Coordinated pre- and postsynaptic protein dynamics underlie rapid Sema4D-induced inhibitory synapse assembly

Inhibitory synapse formation is poorly understood compared to 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 minutes 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 minutes 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 observe 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. Significance Statement The assembly of new synaptic contacts requires precise coordination of specialized proteins in pre- and postsynaptic neurons. Inhibitory synapses, which suppress neuronal activity and are essential for circuit stability, contain distinct molecular components, yet the mechanisms governing their assembly remain poorly understood. We used Sema4D, a protein that rapidly induces inhibitory synapse formation, as a molecular tool to dissect how synaptic proteins on either side of the synaptic cleft are coordinated in space and time. Using live imaging we show that Sema4D acts on both pre- and postsynaptic compartments to recruit synaptic proteins with spatiotemporal precision. Together, these findings define the sequence of molecular events underlying inhibitory synapse assembly and have implications for neurodevelopmental disorders in which inhibition is disrupted.