Regulated Dynamic Trafficking of Neurexins Inside and Outside of Synaptic Terminals

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 alpha Nrxn molecules are more mobile in the plasma membrane than smaller beta Nrxns because alpha 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 alpha Nrxns when both molecules were coexpressed. Conversely, impeding other interactions by insertion of splice sequence #4 or removal of extracellular Ca2+ augmented the mobility of alpha Nrxns and beta 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 beta Nrxn(+) STVs was faster than that of alpha Nrxns, but both depended on the microtubule motor protein KIF1A and neuronal activity regulated the velocity. Large spontaneous fusion of Nrxn(+) STVs occurred simultaneously with synaptophysin on axonal membranes mostly outside of active presynaptic terminals. Surface Nrxns enriched at synaptic terminals where alpha Nrxns and Nxph1/alpha 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.