Molecular mechanism of Netrin-1–dependent axon branching. (top left) In the absence of Netrin-1, TRIM9 (orange) is bound to SNAP25 (green). The TRIM9 interaction blocks SNAP25 binding to VAMP2 or VAMP7 (blue), which inhibits SNARE complex formation and exocytic vesicle fusion. Prevention of plasma membrane expansion constrains basal levels of axon branching. (top right) After Netrin-1 stimulation, TRIM9 releases SNAP25, which promotes SNARE complex formation, vesicle fusion, local plasma membrane expansion, and axon branching. (bottom) Genetic deletion of TRIM9 renders cells nonresponsive to Netrin-1. As the interaction between SNAP25 and VAMP2 or VAMP7 is no longer inhibited, constitutive levels of SNARE complex formation, exocytosis, and axon branching increase. To reduce and rescue the exocytosis and axon branching phenotypes associated with loss of TRIM9, introduction of TRIM9 containing the SNAP25 binding domain is required. To rescue Netrin-1 sensitivity, TRIM9 containing ubiquitin ligase activity must be introduced.