Loss- and gain-of-function analyses of <i>vacuolar protein sorting 2</i> in Notch signaling of <i>Drosophila melanogaster</i>

Abstract

Notch signaling is an evolutionarily conserved mechanism that controls many cell-fate specifications through local cell-cell interactions. The core mechanisms of Notch activation and its subsequent intracellular signaling are well understood. Various cellular functions are required for the activation and regulation of Notch signaling. Among them, the endocytosis of Notch and its ligands is important for the activation and suppression of Notch signaling. The endosomal sorting complex required for transport (ESCRT) proteins are required to sort ubiquitinated membrane proteins, such as Notch, into early endosomes. A loss-of-function allele of vacuolar protein sorting 2 (vps2), which encodes a component of ESCRT-III, has been reported. However, this vps2 mutant still produces the N-terminal half of the protein, and its phenotypes were studied in only a few organs. Here, we generated the first null mutant allele of Drosophila vps2, designated vps2 2 , to better understand the function of this gene. In Drosophila wing imaginal discs homozygous for the vps2 2 allele, early endosomes and multivesicular bodies (MVBs) were enlarged, and Notch and Delta accumulated inside them. As reported for the previous vps2 mutant, the epithelium grew excessively under this condition. We further studied the roles of vps2 by RNA interferenceknockdown. These experiments revealed that a partial reduction of vps2 attenuated Notch signaling; in contrast, the loss-of-function vps2 mutant is reported to up-regulate the Notch signaling in eye imaginal disc cells. These results suggest that Notch signaling can be up-or down-regulated, depending on the level of vps2 expression. Finally, we found that vps2 overexpression also resulted in earlyendosome enlargement and the accumulation of Notch and Delta. In these cells, a portion of the Vps2 protein was detected in MVBs and colocalized with Notch. These data indicate that the expression of vps2 must be precisely regulated to maintain the normal structure of early endosomes.

Drosophila Vps4 promotes Epidermal growth factor receptor signaling independently of its role in receptor degradation

“…Internalization of these receptors into the ILVs of MVBs segregates their intracellular domains from cytoplasmic effectors and should thus terminate signaling (Piper and Lehner, 2011). This model is consistent with the excessive Notch and EGFR signaling observed in eye and wing discs in the absence of many ESCRT proteins (Aoyama et al, 2013;Moberg et al, 2005;Rodahl et al, 2009b;Thompson et al, 2005;Vaccari and Bilder, 2005;Vaccari et al, 2009). This increased signaling has been attributed to the lack of lysosomal degradation of these receptors and their continued activity on the endosomal membrane.…”

Ligand-Independent Mechanisms of Notch Activity

“…In lgd and dx tissues, a lysosome-specific environmental factor produces the membrane tethered Notch fragment. In ESCRT mutant tissue, Notch does not reach the lysosome, but becomes trapped in high quantities in large irregular endosomes [44–49]. Therefore, it could be imagined that a high quantity of closely contained membrane proteins might alter intra-protein kinetics resulting in auto-activation.…”

Expression of mutant CHMP2B, an ESCRT‐III component involved in frontotemporal dementia, causes eye deformities due to Notch misregulation inDrosophila

“…The ey> CHMP2B Intron5 eye phenotype was similar to the eye tumorigenesis observed in ey ‐Gal4 driven constitutive active Notch ( ey>N act ; ref. 26) and flies with mutations in ESCRT‐I ( vps23/erupted/tsg101, vps28 ), ESCRT‐II ( vps22 and vps25 ) and ESCRT‐III ( vps2 and vps20 ) components due to Notch accumulation (27–32). Therefore, we investigated whether eye deformities in ey> CHMP2B Intron5 flies was due to Notch up‐regulation.…”

“…4 ). To determine the vesicular identity of the enlarged Notch punctae, we colocalized Notch with Rab5 and Rab7 as markers of early endosomes and late endosomes/MVBs respectively (32–35). ey> CHMP2B Intron5 eyes showed enlarged Rab5 and Rab7 positive endosomes, which showed partial colocalization with Notch positive puncta relative to ey> CHMP2B WT eyes (Fig.…”