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

Abstract

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 human embryonic kidney 293 (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 <i>synthetic</i> ribbons or <i>SyRibbons</i>. Super-resolution stimulated emission depletion (STED) microscopy and cryo-correlative electron tomography revealed <i>SyRibbons</i> were similar to native ribbons at AZs of cochlear inner hair cells in shape and size. <i>SyRibbons</i> with 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 with <i>SyRibbons</i> were larger than ribbonless Ca_V1.3 Ca²⁺ channel clusters, and functional analysis by Ca²⁺ imaging in combination with patch clamp showed partial confinement of the Ca²⁺ signal at <i>SyRibbons</i>. In summary, we identify Ca²⁺ channels, RBP, membrane-anchored Bassoon, and RIBEYE as minimal components for reconstituting a basic ribbon-type AZ. <i>SyRibbons</i> might complement animal studies on molecular interactions of AZ proteins.