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During the previous funding period, we verified the hypothesis that synaptic vesicles bind soluble proteins, including exo- and endocytosis cofactors, and maintain them in the synapse. We now plan to follow up on these findings by testing the link between the synaptic vesicle cluster and the cytoskeleton of the presynapse. We will differentiate between two hypotheses of presynaptic organization: 1) the synaptic cytoskeleton serves to organize the vesicle clusters, and 2) alternatively, the vesicles organize the synaptic cytoskeleton. We will use a combination of in vitro imaging and in vitro manipulation tools.