Protein Composition of Immunoprecipitated Synaptic Ribbons

Abstract

The synaptic ribbon is an electron-dense structure found in hair cells and photoreceptors. The ribbon is surrounded by neurotransmitter-filled vesicles and considered to play a role in vesicle release. We generated an objective, quantitative analysis of the protein composition of the ribbon complex using a mass spectrometry-based proteomics analysis. Our use of affinity-purified ribbons and control IgG immunoprecipitations ensure that the identified proteins are indeed associated with the ribbon complex. The use of mouse tissue, where the proteome is complete, generated a comprehensive analysis of the candidates. We identified 30 proteins (comprising 56 isoforms and subunits) associated with the ribbon complex. The ribbon complex primarily comprises proteins found in conventional synapses, which we categorized into 6 functional groups: vesicle handling (38.5%), scaffold (7.3%), cytoskeletal molecules (20.6%), phosphorylation enzymes (10.6%), molecular chaperones (8.2%), and transmembrane proteins from the presynaptic membrane firmly attached to the ribbon (11.3%). The 3 CtBP isoforms represent the major protein in the ribbon whether calculated by molar amount (30%) or by mass (20%). The relatively high quantity of phosphorylation enzymes suggests a very active and regulated structure. The ribbon appears to comprise a concentrated cluster of proteins dealing with vesicle creation, retention and distribution, and consequent exocytosis.

Phosphorylation of syntaxin 3B by CaMKII regulates the formation of t-SNARE complexes

“…What could be the functional relevance of the phosphorylation of syntaxin 3B by CaMKII in ribbon synapses? Different studies have shown that ribbon synapses are enriched in CaMKII (Bronstein et al, 1988; Ullrich and Südhof, 1994) and CaMKII has also recently been identified in a ribbon associated complex of proteins (Uthaiah and Hudspeth, 2010; Kantardzhieva et al, 2012). CaMKII has been implicated in regulating synaptic signal transmission in conventional synapses on the presynaptic (reviewed in Wang, 2008) and post synaptic side (reviewed in Lisman et al, 2012).…”

“…CaMKII is part of a ribbon associated complex of proteins (Uthaiah and Hudspeth, 2010; Kantardzhieva et al, 2012) and presynaptic CaMKII is thought to be activated by the influx of Ca 2+ through voltage dependent Ca 2+ channels during synaptic activity (Gorelick et al, 1988). In ribbon synapses the majority of the channel mediated Ca 2+ influx is located to the proximity of the synaptic ribbon probably due to a clustering of voltage dependent Ca2+ channels at the ribbon (Matthews and Fuchs, 2010).…”

Calmodulin enhances ribbon replenishment and shapes filtering of synaptic transmission by cone photoreceptors

“…However, deletion of Munc13 in photoreceptors had no effect on vesicle tethering to the ribbon and only modest effects on synaptic transmission measured with ERG recordings (Cooper et al, 2012), pointing toward a limited involvement of Munc13 in vesicle replenishment in photoreceptors. Synaptotagmin (syt) 7 complexes with CaM to accelerate replenishment in hippocampal neurons (Liu et al, 2014), but there is no evidence for syt 7 at retinal ribbon synapses (Kantardzhieva et al, 2012). Another potential CaM target is Rab3a, a vesicle-associated protein involved in both long-and short-term plasticity (Castillo et al, 1997;Geppert et al, 1997;Nonet et al, 1997;Leenders et al, 2001;Schlüter et al, 2004Schlüter et al, , 2006 accelerate replenishment by quickening the transition of vesicles to release sites at the ribbon base.…”

C-terminus of Hsc70-interacting protein regulates C-terminal binding protein 2 and the expression of its target genes

“…Recently, CtBP2 and Hsp70 were shown to be associated, together with synaptic ribbons in photoreceptors and in hair cells [23]. CHIP is a co-chaperone E3 ligase that regulates the client proteins of Hsp70.…”