Drosophila Ndfip is a novel regulator of Notch signaling

Abstract

In the Drosophila wing, the Nedd4 ubiquitin ligases (E3s), dNedd4 and Su(dx), are important negative regulators of Notch signaling; they ubiquitinate Notch, promoting its endocytosis and turnover. Here, we show that Drosophila Nedd4 family interacting protein (dNdfip) interacts with the Drosophila Nedd4-like E3s. dNdfip expression dramatically enhances dNedd4 and Su(dx)-mediated wing phenotypes and further disrupts Notch signaling. dNdfip colocalizes with Notch in wing imaginal discs and with the late endosomal marker Rab7 in cultured cells. In addition, dNdfip expression in the wing leads to ectopic Notch signaling. Supporting this, expression of dNdfip suppressed Notch(+/-) wing phenotype and knockdown of dNdfip enhanced the Notch(+/-) wing phenotype. The increase in Notch activity by dNdfip is ligand independent as dNdfip expression also suppressed deltex RNAi and Serrate(+/-) wing phenotypes. The opposing effects of dNdfip expression on Notch signaling and its late endosomal localization support a model whereby dNdfip promotes localization of Notch to the limiting membrane of late endosomes allowing for activation, similar to the model previously shown with ectopic Deltex expression. When dNedd4 or Su(dx) are also present, dNdfip promotes their activity in Notch ubiquitination and internalization to the lysosomal lumen for degradation.

Figure 1

dNdfip localizes to the endosomal compartment, including the late endosome. (a) Schematic of Ndfip proteins showing the locations of PY motifs and transmembrane domains. PY motif sequences are also indicated. (b) Formaldehyde-fixed SL2 cells stained for HA-tagged dNdfip and V5-tagged late endosomal marker Rab7. (c) Live SL2 cells with GFP-tagged dNdfip (green) and the lysosome marker Lysostracker Red DND-99 (red). (d) Formaldehyde-fixed SL2 cells stained for dNdfip and early endosomal marker Rab5. (e) Formaldehyde-fixed SL2 cells stained for dNdfip and recycling endosomal marker Rab11. Insets are a × 2 magnification of the corresponding box indicated. Scale bars=5 μm

Figure 2

dNdfip interacts with the Drosophila E3s dNedd4, Su(dx) and dSmurf in a PY-dependent manner. (a–c) Lysates from SL2 cells co-transfected with wild-type HA-tagged dNdfip and Flag-tagged E3 ligases (a, dNedd4; b, Su(dx); c, dSmurf) were subjected to immunoprecipitation with 5 μg of control (equal mix of anti-Flag and anti-HA), anti-Flag or anti-HA antibodies as indicated (C, F and H, respectively). Input controls were 5% of each protein lysate. Proteins were separated by SDS-PAGE and immunoblotted for Flag or HA. (d) SL2 cells were co-transfected with wild-type Flag-tagged dNedd4 and HA-tagged dNdfip (wild type (+) or PY mutants as indicated). Immunoprecipitations were carried out as in (a–c). Relative binding indicates the normalized protein levels of dNdfip binding to dNedd4

Figure 3

dNdfip partially colocalizes with the Drosophila E3s dNedd4, Su(dx) and dSmurf. (a–c) Transfected SL2 cells were fixed and stained for Flag-tagged E3 ligases (a, dNedd4; b, Su(dx); c, dSmurf) alone (left hand side panel) or with HA-tagged dNdfip (other panels). Insets are × 2 magnifications of the marked boxes that highlight points of colocalization, see right hand side merge panels. Scale bars=5 μm

Figure 4

dNdfip enhances wing phenotypes conferred by dNedd4 and Su(dx). The ptc-GAL4 driver control (a, g and m) and driving expression of UAS-dNedd4GS alone (b, h and n), UAS-Su(dx) alone (c, i and o) and UAS-HA-dNdfip alone (d, j and p) and co-overexpression of UAS-dNedd4GS with UAS-HA-dNdfip (e, k and q) and UAS-Su(dx) with UAS-HA-dNdfip (f, l and r). (a–f) Adult wings from the indicated genetic combinations. (g–l) Wing discs from third instar larvae fixed and stained for Wg as a marker of Notch activity. (m–r) Wing discs from third instar larvae fixed and stained for Cut as a marker of Notch activity. Scale bar=100 μm

Figure 5

dNdfip enhances wing and wing disc phenotypes conferred by dNedd4 and Su(dx). The wing-specific driver MS1096-GAL4 control (a, g and m) and with UAS-dNedd4GS alone (b, h and n), UAS-Su(dx) alone (c, i and o), UAS-HA-dNdfip alone (d, j and p) and co-overexpression of UAS-dNedd4GS with UAS-HA-dNdfip (e, k and q) and UAS-Su(dx) with UAS-HA-dNdfip (f, l and r).(a–f) Adult wings from the indicated genetic combinations. (g–l) Wing discs from third instar larvae fixed and stained for Wg as a marker of Notch activity. (m–r) Wing discs from third instar larvae fixed and stained for Cut as a marker of Notch activity. Scale bar=100 μm

Figure 6

Expression of dNdfip in the wing disrupts wing structure, increases Wg expression and modifies Notch-dependent wing phenotypes. (a–d) MS1096-GAL4 driver control (a–b′) and with two-copies UAS-HA-dNdfip (c–d′) showing adult wing phenotypes (a and c) and Wg expression in third instar larval wing discs as a measure of Notch activation (b, b′, d and d′). Scale Bars=100 μm (b); 5 μm (b′). b And d are maximum pixel intensity merges of six z-stack images each, 2.0 μm apart. b′ And d′ are merges with maximum pixel intensity of four z-stack images each, 1.0 μm apart. (e and f) Adult wings from MS1096-GAL4 UAS-Dcr driver control (e) and with UAS-dNdfipIR (f). To improve the knockdown efficiency of dNdfip by RNAi Dicer (Dcr) was co-expressed. (g) Real-time PCR analysis of the knock down of dNdfip in Act5c-GAL4UAS-dNdfipIR flies. (h–j) Adult wings from N^264−39/+ flies carrying the MS1096-GAL4 driver control (h) with UAS-HA-dNdfip (i) and with UAS-dNdfipIR (j). (k and l) Adult wings from flies carrying the MS1096-GAL4 driver and UAS-deltexIR RNAi (k) and with UAS-HA-dNdfip (l). (m) Quantification of the variation in the Notch wing phenotypes observed. The phenotypes were classed as mild with mostly normal wings but having minor vein defects, thick vein (arrow in h) at margin without wing notching and notched (arrow in j) with wing margin notching and vein thickening. (n and o) Adult wings from Ser^1/+ flies with MS1096-GAL4 driver control (n) and with UAS-HA-dNdfip (o). Arrows indicate various wing defects

Figure 7

Endogenous dNdfip colocalizes with Notch and apposes Wg at the dorso-ventral wing boundary. (a–c) Wild-type third instar larval wing discs stained for dNdfip (a, c; green) and Notch intracellular domain (b, c; red, Notch-icd). (d and e) Wild-type third instar larval wing discs stained for dNdfip (d, f; green) and Notch extracellular domain (e, f; red, Notch-ecd). Images are a maximum pixel intensity merge of four z-stack images each, 0.10-μm apart. Insets are × 2 magnifications of the boxes indicated, colocalization of Notch and dNdfip can be seen as yellow puncta in inset box c. Scale bar=5 μm. (g–i′) Wild-type third instar larval wing discs stained for dNdfip (g, h, i; green) and Wg (g', h', i′; red). h, h′, i and i′ are a maximum pixel intensity merge of five z-stack images, 0.500 μm apart. Scale bars=100 μm (g′); 40 μm (h′); 10 μm (i′)