The cleft is an integral part of synapses, yet its macromolecular organization remains unclear. We show here that the cleft of excitatory synapses exhibits a distinct density profile as measured by cryoelectron tomography (cryo-ET). Aiming for molecular insights, we analyzed the synapse-organizing proteins Synaptic Cell Adhesion Molecule 1 (SynCAM 1) and EphB2. Cryo-ET of SynCAM 1 knockout and overexpressor synapses showed that this immunoglobulin protein shapes the cleft's edge. SynCAM 1 delineates the postsynaptic perimeter as determined by immunoelectron microscopy and super-resolution imaging. In contrast, the EphB2 receptor tyrosine kinase is enriched deeper within the postsynaptic area. Unexpectedly, SynCAM 1 can form ensembles proximal to postsynaptic densities, and synapses containing these ensembles were larger. Postsynaptic SynCAM 1 surface puncta were not static but became enlarged after a long-term depression paradigm. These results support that the synaptic cleft is organized on a nanoscale into sub-compartments marked by distinct trans-synaptic complexes.
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