Characterization of ribeye subunits in zebrafish hair cells reveals that exogenous ribeye b-domain and ctbp1 localize to the basal ends of synaptic ribbons

Synaptic ribbons are presynaptic structures formed by the self-Association of RIBEYE-the main structural component of ribbon synapses. RIBEYE consists of two domains: A unique N-Terminal A-domain and a C-Terminal B-domain that is identical to the transcription co-repressor C-Terminal binding protein 2 (CtBP2). Previous studies in cell lines have shown that RIBEYE A-domain alone is sufficient to form ribbon-like aggregates and that both A-and B-domains form homo-And heterotypic interactions. As these interactions are likely the basis for synaptic-ribbon assembly and structural plasticity, we wanted to examine how zebrafish Ribeye A-and B-domains interact with synaptic ribbons in vivo. To that end, we characterized the localization of exogenously expressed Ribeye A-and B-domains and the closely related protein, CtBP1, in the hair cells of transgenic zebrafish larvae. Unexpectedly, exogenously expressed Ribeye A-domain showed variable patterns of localization in hair cells; one zebrafish paralog of A-domain failed to self-Associate or localize to synaptic ribbons, while the other selfassembled but sometimes failed to localize to synaptic ribbons. By contrast, Ribeye B-domain/CtBP2 was robustly localized to synaptic ribbons. Moreover, both exogenously expressed B-domain/CtBP2 and CtBP1 were preferentially localized to the basal end of ribbons adjacent to the postsynaptic density. Overexpression of B-domain/CtBP2 also appeared to affect synaptic-ribbon composition; endogenous levels of ribbon-localized Ribeye were significantly reduced as hair cells matured in B-domain/CtBP2 transgenic larvae compared to wild-Type. These results reveal how exogenously expressed Ribeye domains interact with synaptic ribbons, and suggest a potential organization of elements within the ribbon body.