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 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 callsyntheticribbons orSyRibbons.SyRibbonswith Ca²⁺channel clusters formed upon additional expression of Ca_V1.3 Ca²⁺channels and RIM-binding protein 2 (RBP2), known to promote presynaptic Ca²⁺channel clustering. Confocal and super-resolution microscopy along with functional analysis by patch-clamp and Ca²⁺-imaging revealed striking similarities and interesting differences ofSyRibbonsin comparison to native IHC ribbon-type AZs. In summary, we identify Ca²⁺channels, RBP, membrane-anchored Bassoon, and RIBEYE as minimal components for reconstituting a basic ribbon-type AZ.SyRibbonsmight complement animal studies on molecular interactions of AZ proteins.