Abstract
Encoding of several sensory modalities into neural signals is mediated by ribbon synapses. The synaptic ribbon tethers synaptic vesicles at the presynaptic active zone (AZ) and may act as a super-scaffold organizing AZ topography. Here, we employed a synthetic biology approach to reconstitute structures mimicking ribbon-type AZs in human embryonic kidney 293 (HEK293) cells for probing minimal molecular requirements and studying presynaptic Ca2+ channel clustering. Co-expressing a membrane-targeted version of the AZ protein Bassoon and the ribbon core protein RIBEYE, we observed structures recapitulating basic aspects of ribbon-type AZs, which we call synthetic ribbons or SyRibbons. Super-resolution stimulated emission depletion (STED) microscopy and cryo-correlative electron tomography revealed SyRibbons were similar to native ribbons at AZs of cochlear inner hair cells in shape and size. SyRibbons with Ca2+ channel clusters formed upon additional expression of CaV1.3 Ca2+ channels and RIM-binding protein 2 (RBP2). CaV1.3 Ca2+ channel clusters associated with SyRibbons were larger than ribbonless CaV1.3 Ca2+ channel clusters, and functional analysis by Ca2+ imaging in combination with patch clamp showed partial confinement of the Ca2+ signal at SyRibbons. In summary, we identify Ca2+ channels, RBP, membrane-anchored Bassoon, and RIBEYE as minimal components for reconstituting a basic ribbon-type AZ. SyRibbons might complement animal studies on molecular interactions of AZ proteins.