The functions of auxilin and Rab11 in Drosophila suggest that the fundamental role of ligand endocytosis in notch signaling cells is not recycling

Abstract

Notch signaling requires ligand internalization by the signal sending cells. Two endocytic proteins, epsin and auxilin, are essential for ligand internalization and signaling. Epsin promotes clathrin-coated vesicle formation, and auxilin uncoats clathrin from newly internalized vesicles. Two hypotheses have been advanced to explain the requirement for ligand endocytosis. One idea is that after ligand/receptor binding, ligand endocytosis leads to receptor activation by pulling on the receptor, which either exposes a cleavage site on the extracellular domain, or dissociates two receptor subunits. Alternatively, ligand internalization prior to receptor binding, followed by trafficking through an endosomal pathway and recycling to the plasma membrane may enable ligand activation. Activation could mean ligand modification or ligand transcytosis to a membrane environment conducive to signaling. A key piece of evidence supporting the recycling model is the requirement in signaling cells for Rab11, which encodes a GTPase critical for endosomal recycling. Here, we use Drosophila Rab11 and auxilin mutants to test the ligand recycling hypothesis. First, we find that Rab11 is dispensable for several Notch signaling events in the eye disc. Second, we find that Drosophila female germline cells, the one cell type known to signal without clathrin, also do not require auxilin to signal. Third, we find that much of the requirement for auxilin in Notch signaling was bypassed by overexpression of both clathrin heavy chain and epsin. Thus, the main role of auxilin in Notch signaling is not to produce uncoated ligand-containing vesicles, but to maintain the pool of free clathrin. Taken together, these results argue strongly that at least in some cell types, the primary function of Notch ligand endocytosis is not for ligand recycling.

Figure 1. Rab11 is not required for Notch signaling in eye discs.

Confocal microscope images of third instar larval eye discs with clones of mutant cells are shown. The discs are immunolabeled to reveal Notch activation (anti-ßgal), photoreceptor cell nuclei (anti-Elav), and F-actin (phalloidin). Homozygous mutant cell clones are marked by the absence of nuclear GFP expression. Clones are outlined in white. Arrow heads point to some of the mutant cells within the clones that express ß-gal, indicating that Notch is activated. (A,A’) A Notch null (N-) clone was generated in larvae of the genotype N^55e11 FRT19A/ubi-ngfp FRT19A; ey-gal4, UAS-flp/+; m∂-lacZ/+. (B,B’) A Delta null (Dl-) clone was generated in larvae of the genotype ey-flp;m∂-lacZ/+; FRT82B Dl^rev10/FRT82B ubi-ngfp (C,C’) lqf- clones generated in larvae of the genotype eyflp; m∂-lacZ/+; lqf^ARI FRT80B/ubi-ngfp FRT80B. (D,D’) aux- clones were generated in larvae of the genotype ey-flp; m∂-lacZ/+; FRT^5-5Z3515 aux^F956*/FRT^5-5Z3515 tub-ngfp. (E-E3’) The same Rab11- clone is shown in all panels, generated in larvae of the genotype ey-flp; m∂-lacZ/+; Rab11^ΔFRT/FRT5377 Hrb98DE::GFP. Scale bar 20 µm.

Figure 2. Female germline cells do not require auxilin to send Delta signals to follicle cells.

(A) A diagram of an oocyte/nurse cell complex (stage 6–7) is shown. The fifteen nurse cells are diploid, and the cytoplasms of the nurse cells and the oocyte are interconnected by cytoplasmic bridges. (B–D’) Confocal microscope images of oocyte/nurse cell complexes are shown. The complexes were immunolabeled to reveal Notch activation in the follicle cells (anti-Hnt) and F-actin (phalloidin). Homozygous mutant cell nuclei are marked by the absence of GFP. (B,B’) Wild-type (WT) complexes are shown. Notch is activated in the follicle cells. (C,C’) A mosaic complex with aux- germ-line cells and aux+ follicle cells is shown. Notch was activated in the follicle cells. The clone was generated in females of the genotype hs-flp/+; ubi-gfp tub-aux FRT40A/FRT40A; aux¹³⁶/aux⁷²⁷. (D,D’) As in (C,C’), except the genotype was hs-flp/+; FRT^5-5Z3515, aux^F956*/FRT^5-5Z3515, ubi-ngfp. Reduced levels of Hnt were seen at the poles of the aux+/aux- mosaic oocyte/nurse cell complexes, as was also observed in Chc+/Chc- mosaics [SLW and DB, unpublished observation]. This is quite distinct, however, from the absence of Hnt throughout the follicle epithelium observed with lqf- or Dl- germ line clones . Scale bar 20 µm.

Figure 3. Overexpression of clathrin heavy chain and/or epsin suppresses the adult eye defects in aux loss-of-function mutants.

(A–E) Light micrographs of adult external eyes of the genotypes indicated beneath are shown. (F) A diagram of an apical tangential section of a single ommatidium is shown. The numbers are photoreceptor cells R1 – R7. The black circular projections from each cells are the light-gathering organelles called rhabdomeres. The hexagonal shape is formed by pigment cells. (G–K) Small fields of apical tangential sections of adult eyes are shown. (H) Ommatidia of aux hypomorphs are usually disorganized, and often have extra photoreceptors. (I–K) Addition of genomic DNA transgenes that express Chc+ or lqf+ suppresses the eye morphology defects of aux hypomorphs. The fraction of phenotypically wild-type (wt) ommatidia was determined by observing 300–500 ommatidia in 4–5 eyes of each genotype. The error is one standard deviation. Scale bar 10 µm (G–K) and 60 µm (A–E).