Many endocytic proteins accumulate in the reserve pool of synaptic vesicles 
(SVs) in synapses and relocalize to the endocytic periactive zone during 
neurotransmitter release. Currently little is known about their functions 
outside the periactive zone. Here we show that in the Drosophila neuromuscular 
junction (NMJ), the endocytic scaffolding protein Dap160 colocalizes during the 
SV cycle and forms a functional complex with the SV-associated phosphoprotein 
synapsin, previously implicated in SV clustering. This direct interaction is 
strongly enhanced under phosphorylation-promoting conditions and is essential 
for proper localization of synapsin at NMJs. In a dap160 rescue mutant lacking 
the interaction between Dap160 and synapsin, perturbed reclustering of SVs 
during synaptic activity is observed. Our data indicate that in addition to the 
function in endocytosis, Dap160 is a component of a network of protein-protein 
interactions that serves for clustering of SVs in conjunction with synapsin. 
During the SV cycle, Dap160 interacts with synapsin dispersed from SVs and helps 
direct synapsin back to vesicles. The proteins function in synergy to achieve 
efficient clustering of SVs in the reserve pool.
SIGNIFICANCE STATEMENT: We provide the first evidence for the function of the 
SH3 domain interaction in synaptic vesicle (SV) organization at the synaptic 
active zone. Using Drosophila neuromuscular junction as a model synapse, we 
describe the molecular mechanism that enables the protein implicated in SV 
clustering, synapsin, to return to the pool of vesicles during neurotransmitter 
release. We also identify the endocytic scaffolding complex that includes Dap160 
as a regulator of the events linking exocytosis and endocytosis in synapses.