Establishing synthetic ribbon-type active zones in a heterologous expression system

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 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 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+ channels 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. Significance StatementEncoding of sensory information in our eyes and ears builds on specialized ribbon synapses of sensory cells. Elucidating the molecular underpinning of their fascinating structure and function is an ongoing effort to which we add a bottom-up reconstitution approach in cultured cells. Aiming to recapitulate basic properties of ribbon-type presynaptic active zones of cochlear inner hair cells, we identified a minimal set of proteins that assemble in cellular nanodomains, structurally and functionally alike active zones. While not yet reconstituting synaptic vesicle exocytosis, we consider the established synthetic ribbon-type active zones a valuable platform for studying molecular interactions of active zone proteins. We expect the approach to complement and reduce experiments on native ribbon synapses asserted from animals.