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 callsyntheticribbons orSyRibbons. Super-resolution STED microscopy and cryo-correlative electron tomography revealedSyRibbonswere similar to native ribbons at AZs of cochlear inner hair cells in shape and size.SyRibbonswith Ca²⁺channel clusters formed upon additional expression of Ca_V1.3 Ca²⁺channels and RIM-binding protein 2 (RBP2). Ca_V1.3 Ca²⁺channel clusters associated withSyRibbonswere larger than ribbonless Ca_V1.3 Ca²⁺channels clusters and functional analysis by Ca²⁺-imaging in combination with patch clamp showed partial confinement of the Ca²⁺signal atSyRibbons. 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.