ELKS1 localizes the synaptic vesicle priming protein bMunc13-2 to a specific subset of active zones.

Presynaptic active zones (AZs) are unique subcellular structures at neuronal synapses, which contain a network of specific proteins that control synaptic vesicle (SV) tethering, priming, and fusion. Munc13s are core AZ proteins with an essential function in SV priming. In hippocampal neurons, two different Munc13s-Munc13-1 and bMunc13-2-mediate opposite forms of presynaptic short-term plasticity and thus differentially affect neuronal network characteristics. We found that most presynapses of cortical and hippocampal neurons contain only Munc13-1, whereas ∼10% contain both Munc13-1 and bMunc13-2. Whereas the presynaptic recruitment and activation of Munc13-1 depends on Rab3-interacting proteins (RIMs), we demonstrate here that bMunc13-2 is recruited to synapses by the AZ protein ELKS1, but not ELKS2, and that this recruitment determines basal SV priming and short-term plasticity. Thus, synapse-specific interactions of different Munc13 isoforms with ELKS1 or RIMs are key determinants of the molecular and functional heterogeneity of presynaptic AZs.

The Munc13 proteins differentially regulate readily releasable pool dynamics and calcium-dependent recovery at a central synapse.

The Munc13 gene family encodes molecules located at the synaptic active zone that regulate the reliability of synapses to encode information over a wide range of frequencies in response to action potentials. In the CNS, proteins of the Munc13 family are critical in regulating neurotransmitter release and synaptic plasticity. Although Munc13-1 is essential for synaptic transmission, it is paradoxical that Munc13-2 and Munc13-3 are functionally dispensable at some synapses, although their loss in other synapses leads to increases in frequency-dependent facilitation. We addressed this issue at the calyx of Held synapse, a giant glutamatergic synapse that we found to express all these Munc13 isoforms. We studied their roles in the regulation of synaptic transmission and their impact on the reliability of information transfer. Through detailed electrophysiological analyses of Munc13-2, Munc13-3, and Munc13-2-3 knock-out and wild-type mice, we report that the combined loss of Munc13-2 and Munc13-3 led to an increase in the rate of calcium-dependent recovery and a change in kinetics of release of the readily releasable pool. Furthermore, viral-mediated overexpression of a dominant-negative form of Munc13-1 at the calyx demonstrated that these effects are Munc13-1 dependent. Quantitative immunohistochemistry using Munc13-fluorescent protein knock-in mice revealed that Munc13-1 is the most highly expressed Munc13 isoform at the calyx and the only one highly colocalized with Bassoon at the active zone. Based on these data, we conclude that Munc13-2 and Munc13-3 isoforms limit the ability of Munc13-1 to regulate calcium-dependent replenishment of readily releasable pool and slow pool to fast pool conversion in central synapses.

Munc13-independent vesicle priming at mouse photoreceptor ribbon synapses.

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.

Molecular dynamics of a presynaptic active zone protein studied in Munc13-1-enhanced yellow fluorescent protein knock-in mutant mice.

GFP (green fluorescent protein) fusion proteins have revolutionized research on protein dynamics at synapses. However, corresponding analyses usually involve protein expression methods that override endogenous regulatory mechanisms, and therefore cause overexpression and temporal or spatial misexpression of exogenous fusion proteins, which may seriously compromise the physiological validity of such experiments. These problems can be circumvented by using knock-in mutagenesis of the endogenous genomic locus to tag the protein of interest with a fluorescent protein. We generated knock-in mice expressing a fusion protein of the presynaptic active zone protein Munc13-1 and enhanced yellow fluorescent protein (EYFP) from the Munc13-1 locus. Munc13-1-EYFP-containing nerve cells and synapses are functionally identical to those of wild-type mice. However, their presynaptic active zones are distinctly fluorescent and readily amenable for imaging. We demonstrated the usefulness of these mice by studying the molecular dynamics of Munc13-1-EYFP at individual presynaptic sites. Fluorescence recovery after photobleaching (FRAP) experiments revealed that Munc13-1-EYFP is rapidly and continuously lost from and incorporated into active zones (tau1 approximately 3 min; tau2 approximately 80 min). Munc13-1-EYFP steady-state levels and exchange kinetics were not affected by proteasome inhibitors or acute synaptic stimulation, but exchange kinetics were reduced by chronic suppression of spontaneous activity. These experiments, performed in a minimally perturbed system, provide evidence that presynaptic active zones of mammalian CNS synapses are highly dynamic structures. They demonstrate the usefulness of the knock-in approach in general and of Munc13-1-EYFP knock-in mice in particular for imaging synaptic protein dynamics.