Munc13-Independent Vesicle Priming at Mouse Photoreceptor Ribbon Synapses

Abstract

Munc13 proteins are essential regulators of exocytosis. In hippocampal glutamatergic neurons, the genetic deletion of Munc13s results in the complete loss of primed synaptic vesicles (SVs) in direct contact with the presynaptic active zone membrane, and in a total block of neurotransmitter release. Similarly drastic consequences of Munc13 loss are detectable in hippocampal and striatal GABAergic neurons. We show here that, in the adult mouse retina, the two Munc13-2 splice variants bMunc13-2 and ubMunc13-2 are selectively localized to conventional and ribbon synapses, respectively, and that ubMunc13-2 is the only Munc13 isoform in mature photoreceptor ribbon synapses. Strikingly, the genetic deletion of ubMunc13-2 has little effect on synaptic signaling by photoreceptor ribbon synapses and does not prevent membrane attachment of synaptic vesicles at the photoreceptor ribbon synaptic site. Thus, photoreceptor ribbon synapses and conventional synapses differ fundamentally with regard to their dependence on SV priming proteins of the Munc13 family. Their function is only moderately affected by Munc13 loss, which leads to slight perturbations of signal integration in the retina.

Functional Roles of Complexin 3 and Complexin 4 at Mouse Photoreceptor Ribbon Synapses

“…It is generally assumed that unique molecular constituents facilitate the extraordinary performance of photoreceptor ribbon synapses. Such constituents include the ribbon synapse specific proteins RIBEYE (Schmitz et al, 2000;Maxeiner et al, 2016) and Piccolino (Regus-Leidig et al, 2013, 2014a, the active zone protein ubMunc13-2 (Cooper et al, 2012), and Cplx3 and Cplx4 (Reim et al, 2005). Conventional chemical synapses contain the essential SNARE regulators Cplx1 and Cplx2 (Reim et al, 2001;Brose, 2008).…”

Nonconserved Ca2+/Calmodulin Binding Sites in Munc13s Differentially Control Synaptic Short-Term Plasticity

“…However, we could not test this hypothesis, because Ca 2ϩ -phosphate transfection of primary neurons results in very low transfection efficiencies that are incompatible with Western blot detection. Moreover, immunocytochemical comparison using a bMunc13-2 antibody (52) was inconclusive due to somatic background staining in wild-type and Munc13-1/2 DKO neurons (not shown) similar to that observed in the retina (10), and immunostaining of the C-terminal VENUS tag would be masked by the free EGFP derived from an EGFP cassette in the pCAGIG plasmid. In any case, the analysis of amplitude-matched neurons provided a sub-data set in which the characteristics of basal neurotransmission mediated by bMunc13-2 WT and bMunc13-2 CaM* were comparable and enabled us to dissect the specific effect of CaM binding on STP.…”

“…While Munc13-1 is expressed throughout the rodent brain, Munc13-2 and Munc13-3 exhibit distinct and neuronal subtypespecific expression patterns and likely modulate neurotransmitter release in concert with Munc13-1 (2,3,10,42). However, the priming function of bMunc13-2 and Munc13- 3 has not yet been proven experimentally.…”

Unconventional molecular regulation of synaptic vesicle replenishment in cochlear inner hair cells

“…Therefore, we performed immunostainings with extensively tested antibodies for all Munc13 and CAPS isoforms (Cooper et al, 2012) on acutely dissected organs of Corti from hearing P16-P17 WT mice (Fig. 3).…”

“…S1A) were used for immunolabellings: polyclonal rabbit anti-Munc13-1, anti-bMunc13-2 (brain-specific Munc13-2 isoform), anti-ubMunc13-2 (ubiquitous Munc13-2 isoform), anti-Munc13-3 antibodies (Cooper et al, 2012;Varoqueaux et al, 2005), anti-CAPS1, anti-CAPS2 (Synaptic Systems), guinea pig antibody against both synapsin 1 and 2 (Synaptic Systems), mouse anti-calbindin D28k (Swant) and mouse or rabbit anti-otoferlin antibodies (Abcam/Synaptic Systems), detected by species-specific secondary anti-IgG antibodies conjugated to AlexaFluorH 488, 568 and 647 (Life technologies). PhalloidinAlexaFluorH568 (Life technologies) was used to label actincontaining stereocilia.…”