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

Abstract

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 ( D Ecad) knock down caused N and Dl localization to the SAC/AJs to fail. N, but not Dl, formed a specific complex with D Ecad in vivo . Finally, our results suggest that juxtacrine signaling in epithelia generally depends on the apicobasally polarized structure of epithelial cells.

O-Fucose Monosaccharide of Drosophila Notch Has a Temperature-sensitive Function and Cooperates with O-Glucose Glycan in Notch Transport and Notch Signaling Activation

“…6). In epithelial cells in wild-type wing discs, most of the N protein is detected in the sub-apical region and the adherence junctions at 25°C, although it is also detected in intracellular vesicles (26,64).…”

“…O-Fut1 has two O-fucosyltransferase activity-independent functions (23)(24)(25)(26). First, it is an endoplasmic reticulum (ER) resident protein and functions as a N-specific chaperon (23).…”

Cell and molecular biology of Notch

“…Notch and Delta are located in the apical region of the cells restricting the signalling activation to this region. The role and mediators of Notch and Delta apical localization remain elusive (Sasaki et al 2007). However, Par-1 (a polarity protein) has been suggested to be a mediator of Delta localization and Par-1 loss of function (induced through RNAi) has a neurogenic phenotype (Bayraktar et al 2006).…”

Regulation of Notch signaling by glycosylation

“…Clearly none of these strategies uniquely targets Notch receptors since a number of proteins have EGF repeats with the consensus recognized by OFUT1/Pofut1 [45], and all glycoproteins acted on by any fucosyltransferase will be affected by the loss of GDP-Fucose or the GDP-Fucose transporter. In addition, elimination of a glycosyltransferase may have pleiotrophic consequences if it has more than one function, or it acts in both a cell-autonomous and non cell-autonomous fashion [13][14][15]. Overexpression artefacts are a concern when transgenes are expressed under the control of exogenous promoters and/or in an ectopic location, since regulated Notch signaling is notoriously sensitive to the level of Notch receptor activity [46,47].…”

“…Exogenously added OFUT1 reduces Notch accumulation and the authors suggest that secreted OFUT1 bound to cell surface Notch regulates constitutive endocytosis of Notch receptors [14]. In a related paper, however, the same authors conclude that OFUT1 is necessary for transcytosis of Notch at the cell surface to the subapical complex of the adherens junctions [15]. Clearly, technical issues regarding the basis for an antibody to accumulate in a particular compartment in a live cell, and the resolving power of confocal images for localization to intracellular compartments of small Drosophila cells, need to be further investigated.…”