A challenge in neuroscience is to understand the mechanisms underlying synapse formation. Most excitatory synapses in the brain are built on spines, which are actin-rich protrusions from dendrites. Spines are a major substrate of brain plasticity, and spine pathologies are observed in various mental illnesses. Here we investigate the role of neurobeachin (Nbea), a multidomain protein previously linked to cases of autism, in synaptogenesis. We show that deletion of Nbea leads to reduced numbers of spinous synapses in cultured neurons from complete knockouts and in cortical tissue from heterozygous mice, accompanied by altered miniature postsynaptic currents. In addition, excitatory synapses terminate mostly at dendritic shafts instead of spine heads in Nbea mutants, and actin becomes less enriched synaptically. As actin and synaptopodin, a spine-associated protein with actin-bundling activity, accumulate ectopically near the Golgi apparatus of mutant neurons, a role emerges for Nbea in trafficking important cargo to pre- and postsynaptic compartments.

Synapses depend on trafficking of key membrane proteins by lateral diffusion from surface populations and by exocytosis from intracellular pools. The cell adhesion molecule neurexin (Nrxn) plays essential roles in synapses, but the dynamics and regulation of its trafficking are unknown. Here, we performed single-particle tracking and live imaging of transfected, epitope-tagged Nrxn variants in cultured rat and mouse wild-type or knock-out neurons. We observed that structurally larger ␣Nrxn molecules are more mobile in the plasma membrane than smaller ␤Nrxns because ␣Nrxns displayed higher diffusion coefficients in extrasynaptic regions and excitatory or inhibitory terminals. We found that well characterized interactions with extracellular binding partners regulate the surface mobility of Nrxns. Binding to neurexophilin-1 (Nxph1) reduced the surface diffusion of ␣Nrxns when both molecules were coexpressed. Conversely, impeding other interactions by insertion of splice sequence #4 or removal of extracellular Ca 2ϩ augmented the mobility of ␣Nrxns and ␤Nrxns. We also determined that fast axonal transport delivers Nrxns to the neuronal surface because Nrxns comigrate as cargo on synaptic vesicle protein transport vesicles (STVs). Unlike surface mobility, intracellular transport of ␤Nrxn ϩ STVs was faster than that of ␣Nrxns, but both depended on the microtubule motor protein KIF1A and neuronal activity regulated the velocity. Large spontaneous fusion of Nrxn ϩ STVs occurred simultaneously with synaptophysin on axonal membranes mostly outside of active presynaptic terminals. Surface Nrxns enriched at synaptic terminals where ␣Nrxns and Nxph1/␣Nrxns recruited GABA A R subunits. Therefore, our results identify regulated dynamic trafficking as an important property of Nrxns that corroborates their function at synapses.