Synj’s 5′-PPase suppresses ADBE, while its SAC activity is required for normal ADBE. (A) Schematic of Synj domains and their known functions. Synj functional domain mutants are highlighted by orange boxes. (B) Images of NMJ loaded with dextran (red) and stained with HRP (green) following 10-Hz stimulation for 10 min. (C) Fold change in dextran uptake. For control, n = 30; synj mutant, n = 28. In synj mutant background: synj^5′-AD, n = 13, synj^SAC-AD, n = 16; synj^SAC-R258Q, n = 14; synj ^ΔPRD, n = 16. *, P < 0.05 compared with control. (D) Acute inhibition of Mnb by proINDY enhances dextran uptake in Synj^WT but not in flies with deficient SAC domain. For DMSO treatment: control, n = 25. In synj mutant background: synj^WT, n = 7; synj^5′-AD, n = 7; synj^SAC-AD, n = 7. For proINDY treatment: control, n = 9. In synj mutant background: synj^WT, n = 7; synj^5′-AD, n = 7; synj^SAC-AD, n = 8. In B and D, scale bar = 2 µm. n value indicates the number of NMJs examined. All values represent mean ± SEM. (E) Model depicting Mnb- and CaN-dependent coordination of endocytosis modes through phosphorylation of Synj. Mnb phosphorylates Synj to enhance Synj’s 5′-PPase activity, thereby facilitating CME but suppressing ADBE during moderate activity. During prolonged intense activity, higher intracellular Ca²⁺ activates CaN, which further dephosphorylates Synj to promote ADBE. This may or may not be coupled with Mnb inactivation during prolonged activity. Dephosphorylation of Synj reduces its 5′-PPase activity, and ADBE is enhanced through PI(4,5)P₂ accumulation and actin polymerization. Normal ADBE also requires Synj’s SAC domain (not depicted).