Coordinated Pre- and Postsynaptic Protein Dynamics Underlie Rapid Sema4D-Induced Inhibitory Synapse Assembly

Inhibitory synapse formation is poorly understood compared with excitatory synaptogenesis, in part because the molecular events underlying assembly occur asynchronously and on timescales that have been difficult to resolve. Here, we exploit the ability of Semaphorin 4D (Sema4D) to rapidly and selectively induce GABAergic synapse formation in cultured hippocampal neurons, synchronizing these events to enable direct observation of pre- and postsynaptic protein dynamics by two-channel live imaging. We find that Sema4D promotes a population-wide increase in the mobility of GAD65-containing presynaptic boutons within 20 min of treatment while postsynaptic gephyrin scaffolds are mobilized only locally in a proximity-dependent manner, consistent with a presynapse-first model of inhibitory synapse assembly. Sema4D also drives recruitment of GABA A Rγ2 subunits to receptor-poor postsynaptic gephyrin scaffolds within 10 min of treatment, prior to detectable changes in GAD65–gephyrin colocalization, suggesting that postsynaptic scaffolds are primed for receptor capture before alignment with a presynaptic partner. Finally, we observed new colocalization events between established gephyrin and GABA A R protein assemblies, suggesting that clustering of either the gephyrin scaffold or GABA A Rs alone is sufficient to nucleate assembly of the postsynaptic specialization. Together, these results reveal a temporally ordered, spatially constrained mechanism by which Sema4D coordinates pre- and postsynaptic protein dynamics to assemble inhibitory synapses on the timescale of minutes.