Polarized exocytosis and transcytosis of Notch during its apical localization in Drosophila epithelial cells

Department of Biophysics and Biochemistry, The University of Tokyo, 白山, Tōkyō, Japan
Genes to Cells (Impact Factor: 2.81). 02/2007; 12(1):89-103. DOI: 10.1111/j.1365-2443.2007.01037.x
Source: PubMed


Notch (N) and its ligands, Delta (Dl) and Serrate (Ser), are transmembrane proteins that mediate the cell-cell interactions necessary for many cell-fate decisions. In Drosophila, N is predominantly localized to the apical portion of epithelial cells, but the mechanisms and functions of this localization are unknown. Here, we found N, Dl, and Ser were mostly located in the region from the subapical complex (SAC) to the apical portion of the adherens junctions (AJs) in wing disc epithelium. N was delivered to the SAC/AJs in two phases. First, polarized exocytosis specifically delivered nascent N to the apical plasma membrane and AJs in an O-fut1-independent manner. Second, N at the plasma membrane was relocated to the SAC/AJs by Dynamin- and Rab5-dependent transcytosis; this step required the O-fut1 function. Disruption of the apical polarity by Drosophila E-cadherin (DEcad) knock down caused N and Dl localization to the SAC/AJs to fail. N, but not Dl, formed a specific complex with DEcad in vivo. Finally, our results suggest that juxtacrine signaling in epithelia generally depends on the apicobasally polarized structure of epithelial cells.

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    • "Such Notch accumulation phenotype in awd mutant resembles that of mutants in avalanche (avl; which encodes Syntaxin) and rab5[34,55,59], two gene functions required for maturation of early endosomes [59], but is different from the phenotype in dynamin mutant (shi ts ), in which Notch accumulates on the cell surface and in very large aggregates on apical and basal sides of the follicle cells (Figure  5D) as noted previously [60]. This pattern is likely because of the failure to deliver Notch to apical membrane via Dynamin-mediated transcytosis [61] as well as to internalize Notch for signaling [55]. Since awd mutant cells do not show these very large aggregates throughout the cells, it is unlikely that awd function completely overlaps with that of dynamin. "
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    ABSTRACT: The Drosophila abnormal wing discs (awd) belongs to a highly conserved family of genes implicated in metastasis suppression, metabolic homeostasis and epithelial morphogenesis. The cellular function of the mammalian members of this family, the Nm23 proteins, has not yet been clearly defined. Previous awd genetic analyses unraveled its endocytic role that is required for proper internalization of receptors controlling different signaling pathways. In this study we analyzed the role of Awd in controlling Notch signaling during development. To study the awd gene function we used genetic mosaic approaches to obtain cells homozygous for a loss of function allele. In awd mutant follicle cells and wing disc cells, Notch accumulates in enlarged early endosomes, resulting in defective Notch signaling. Our results demonstrate that awd function is required before gamma-secretase mediated cleavage since over-expression of the constitutively active form of the Notch receptor in awd mutant follicle cells allows rescue of the signaling. By using markers of different endosomal compartments we show that Notch receptor accumulates in early endosome in awd mutant follicle cells. Trafficking assay in living wing discs also shows that Notch accumulates in early endosomes. Importantly, constitutively active Rab5 cannot rescue the awd phenotype, suggesting that awd is required for Rab5 function in early endosome maturation. In this report we demonstrate that awd is essential for Notch signaling via its endocytic role. In addition we identify the endocytic step at which Awd function is required for Notch signaling and we obtain evidence indicating that Awd is necessary for Rab5 function. These findings provide new insights into the developmental and pathophysiological function of this important gene family.
    Full-text · Article · Feb 2014 · BMC Biology
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    • "To detect Notch expression, we used the anti-Notch monoclonal antibody C17.9C6, which was raised against the intracellular domain of the Notch polypeptide (Notch-ICD) (Fehon et al., 1991). This antibody is reported to stain full-length Notch (Fehon et al., 1991) as well as the Notch-ICD, especially when it has accumulated at a high level (Sasaki et al., 2007). In the NE region of the OOA/OPC, we found that Notch localized to the cellular cortex and accumulated near the AJR (Figs. 2F to H), similar to DEcad. "
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    ABSTRACT: The first step in the development of the Drosophila optic medullar primordia is the expansion of symmetrically dividing neuroepithelial cells (NEs); this step is then followed by the appearance of asymmetrically dividing neuroblasts (NBs). However, the mechanisms responsible for the change from NEs to NBs remain unclear. Here, we performed detailed analyses demonstrating that individual NEs are converted into NBs. We also showed that this transition occurs during an elongated G1 phase. During this G1 phase, the morphological features and gene expressions of each columnar NE changed dynamically. Once the NE-to-NB transition was completed, the former NE changed its cell-cycling behavior, commencing asymmetric division. We also found that Notch signaling pathway was activated just before the transition and was rapidly downregulated. Furthermore, the clonal loss of the Notch wild copy in the NE region near the medial edge caused the ectopic accumulation of Delta, leading to the precocious onset of transition. Taken together, these findings indicate that the activation of Notch signaling during a finite window coordinates the proper timing of the NE-to-NB transition.
    Full-text · Article · Mar 2011 · Developmental Biology
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    • ") and, together with the robust cytoplasmic distribution of both Notch receptors and ligands, is suggestive of an active Notch system [Kopan and Ilagan, 2009]. Apical distribution of Notch receptors has already been described for other epithelia [Sasaki et al., 2007; Tanaka et al., 2007]. Notch1 membrane reinforcement is never seen with the Notch1-IC (bTAN 20) antibody ( fig. 4 a, d, h) which, otherwise, generally behaves as the Notch1- IC (C20) antibody. "
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    ABSTRACT: We portrayed the Notch system in embryonic stem cell (ESC)-derived embryoid bodies (EBs) differentiating under the standard protocols used to assess yolk sac (YS) hematopoiesis in vitro. Notch receptors and Notch ligands were detected in virtually all cells throughout EB development. Notch 1 and Notch 2, but not Notch 4, were visualized in the nucleus of EB cells, and all these receptors were also observed as patent cytoplasmic foci. Notch ligands (Delta-like 1 and 4, Jagged 1 and 2) were immunodetected mostly as cytoplasmic foci. Widespread Notch 1 activation was evident at days 2-4 of EB differentiation, the time window of hemangioblast generation in this in vitro system. EBs experienced major spatial remodeling beyond culture day 4, the time point coincident with the transition between primitive and multilineage waves of YS hematopoiesis in vitro. At day 6, where definitive YS hematopoiesis is established in EBs, these exhibit an immature densely packed cellular region (DCR) surrounded by a territory of mesodermal-like cells and an outer layer of endodermal cells. Immunolabeling of Notch receptors and ligands was usually higher in the DCR. Our results show that Notch system components are continuously and abundantly expressed in the multicellular environments arising in differentiating EBs. In such an active Notch system, receptors and ligands do not accumulate extensively at the cell surface but instead localize at cytoplasmic foci, an observation that fits current knowledge on endocytic modulation of Notch signaling. Our data thus suggest that Notch may function as a territorial modulator during early development, where it may eventually influence YS hematopoiesis.
    Full-text · Article · Nov 2010 · Cells Tissues Organs
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