Ral inhibits ligand-independent Notch signaling in Drosophila

Abstract

We discovered recently that the Drosophila Ral GTPase regulates Notch signaling and thereby affects cell patterning in the eye. Although Ral functions in the ligand signaling cells, Ral does not stimulate ligand signaling directly. Rather, in cells that express both Notch receptor and ligand, Ral activity promotes a cell to become the signaler by inhibiting Notch receptor activation in that cell. Moreover, Ral inhibits a particular pathway of Notch activation-receptor activation that occurs independent of ligand binding. In this Commentary, we discuss the phenomenon of ligand-independent Notch receptor activation and how this event might be regulated by Ral.

Figure 1. Model for Ral function in R3/R4 specification. At left are light microscope images of a single ommatidium. At right are diagrams of the signaling events that distinguish R3 and R4. The top box is wild type (Ral+). Photoreceptors in the wild-type adult Drosophila eye (top) are arranged in a trapezoid with R3 at the apex. The asymmetric arrangement of the originally equivalent R3/R4 pair is due to pre-R3 experiencing more Frizzled (Fz) activation than does pre-R4. This difference in Fz activity is amplified by subsequent Notch activation in pre-R4.^- Fz activation results in increased transcription of Delta and neuralized (neur)^- and also relocalization of a Fz/Dsh (Disheveled) complex that represses Notch receptor at the pre-R3 plasma membrane that faces pre-R4. Thus pre-R3 becomes the Delta signaling cell and Notch is activated in pre-R4. Ral defines a unique pathway for insuring that pre-R3 becomes the Delta signaling cell.Ral transcription also depends on Fz, and Ral activity prevents activation of Notch receptor that is not bound to ligand. Loss of Ral activity (bottom) results in symmetric R3/R4 pairs and ligand-independent Notch activation.

Figure 2. Endosomal trafficking of unliganded Notch receptor. A diagram of the known endosomal routes of the Notch receptor that was not activated by ligand binding. The precise step in the endocytic pathway where the Ubiquitin ligases Su(Dx) and Dx function is not clear. Their locations in the diagram are meant to indicate the process for which each is required. See text for explanation of diagram. ee = early endosome, re = recycling endosome, Hrs protein is required to deliver proteins to MVBs. Other abbreviations are defined in the text.

Figure 3. Ral does not colocalize with Rab5, Rab7, nor Rab11. Confocal microscope images of part of three developing eyes (third instar larval eye discs) are shown. All three discs were labeled with anti-Ral and phalloidin (binds f-actin at the plasma membrane). (A-A”’) A YFP-Rab5 expressing disc is shown. The genotype is ey-gal4, GMR-gal4/ UAS-yfp-rab5. A”’ is a merge of A and A’. The white arrows indicate the few puncta that are simultaneously YFP-Rab5-positive and Ral-positive. The focal plane of this disc image is more basal than that of the other two discs shown below. (B-B”) A Rab7-GFP expressing disc is shown. The genotype is Actin5C-gal4, UAS-rab7-gfp. At the apical focal plane at which there are Rab7-GFP-positive puncta (B), Ral puncta are absent (B’). (C-C”) A wild-type disc also labeled with anti-Rab11 is shown. At the apical focal plane at which Rab11 resides, no Ral is detected (C’).

Figure 4. Rescue of Ral^EE1 eye organization and notal bristle defects by wild-type and mutant Ral transgenes. Adult eyes and nota of flies of Ral^EE1 flies with no transgene (leftmost), or that express the indicated Ral transgenes using the Gal4/UAS system are shown. Act is the Actin5C-gal4 driver and ey is the eyeless-gal4 driver. UAS-Ral^WT has been described. Details of how we constructed UAS-Ral^D46N and UAS-Ral^D46E are available upon request.