Control of basal autophagy rate by vacuolar peduncle

International audienceBasal 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

Starvation-sensitivity assays define the range of autophagy defects in <i>vap</i> flies.

<p>The survival rate of 3 day-old adult males of indicated genotypes was recorded at 25°C in condition of complete food deprivation (see <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0209759#pone.0209759.s004" target="_blank">S4A–S4C Fig</a></b> for initial characterization). (A) The <i>vap</i>-dependent starvation sensitivity (white arrow) was compared to weak (<i>Atg8a</i>^<i>1</i>) and strong (<i>Agt8a</i>^<i>2</i>) alleles of <i>Atg8</i>a. <i>Atg8a</i>² flies showed slightly altered development that might contribute to its greater sensitivity to starvation. (B-B’) Starvation sensitivity effect, as assayed at 25°C, is partially recapitulated by flies that were ectopically expressing an <i>UAS-myc</i>:<i>Atg1</i> transgene (Materials and Methods) along fat cell development performed at 25°C (white arrow in B) when driven by <i>cg-Gal4</i>. As a control, there is no detectable starvation sensitivity (as assayed at 25°C) using identical flies (<i>UAS-myc</i>:<i>Atg1</i> /<i>cg-Gal4</i>) that developed at 18°C to minimized transgene expression (white arrow in B’). Ectopic expression of <i>Atg1</i> during development is therefore responsible for the sensitivity effect found in B. (C) The <i>vap</i>-dependent starvation sensitivity is suppressed (white arrow) by co-expressed <i>Atg5(RI)</i> using the broadly expressed <i>arm-Gal4</i> driver. Genotypes. (A) Control: <i>w</i>^<i>1118</i><i>/Y</i>. Assay <i>vap</i>^<i>1</i><i>/Y</i>. <i>Atg8a</i>^<i>1</i><i>/Y</i>. <i>Atg8a</i>^<i>2</i><i>/Y</i>. (B, B’) Control: <i>UAS-myc</i>:<i>Atg1/+</i> and <i>vap</i>^<i>1</i><i>/Y</i> and <i>vap</i>^<i>1</i><i>/Y; cg-GAL4/+</i>. Assay: <i>vap</i>^<i>1</i><i>/Y</i>. <i>cg-GAL4/ UAS-myc</i>:<i>Atg1(RI)/+</i>. (C) Control: <i>arm-GAL4/+</i> and <i>vap</i>^<i>1</i><i>/Y; arm-GAL4/+</i> and <i>arm-GAL4/ UAS-Atg5(RI)/+</i>. Assay: <i>vap</i>^<i>1</i><i>/Y; arm-GAL4/ UAS-Atg5(RI)/+</i>.</p

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

International audienceNotch signaling 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 spatio temporal regulation are largely unknown.In order to identify Notch signaling intracellular trafficking regulators, we have undertaken a tissue-specific dsRNA genetic screen against candidates potentially involved in endocytosis and recycling within the endolysosomal pathway. dsRNA against 418 genes was induced in Drosophila melanogaster sensory organ lineage in which Notch signaling regulates binary cell fate acquisition. Gain- or loss-of Notch signaling phenotypes were observed in adult sensory organs for 113 of them. Furthermore, 26 genes presented a change in the steady state localization 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 Drosophila melanogaster intracellular trafficking. Among them, we identified CG2747 and show that it regulates the localization of clathrin adaptor AP-1 complex, a negative regulator of Notch signaling. All together, our results further demonstrate the essential function of intracellular trafficking in regulating Notch signaling-dependent binary cell fate acquisition and constitute an additional step toward the elucidation of the routes followed by Notch receptor and ligands to signal

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

International audienceIn 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.: Neuralized-mediated Transcytosis of Delta

International audienceNotch 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