Draper-dependent glial phagocytic activity is mediated by Src and Syk family kinase signalling

Abstract

The cellular machinery promoting phagocytosis of corpses of apoptotic cells is well conserved from worms to mammals. An important component is the Caenorhabditis elegans engulfment receptor CED-1 (ref. 1) and its Drosophila orthologue, Draper. The CED-1/Draper signalling pathway is also essential for the phagocytosis of other types of 'modified self' including necrotic cells, developmentally pruned axons and dendrites, and axons undergoing Wallerian degeneration. Here we show that Drosophila Shark, a non-receptor tyrosine kinase similar to mammalian Syk and Zap-70, binds Draper through an immunoreceptor tyrosine-based activation motif (ITAM) in the Draper intracellular domain. We show that Shark activity is essential for Draper-mediated signalling events in vivo, including the recruitment of glial membranes to severed axons and the phagocytosis of axonal debris and neuronal cell corpses by glia. We also show that the Src family kinase (SFK) Src42A can markedly increase Draper phosphorylation and is essential for glial phagocytic activity. We propose that ligand-dependent Draper receptor activation initiates the Src42A-dependent tyrosine phosphorylation of Draper, the association of Shark and the activation of the Draper pathway. These Draper-Src42A-Shark interactions are strikingly similar to mammalian immunoreceptor-SFK-Syk signalling events in mammalian myeloid and lymphoid cells. Thus, Draper seems to be an ancient immunoreceptor with an extracellular domain tuned to modified self, and an intracellular domain promoting phagocytosis through an ITAM-domain-SFK-Syk-mediated signalling cascade.

Figure 1. Shark binds an ITAM in the Draper intracellular domain

a, Draper contains an ITAM domain from Y934-L952 (YXXI-X₁₁-YXXL). The requirements for the five tyrosine residues within and adjacent to this domain (shown) and Src were assayed in the yeast two-hybrid system. b, Lysates from yeast cultures in a were tested in quantitative β-galactosidase (β-Gal) assays. Error bars represent s.e.m.; n = 3; *, P < 0.05. c, S2 cells were transfected with pMT-Myc::Shark and pMT-Drpr-I constructs. Draper immunoprecipitates (IP) were analysed by western blotting (WB) with anti-phosphotyrosine (pTyr), anti-Myc and anti-HA antibodies. Vec., vector.

Figure 2. Shark is required for recruitment of Draper and glial membranes to severed axons

a, Control animals (yw;+/UAS-shark^RNAi) and those with glia-specific knockdown of shark (yw;repo-Gal4/UAS-shark^RNAi) were assayed for expression of Draper (red). Glial nuclei were stained with Repo (blue). Left, uninjured; centre, maxillary palp ablation (day 1); right, antennal ablation (day 1). Outlined, example of a maxillary palp-innervated glomerulus; arrow, nerve containing severed maxillary palp ORN axons; open arrowhead, antennal lobe glial cell; boxes, areas quantified in b. b, Quantification of data from a. Error bars represent s.e.m.; n ≥ 10. c, Glial membranes were labelled in control (yw;UAS-GFPS65T/+;repo-Gal4/+) or glial shark^RNAi animals (yw;UAS-GFPS65T/+;repo-Gal4/UAS-shark^RNAi) and assayed for morphology before or after injury (panel order as in a). Outlined, maxillary palp-innervated glomerulus; arrow, nerve containing severed maxillary palp ORN axons; boxes, areas used to quantify glial hypertrophy in d. d, Quantification data from c. Error bars represent s.e.m.; n ≥ 10.

Figure 3. Shark is required for glial clearance of severed axons from the CNS

a, The axons of OR85e-expressing ORNs were labelled with mCD8::GFP in control (yw;OR85e-mCD8::GFP/+;repo-Gal4/+) and glial shark^RNAi (yw;OR85e-mCD8::GFP/+;repo-Gal4/UAS-shark^RNAi) animals, maxillary palps were ablated, and the clearance of severed ORN axons from the CNS was assayed with anti-GFP antibody stains (green). Maxillary nerves are indicated (arrowheads). b, Quantification of data from a. Error bars represent s.e.m.; n ≥ 10. c, OR85e-expressing ORN axons were labelled in control (yw;OR85e-mCD8::GFP/+) animals and in shark¹ or draper^Δ5 null mutant backgrounds, maxillary palps were ablated, and clearance was assayed at 5 days. d, Quantification of data from c. Error bars represent s.e.m.; n ≥ 10; *, P < 0.05; **, P < 0.001; ***, P < 0.0001. e, Quantification of brain hemispheres containing GFP-labelled ORN axonal debris along the maxillary nerve for genotypes described in c.

Figure 4. Src42Aa is required for glial responses to axon injury and modulates Draper phosphorylation status

a, Control animals (yw;UAS-src42A^RNAi/+, no driver) and those with glia-specific knockdown of src42A (yw;UAS-src42A^RNAi/+;repo-Gal4/+) were assayed for injury-induced changes in glial Draper expression and for recruitment of Draper to severed axons (red). b, c, Quantification of data from a for palp-innervated glomeruli (b) and antennal lobe glia (c). Error bars represent s.e.m.; n ≥ 10. d, The axons of OR85e-expressing ORNs were labelled with mCD8::GFP in control (yw;OR85e-mCD8::GFP/+) and glial src42A^RNAi (yw;OR85e-mCD8::GFP/UAS-src42A^RNAi;repo-Gal4/+) animals, maxillary palps were ablated, and the clearance of severed ORN axons from the CNS was assayed with anti-GFP antibody stains (green) 5 days after injury. e, Quantification of data from d. Error bars represent s.e.m.; n ≥ 10. f, S2 cells were co-transfected with pMT-HA::Draper, pMT-Myc::Shark, pMT-Flag::Src42A, pMT-Myc::Shark K698R (kinase-dead) and pMT vector. After transfection and expression, some cells were incubated for 60 min with the SFK inhibitor PP2 (10 μM) before cell lysis. Anti-Draper and IgG control immunoprecipitates (IP) from cells were analysed by SDS-PAGE and western blotted (WB) with antibodies against pTyr, Draper, Myc and Flag.