Synergy between the ESCRT-III complex and Deltex defines a ligand-independent Notch signal - PubMed (original) (raw)

Synergy between the ESCRT-III complex and Deltex defines a ligand-independent Notch signal

Kazuya Hori et al. J Cell Biol. 2011.

Abstract

The Notch signaling pathway defines a conserved mechanism that regulates cell fate decisions in metazoans. Signaling is modulated by a broad and multifaceted genetic circuitry, including members of the endocytic machinery. Several individual steps in the endocytic pathway have been linked to the positive or negative regulation of the Notch receptor. In seeking genetic elements involved in regulating the endosomal/lysosomal degradation of Notch, mediated by the molecular synergy between the ubiquitin ligase Deltex and Kurtz, the nonvisual β-arrestin in Drosophila, we identified Shrub, a core component of the ESCRT-III complex as a key modulator of this synergy. Shrub promotes the lysosomal degradation of the receptor by mediating its delivery into multivesicular bodies (MVBs). However, the interplay between Deltex, Kurtz, and Shrub can bypass this path, leading to the activation of the receptor. Our analysis shows that Shrub plays a pivotal rate-limiting step in late endosomal ligand-independent Notch activation, depending on the Deltex-dependent ubiquitinylation state of the receptor. This activation mode of the receptor emphasizes the complexity of Notch signal modulation in a cell and has significant implications for both development and disease.

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Figures

Figure 1.

shrub modulates the synergy between dx and krz. (A) Wild-type adult wing. (B) Heterozygous Notch-null allele (N54l9/+) is associated with the typical wing notching. (C) Expression of full-length Notch driven by C96-Gal4 (C96-Gal4/UAS-NFL7) does not affect wing morphology under our experimental conditions. (D and F) Co-expression of Dx and Krz shows wing notching (UAS-Flag:Dx; C96-Gal4, UAS-HA:Krz/+) (D), consistent with Notch loss-of-function, which is rescued by expressing a transgene encoding wild-type Notch (UAS-Flag:Dx/+; C96-Gal4, UAS-HA:Krz/ UAS-NFL7 = 90%, n = 20) (F). (E) N54l9/+ enhances Dx- and Krz-mediated wing notching phenotype (N54l9/+; UAS-Flag:Dx/+; C96-Gal4, UAS-HA:Krz/+ = 100%, n = 17). (G) Wing notching phenotype associated with Dx and Krz is rescued by treatment with chloroquine (52%, n = 23). (H and I) Heterozygote shrub loss-of-function mutations, shrub4/+ (H), or overexpression of Shrub alone by C96-GAL4 in the developing wing (I) does not display wing notching. (J and K) The wing notching phenotype is suppressed by expressing ShrubRNAi (UAS-Flag:Dx/+; C96-Gal4, UAS-HA:Krz/UAS-ShrubRNAi = 76%, n = 33) (J), or by reducing shrub levels in heterozygous animals (shrub4/UAS-Flag:Dx; C96-Gal4, UAS-HA:Krz/+ = 91%, n = 11) (K). (L) The wing notching is enhanced by increasing shrub levels along with Dx and Krz (UAS-Flag:Dx/+; C96-Gal4, UAS-HA:Krz/UAS-Shrub = 83%, n = 23). Bars, 0.2 mm. Representative examples are shown.

Figure 2.

Shrub regulates the synergistic effects of dx and krz on Notch by affecting trafficking. (A–C) In the wing disc expressing Dx driven by C96-Gal4 (UAS-Flag:Dx/+; C96-Gal4/+), Notch (green) is colocalized with Dx (purple) in enriched intracellular vesicles. (D–F) In the wing disc coexpressing Dx and Krz driven by C96-Gal4 (UAS-Flag:Dx/+; C96-Gal4, UAS-HA:Krz/+), Notch (green) does not colocalize with Dx (purple)-positive intracellular vesicles. (G–I) In wing discs expressing ShrubRNAi in addition to Dx and Krz by C96-GAL4 (UAS-Flag:Dx/+; C96-Gal4, UAS-HA:Krz/UAS-ShrubRNAi), Notch (green) colocalizes with Dx (purple). (J–L) Expression of Shrub by ptc-GAL4 driver (ptc-GAL4/+; UAS-Shrub/+) leads to accumulation of Notch (green) in Shrub (purple)-positive enlarged vesicles. Dashed lines indicate the boundary between cells induced to express Shrub versus wild-type cells.

Figure 3.

Shrub antagonizes Dx while it enhances Krz activity. (A and B) Wild-type Cut (purple) expression in larval wing disc, along the DV boundary in ptc-Gal4/UAS-GFP animals. (C and D) Suppression of Cut (purple, arrowhead) is seen when Shrub is expressed by ptc-GAL4 driver (ptc-Gal4/UAS-GFP; UAS-Shrub/+). (E and F) Ectopic Cut expression (purple, arrowhead) in the ventral region of the wing pouch is induced when shrub activity is inhibited through shrub RNAi expression driven by ptc-GAL4 (ptc-Gal4/UAS-GFP; UAS-ShrubRNAi/+) (see also

Fig. S2

). (A–F) Expression of GFP (green) marks ptc-Gal4 expression domain. (G and H) Expression of Dx (green) alone results in ectopic Cut (purple, arrowhead) expression (ptc-GAL4/UAS-Flag:Dx). (I and J) This effect is suppressed (purple, arrowhead) by the co-expression of Shrub with Dx (green) driven by ptc-Gal4 (ptc-Gal4/UAS-Flag:Dx; UAS-Shrub/+). (K and L) When ShrubRNAi is expressed along the AP boundary together with Dx (green) (ptc-Gal4/UAS-Flag:Dx; UAS-ShrubRNAi/+), a dramatic up-regulation of Cut (purple) is seen, albeit in the ventral part of the disc. (M and N) Expression of Krz (green) alone results in a slight but consistent suppression of Cut (purple, arrowhead) (ptc-Gal4/; UAS-HA:Krz/+). (O and P) Co-expression of Krz (green) and Shrub results in an obvious suppression of Cut (purple, arrowhead) (ptc-Gal4/+; UAS-HA:Krz/UAS-Shrub). (Q and R) Co-expression of Krz (green) with ShrubRNAi does not affect endogenous Cut (purple, arrowhead) levels (ptc-Gal4/+; UAS-HA:Krz/UAS-ShrubRANi). All crosses were performed at 18°C.

Figure 4.

Shrub modulates the subcellular distribution of Notch. (A–C) In wing discs expressing Shrub by ptc-GAL4 (ptc-GAL4/UAS-GFP; UAS-Shrub/+), Notch (blue) is accumulated in Rab7 (purple)-positive large vesicles. (D–F) In the wing disc expressing ShrubRNAi by ptc-GAL4 (ptc-GAL4/UAS-GFP; UAS-ShrubRNA/+), Notch (blue) is localized in Rab7 positive vesicles (purple). Dashed lines indicate the boundary of the region (green) that is induced by ptc-GAL4 driver. All crosses were performed at 18°C.

Figure 5.

Shrub modulates endosomal trafficking and activation of Notch. (A–C) Shrub (green) localization partially overlaps with Rab7-positive vesicles (purple) in S2-N cells transfected with Shrub-Flag-HA. (D–F) Localization of Shrub (green) and Notch (purple) in subcellular compartments in S2-N cells transfected with Shrub-Flag-HA. (G–I) Relative localization of Notch (purple) and Shrub (green) on subcellular vesicles in S2-N cells transfected with Shrub-Flag-HA. (J–L) Relative Localization of Shrub (green), Notch (purple), and Dx (blue) on subcellular vesicles of S2-N cells transfected with Shrub-Flag-HA and Dx. M, N, and O are the merged images, respectively, of J and K, K and L, and J–L.

Figure 6.

Activation of Notch induced by Dx and ShrubRNAi is independent of Dl and Ser, but dependent on Psn and Su(H). (A and B) In the mosaic clones expressing Dx and ShrubRNAi (marked by GFP) (hs-FLP, tub-GAL4, UAS-GFP/+; UAS-Flag:Dx/UAS-ShrubRNAi; tub-GAL80, FRT82B/FRT82B), ectopic expression of Cut (purple) is induced in mostly the ventral part of the wing disc. (C and D) Co-expression of Dx and ShrubRNAi in DlREv10 and SerRX82 clones (marked by GFP) maintains the ectopic Cut expression (purple) (hs-FLP, tub-GAL4, UAS-GFP/+; UAS-Flag:Dx/UAS-ShrubRNAi; tub-GAL80, FRT82B/DlREV10, SerRX82, FRT82B). (E and F) In PsnC2 clones (marked by GFP), coexpression of Dx and ShrubRNAi does not induce the expression of Cut (purple) (hs-FLP, tub-GAL4, UAS-GFP/+; UAS-Flag:Dx/UAS-ShrubRNAi; tub-GAL80, FRT2A/PsnC2, FRT2A). (G and H) Su(H)Δ47 clones (marked by GFP), with coexpressing Dx and ShrubRNAi, fail to induce Cut expression (purple) (hs-FLP, tub-GAL4, UAS-GFP/+; tub-GAL80, FRT40A/Su(H)Δ47 FRT40A; UAS-Flag:Dx/UAS-ShrubRNAi).

Figure 7.

Notch signal induced by Dx and ShrubRNAi is dependent on ESCRT-III. (A, D, and G) Expression of double-strand RNA of vps28 (vps28RNAi) (ptc-GAL4/+; UAS-vps28RNAi/+) (A), vps25 (vps25RNAi) (ptc-GAL4/+; UAS-vps25RNAi/+) (D), or vps2 (vps2RNAi) (ptc-GAL4/+; UAS-vps2RNAi/+) (G), by ptc-GAL4 does not show significant effect on endogenous Cut levels (purple). (B, C, E, and F) Co-expression of Dx (green) with vps28RNAi (ptc-GAL4/UAS-Flag:Dx; UAS-vps28RNAi/+) (B and C) or vps25RNAi (ptc-GAL4/UAS-Flag:Dx; UAS-vps25RNAi/+) (E and F) results in the subtle but reproducible reduction of Cut (purple). (H and I) Co-expression of Dx (green) with vps2RNAi results in the ectopic activation of Cut (purple) (ptc-GAL4/UAS-Flag:Dx; UAS-vps2RNAi/+). All crosses were performed at 18°C.

Figure 8.

Shrub and Dx regulate the ubiquitinylation status of Notch. (A) Expression of Shrub by ptc-GAL4 results in the accumulation of Notch (green) in endosomal vesicles marked by FK1 (purple), an antibody that recognizes poly-ubiquitinated proteins (ptc-Gal4/+; UAS-Shrub/+). (B) Expression of ShrubRNAi by ptc-GAL4 results in a significant increase of Notch-containing vesicles (green) that are not marked by FK1 (purple) (ptc-Gal4/+; UAS-ShrubRNAi/+). (C) Co-expression of ShrubRNAi and Dx results in Notch accumulation (green) in subcellular vesicles that are negative for FK1 (purple) (ptc-Gal4/UAS-Flag:Dx; UAS-ShrubRNAi/+). (D–F) Expression of Shrub (D), ShrubRNAi (E), or ShrubRNAi and Dx (F), driven by ptc-GAL4, increases the number of Notch-containing vesicles (green) that are marked by FK2 (purple). (G and H) Ubiquitinylation assay in S2R+ cells transfected with pMT-NFL, pMK33-Shrub-Flag-HA, pMT-Dx, pMT-Flag-Ubwt (G), or pMT-Flag-Ubmono (H) shows a significant increase in Notch ubiquitinylation in the presence of Dx, suggesting that Dx drives Notch to a mono-ubiquitinated form. The cells were treated with shrub dsRNA to deplete shrub levels.

Figure 9.

Shrub-Dx-Krz–dependent modulation of Notch signaling. The ubiquitinylation state of the Notch receptor regulates its activation fate as it enters the endocytic path. Although some steps in this path have been characterized, some simply define working hypothesis. Our studies indicate that Dx in synergy with Krz promotes the poly-ubiquitinated state of the receptor, which leads to the degradation of Notch, through the MVBs, a step regulated by Shrub, a core component of the ESCRT-III complex. The close proximity of the Shrub–ESCRT-III complex with Notch in the cartoon is not meant to imply a direct association of Shrub with Notch, given that we could not find evidence favoring such interaction. Our evidence is consistent with the notion that Shrub “surrounds” the ubiquitinylated receptor, a role compatible with the previously suggested role of the yeast homologue Snf7 (Wollert et al., 2009). The expression of Dx, which physically interacts with Notch, favors a mono-ubiquitinated state of the receptor, which leads to a ligand-independent intracellular activation of Notch (NICD: the cleaved, activated form of Notch).

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Synergy between the ESCRT-III complex and Deltex defines a ligand-independent Notch signal - PubMed (original) (raw)