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

Abstract

AbstractEncoding 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 CaV1.3 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.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, refine, and reduce experiments on native ribbon synapses asserted from animals.