Cellular reconstitution of Draper-dependent engulfment. (A) Schematic of apoptotic cell clearance by Draper (green), which recognizes ligands to promote engulfment of cell corpses (purple). Representative image of an APC Annexin V–labeled cell corpse fully internalized by a Draper-GFP–expressing S2 cell. Overexpression of Draper-GFP in S2 cells results in an approximately fivefold increase in phagocytic proficiency toward apoptotic cell corpses. Draper-GFP⁺ S2 cells were incubated with labeled cell corpses and phagocytic proficiency was assessed after a 45-min incubation at 27°C. Results from three independent biological replicates are shown (mean ± SD). (B) Draper-GFP’s ability to bring Shark to the synapse between cell corpse (cyan) and S2 cell was assessed by live-cell imaging. Draper-GFP signal (left) overlaps with Shark-mCherry localization (middle) at the synapse between corpse and S2 cell. Shark was enriched in 13 of 13 synapses examined. (C) Schematic of reconstitution system to study Draper signaling. The cell corpse was replaced with a silica bead coated with a lipid bilayer containing PS. Representative image of an atto390-labeled 10% PS bead fully internalized by a Draper-GFP expressing S2 cell. Draper-GFP–expressing S2 cells were incubated with beads containing increasing mol % of PS in the lipid bilayer, and phagocytic proficiency was assessed after a 30-min incubation at 27°C. The number of beads fully ingested per cell for three independent biological replicates is shown (mean ± SD). (D) Shark kinase is enriched at the synapse between the 10% PS bead and the S2 cell. S2 cells were cotransfected with Draper-GFP and Shark-mCherry and incubated with 10% PS beads labeled with 0.5% atto390-DOPE. After 15 min, cells with active synapses were imaged by spinning-disk confocal microscopy. The middle slice from a z stack is shown. Shark was recruited to 26 of 30 synapses examined. Bars, 5 µm.