Fig. 4: The endocytic pathway controls crystal cell differentiation.
A, B, C Schematic representation of the Notch pathway. A After Ser binding, Kuzbanian (Kuz) and the γ-secretase complex (γ-sec) cleave Notch, and the Notch Intracellular Domain (NICD) enters the nucleus to regulate gene expression together with Suppressor of Hairless (Su(H)). B After cleavage by Kuz, Notch can undergo endocytosis that depends on the dynamin shibire (shi), and remains inserted in the membrane of early endosomes that mature by the action of Hrs and Rab5 to late endosomes. Formation of intraluminal vesicles of the multivesicular body is mediated by ESCRT complexes 0-III, which include the subunits TSG101, Vps25 and Shrub; Vps4 is the effector ATPase. Notch can be cleaved at the limiting membrane of late endosomes or multivesicular bodies, and enters the nucleus to regulate transcription. Notch present in intraluminal vesicles of the multivesicular body is degraded at the lysosome (not shown in the scheme). C Ligand-independent activation of Notch: Notch can undergo endocytosis without previous ligand binding; in this case, the entire (uncleaved) receptor ends up in the membrane of endosomes. The subsequent steps of the pathway are identical to those described in B. The E3 ubiquitin ligase deltex (dx) stimulates Notch endocytosis; Suppressor of deltex (Su(dx)) stimulates the formation of Notch-containing intraluminal vesicles. D–G Crystal cells visualized by anti-Lozenge (Lz) or anti-prophenoloxidase (PPO) immunofluorescence after knock-down of shi (E), Hrs (F) or Rab5 (G), with a domeMESO-Gal4 driver. White dashed lines mark lymph gland lobes. H Quantification of the results shown in panels (D–G) boxes represent the interquartile range, the central line indicates the median, and whiskers extend to the minimum and maximum values. For wild type, n = 62 primary lobes; for shibireRNAi, n = 24; for HrsRNAi, n = 20; for Rab5DN, n = 22. The statistical analysis was performed in both cases using Kruskal-Wallis test, followed by Dunn’s test for treatment versus control comparisons (p = 0.0017 for Rab5DN Lz+ cells; p < 0.0001 for every other comparison). Source data for plots are provided as a Source Data file. **p < 0.01; ****p < 0.0001.
