Neurotransmitter release: a docking role for UNC‐13 proteins (Commentary on Siksou et al.)

The functioning of a synapse strongly relies on the spatially and temporally precise release of neurotransmitter at the active zone of the presynaptic membrane. Fusion of transmitter-containing synaptic vesicles (SVs) with the presynaptic membrane is triggered within less than one millisecond upon arrival of a depolarizing action potential. In a seminal article, sequential steps of a cycle of membrane trafficking events in the presynaptic bouton – the Sv cycle – have been defined to describe the loading with neurotransmitter, tethering and docking, priming, Cadependent fusion, compensatory endocytosis and recycling of SV (Sudhof, 1995). Recently, however, the view of a docking–priming–fusion pathway with discrete steps has been debated (Wojcik & Brose, 2007; Verhage & Sorensen, 2008). In this issue of EJN, Siksou et al. (2009) describe an experiment that utilized high-pressure freezing of native tissue combined with electron tomography. The results provide strong evidence for the view that SV docking, defined as the close physical attachment of vesicles to the presynaptic membrane, and SV priming, defined as vesicles gaining fusion competence, represent two sides of the same coin and these two processes cannot be dissociated at the molecular level of analysis. Their data indicate that UNC-13 proteins, long known as priming factors for SVs, are simultaneously involved in the docking of SVs. The indispensable role of UNC-13 ⁄ Munc13 proteins for SV exocytosis was established on the basis of the characterization of mutant Caenorhabditis elegans, Drosophila melanogaster and mice which exhibit a complete arrest of spontaneous and evoked vesicular release, i.e. the loss of the release-ready SV pool. However, the number of morphologically docked vesicles is virtually unchanged in these mutants (Aravamudan et al., 1999; Augustin et al., 1999; Richmond et al., 1999; Varoqueaux et al., 2002). Therefore UNC-13 proteins were postulated to function downstream of vesicle docking as ‘priming factors’, to ensure that docked SVs become fusion-competent and thus sensitive to Ca influx through voltage-gated calcium channels situated in presynaptic membrane. These findings supported the view that docking and priming events occur consecutively and rely on separate molecular mechanisms. Technological progress now permits the reexamination of this issue. High-pressure freezing for electron microscopy circumvents the artifacts of tissue shrinkage caused by aldehyde fixatives and allows the examination of samples in a more native state (Rostaing et al., 2006). In the present study, Siksou et al. (2009) have reinvestigated Munc13-1 ⁄ 2 double knock-out (DKO) mice using this method and combining it with electron tomography. Munc13 DKO mice were previously described to form ultra-structurally ‘normal’ synapses in the hippocampus, which, however, remain completely silent (Varoqueaux et al., 2002). The authors now find a complete loss of docked vesicles in absence of the two Munc13 isoforms. Rather, SVs remain situated a short distance away from the active zonal membrane. Importantly, this finding reconciles discrepancies between the original findings and recent studies on the neuromuscular junction of C. elegans unc-13 mutants which reported a dramatic drop of morphologically docked SV numbers in the absence of UNC-13 (Gracheva et al., 2006; Weimer et al., 2006; Hammarlund et al., 2007). Furthermore, the findings of Siksou et al. (2009) clarify the interpretation of molecular mechanisms underlying the tethering–docking–priming– fusion pathway. The active zone harbors a complex network of proteins involved in the performance and regulation of these processes, and UNC13 ⁄ Munc13 is tightly embedded into this network (Fejtova & Gundelfinger, 2006; Schoch & Gundelfinger, 2006). Based on interactions between the Rab3-interacting molecule RIM, the target membrane SNARE proteins syntaxin and SNAP-25 and ⁄ or tomosyn, a negative regulator of SNARE-complex formation, UNC-13 regulates fusion-competence of SVs (Gracheva et al., 2006; Weimer et al., 2006; Hammarlund et al., 2007) and, as is deduced from the evidence described by Siksou et al. (2009), their physical docking. The question remains as to whether tethering and docking – terms that have been considered to refer to identical processes – represent distinct steps of the SV cycle. In Munc13-deficient synapses, Siksou et al. (2009) found that the number of SVs within the first 40 nm from the active zone is unchanged. Furthermore, they observed small filaments connecting vesicles to the plasma membrane. These findings suggest that prior to physical docking to their release sites, these vesicles are tied in the vicinity of the active zone. Indeed, findings from experiments in an endocrine cell line argue for a discrete 20-nm step towards the cell membrane of stimulated vesicles shortly before exocytosis (Karatekin et al., 2008). Candidate molecules for a tethering function have been identified. They include RIM ⁄ UNC-10 proteins (Weimer et al., 2006; Gracheva et al., 2008) as well as UNC-18 ⁄ Munc18 (Toonen et al., 2006). However, both molecules have also been implicated in processes downstream of tethering (Schoch & Gundelfinger, 2006; Wojcik & Brose, 2007). The present evidence described by Siksou et al. (2009) makes an important contribution to the field. However, we still do not understand how the various concepts of a tethering–docking–priming–fusion pathway for SVs can be incorporated into the protein network at the active zone. European Journal of Neuroscience, Vol. 30, pp. 47–48, 2009 doi:10.1111/j.1460-9568.2009. 06832.x