Abstract In mature neurons, excitatory synapses are formed on the dendritic spine, whereas inhibitory synapses are formed on the dendritic shaft. Thus, it is primarily the accumulation of synaptic proteins that characterizes inhibitory synapses as distinct from non-synaptic regions. Protein accumulation is achieved by a combination of microtubule (MT)-based transport by kinesins and lateral diffusion across the plasma membrane; however, how and when proteins are released from kinesins remains unclear. Using primary cultured hippocampal neurons, we found that Teneurin-2 (TEN2) promotes synaptic protein accumulation by recruiting MTs via the representative MT plus end-tracking protein, EB1. MTs recruitment was enhanced when the extracellular domain of TEN2 successfully chose partners, and the lateral diffusion of TEN2 was inhibited. Conversely, if TEN2 partner choice is not achieved, MTs are not recruited, and thus synaptogenesis is not followed. Our study revealed that cargo release from kinesins through TEN2-MTs interactions supports the continuity from partner choice to synaptogenesis, which is a critical step in synaptic maturation.

Teneurin-2 at the Synapse Construction Site is a Signpost for Cargo Unloading from Motor Proteins