitute an additional step toward the elucidation of the routes followed by Notch receptor and ligands during signalling.

Control of basal autophagy rate by vacuolar peduncle.

Basal autophagy is as a compressive catabolic mechanism engaged in the breakdown of damaged macromolecules and organelles leading to the recycling of elementary nutrients. Thought essential to cellular refreshing, little is known about the origin of a constitutional rate of basal autophagy. Here, we found that loss of Drosophila vacuolar peduncle (vap), a presumed GAP enzyme, is associated with enhanced basal autophagy rate and physiological alterations resulting in a wasteful cell energy balance, a hallmark of overactive autophagy. By contrast, starvation-induced autophagy was disrupted in vap mutant conditions, leading to a block of maturation into autolysosomes. This phenotype stem for exacerbated biogenesis of PI(3)P-dependent endomembranes, including autophagosome membranes and ectopic fusions of vesicles. These findings shed new light on the neurodegenerative phenotype found associated to mutant vap adult brains in a former study. A partner of Vap, Sprint (Spri), acting as an endocytic GEF for Rab5, had the converse effect of leading to a reduction in PI(3)P-dependent endomembrane formation in mutants. Spri was conditional to normal basal autophagy and instrumental to the starvation-sensitivity phenotype specific of vap. Rab5 activity itself was essential for PI(3)P and for pre-autophagosome structures formation. We propose that Vap/Spri complexes promote a cell surface-derived flow of endocytic Rab5-containing vesicles, the traffic of which is crucial for the implementation of a basal autophagy rate.

Components of Intraflagellar Transport Complex A Function Independently of the Cilium to Regulate Canonical Wnt Signaling in Drosophila.

The development of multicellular organisms requires the precisely coordinated regulation of an evolutionarily conserved group of signaling pathways. Temporal and spatial control of these signaling cascades is achieved through networks of regulatory proteins, segregation of pathway components in specific subcellular compartments, or both. In vertebrates, dysregulation of primary cilia function has been strongly linked to developmental signaling defects, yet it remains unclear whether cilia sequester pathway components to regulate their activation or cilia-associated proteins directly modulate developmental signaling events. To elucidate this question, we conducted an RNAi-based screen in Drosophila non-ciliated cells to test for cilium-independent loss-of-function phenotypes of ciliary proteins in developmental signaling pathways. Our results show no effect on Hedgehog signaling. In contrast, our screen identified several cilia-associated proteins as functioning in canonical Wnt signaling. Further characterization of specific components of Intraflagellar Transport complex A uncovered a cilia-independent function in potentiating Wnt signals by promoting ß-catenin/Armadillo activity.

The clathrin adaptor AP-1 complex and Arf1 regulate planar cell polarity in vivo.

A key step in generating planar cell polarity (PCP) is the formation of restricted junctional domains containing Frizzled/Dishevelled/Diego (Fz/Dsh/Dgo) or Van Gogh/Prickle (Vang/Pk) complexes within the same cell, stabilized via Flamingo (Fmi) across cell membranes. Although models have been proposed for how these complexes acquire and maintain their polarized localization, the machinery involved in moving core PCP proteins around cells remains unknown. We describe the AP-1 adaptor complex and Arf1 as major regulators of PCP protein trafficking in vivo. AP-1 and Arf1 disruption affects the accumulation of Fz/Fmi and Vang/Fmi complexes in the proximo-distal axis, producing severe PCP phenotypes. Using novel tools, we demonstrate a direct and specific Arf1 involvement in Fz trafficking in vivo. Moreover, we uncover a conserved Arf1 PCP function in vertebrates. Our data support a model whereby the trafficking machinery plays an important part during PCP establishment, promoting formation of polarized PCP-core complexes in vivo.

AP-1 controls the trafficking of Notch and Sanpodo toward E-cadherin junctions in sensory organ precursors.

In Drosophila melanogaster, external sensory organs develop from a single sensory organ precursor (SOP). The SOP divides asymmetrically to generate daughter cells, whose fates are governed by differential Notch activation. Here we show that the clathrin adaptor AP-1 complex, localized at the trans Golgi network and in recycling endosomes, acts as a negative regulator of Notch signaling. Inactivation of AP-1 causes ligand-dependent activation of Notch, leading to a fate transformation within sensory organs. Loss of AP-1 affects neither cell polarity nor the unequal segregation of the cell fate determinants Numb and Neuralized. Instead, it causes apical accumulation of the Notch activator Sanpodo and stabilization of both Sanpodo and Notch at the interface between SOP daughter cells, where DE-cadherin is localized. Endocytosis-recycling assays reveal that AP-1 acts in recycling endosomes to prevent internalized Spdo from recycling toward adherens junctions. Because AP-1 does not prevent endocytosis and recycling of the Notch ligand Delta, our data indicate that the DE-cadherin junctional domain may act as a launching pad through which endocytosed Notch ligand is trafficked for signaling.

Neuralized promotes basal to apical transcytosis of delta in epithelial cells.

Notch receptors mediate short-range signaling controlling many developmental decisions in metazoans. Activation of Notch requires the ubiquitin-dependent endocytosis of its ligand Delta. How ligand endocytosis in signal-sending cells regulates receptor activation in juxtaposed signal-receiving cells remains largely unknown. We show here that a pool of Delta localizes at the basolateral membrane of signal-sending sensory organ precursor cells in the dorsal thorax neuroepithelium of Drosophila and that Delta is endocytosed in a Neuralized-dependent manner from this basolateral membrane. This basolateral pool of Delta is segregated from Notch that accumulates apically. Using a compartimentalized antibody uptake assay, we show that murine Delta-like 1 is similarly internalized by mNeuralized2 from the basolateral membrane of polarized Madin-Darby canine kidney cells and that internalized ligands are transcytosed to the apical plasma membrane where mNotch1 accumulates. Thus, endocytosis of Delta by Neuralized relocalizes Delta from the basolateral to the apical membrane domain. We speculate that this Neuralized-dependent transcytosis regulates the signaling activity of Delta by relocalizing Delta from a membrane domain where it cannot interact with Notch to another membrane domain where it can bind and activate Notch.