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 might act as a super-scaffold organizing AZ topography. Here we employed a synthetic biology approach to reconstitute ribbon-type AZs in HEK293 cells for probing their minimal molecular requirements and studying presynaptic Ca ²⁺ 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 . SyRibbons with Ca ²⁺ channel clusters formed upon additional expression of Ca _V 1.3 Ca ²⁺ channels and RIM-binding protein 2 (RBP2), known to promote presynaptic Ca ²⁺ channel clustering. Confocal and super-resolution microscopy along with functional analysis by patch-clamp and Ca ²⁺ -imaging revealed striking similarities and interesting differences of SyRibbons in comparison to native IHC ribbon-type AZs. In summary, we identify Ca ²⁺ 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.