Ribeye is required for presynaptic CaV1.3a channel localization and afferent innervation of sensory hair cells

Abstract

SUMMARYRibbon synapses of the ear, eye and pineal gland contain a unique protein component: Ribeye. Ribeye consists of a novel aggregation domain spliced to the transcription factor CtBP2 and is one of the most abundant proteins in synaptic ribbon bodies. Although the importance of Ribeye for the function and physical integrity of ribbon synapses has been shown, a specific role in synaptogenesis has not been described. Here, we have modulated Ribeye expression in zebrafish hair cells and have examined the role of Ribeye in synapse development. Knockdown of ribeye resulted in fewer stimulus-evoked action potentials from afferent neurons and loss of presynaptic Ca V 1.3a calcium channel clusters in hair cells. Additionally, afferent innervation of hair cells was reduced in ribeye morphants, and the reduction was correlated with depletion of Ribeye punctae. By contrast, transgenic overexpression of Ribeye resulted in Ca V 1.3a channels colocalized with ectopic aggregates of Ribeye protein.Overexpression of Ribeye, however, was not sufficient to create ectopic synapses. These findings reveal two distinct functions of Ribeye in ribbon synapse formation -clustering Ca V 1.3a channels at the presynapse and stabilizing contacts with afferent neurons -and suggest that Ribeye plays an organizing role in synaptogenesis.

Clarin-1 acts as a modulator of mechanotransduction activity and presynaptic ribbon assembly

“…Similar results, regarding mislocalized ribbons that do not form synapses, were reported in a transgenic zebrafish line overexpressing ribeye b (Sheets et al, 2011). Those results, as well as our findings, suggest that if hair cell presynaptic assembly is altered, either by overexpression of presynaptic components PCR product was sequence verified and used for in vitro transcription using the mMESSAGE mMACHINE T7 Ultra kit (Life Technologies) according to the manufacturer's instructions.…”

“…The ribeye b antibody, developed and qualified by our laboratory (Fig. S5), showed the characteristic puncta staining at the base of the hair cells (Sheets et al, 2011) in controls (Fig. S5, A and C) but not hair cells (Söllner et al, 2004;Seiler et al, 2005).…”

The Synaptic Ribbon Is a Site of Phosphatidic Acid Generation in Ribbon Synapses

“…To address this question, we cotransfected COS cells, i.e., nonneuronal cells that do not contain synaptic ribbons, with full-length RIBEYE(AB)-and PAsensor protein constructs. Heterologously expressed full-length RIBEYE(AB) forms electron-dense protein aggregates Sheets et al, 2011) that resemble synaptic ribbons and that are denoted as artificial synaptic ribbons in the following text. For these COS cell experiments, we used mCherry-tagged Raf-1 as PA-sensor protein because Raf-1 does not require high intracellular Ca 2ϩ levels for PA binding, a situation that may not occur in COS cells (Gosh et al, 1996(Gosh et al, , 2003Baillie et al, 2002) (data not shown).…”

Deletion of the Presynaptic Scaffold CAST Reduces Active Zone Size in Rod Photoreceptors and Impairs Visual Processing

“…The hierarchy of regulation is likely to differ between synapse formation and structural plasticity and among different synapses. Active zone size in sensory cells may additionally be governed by expression levels (Sheets et al, 2011) the effect of CAST/ERC2 deletion. Synapses were formed, showed a generally preserved molecular presynaptic and postsynaptic organization, and were functional.…”

“…The mechanisms regulating active zone size are not well understood but likely to involve Liprins/Syd proteins (Stigloher et al, 2011), cytomatrix of the active zone (CAZ) proteins, such as CAST/ELKS (Kittel et al, 2006), Bassoon and Piccolo (Zhai et al, 2000(Zhai et al, , 2001, and, at ribbon synapses, also RIBEYE (Sheets et al, 2011). Some synapses, such as the neuromuscular junction or ribbon synapses of photoreceptors, and hair cells use large active zones to accommodate high rates of synaptic transmission.…”