Stable Positioning of Unc13 Restricts Synaptic Vesicle Fusion to Defined Release Sites to Promote Synchronous Neurotransmission

Abstract

Neural information processing depends on precisely timed, Ca-activated synaptic vesicle exocytosis from release sites within active zones (AZs), but molecular details are unknown. Here, we identify that the (M)Unc13-family member Unc13A generates release sites and show the physiological relevance of their restrictive AZ targeting. Super-resolution and intravital imaging of Drosophila neuromuscular junctions revealed that (unlike the other release factors Unc18 and Syntaxin-1A) Unc13A was stably and precisely positioned at AZs. Local Unc13A levels predicted single AZ activity. Different Unc13A portions selectively affected release site number, position, and functionality. An N-terminal fragment stably localized to AZs, displaced endogenous Unc13A, and reduced the number of release sites, while a C-terminal fragment generated excessive sites at atypical locations, resulting in reduced and delayed evoked transmission that displayed excessive facilitation. Thus, release site generation by the Unc13A C terminus and their specific AZ localization via the N terminus ensure efficient transmission and prevent ectopic, temporally imprecise release.

Active Zone Scaffold Protein Ratios Tune Functional Diversity across Brain Synapses

“…A very similar pheno-type was observed in flies overexpressing an N-terminal fragment of Unc13A (Figure S5), which lacks the catalytic MUN domain, in ORNs. We have previously shown that this fragment displays a dominant-negative phenotype at NMJ synapses (Reddy-Alla et al, 2017). These data provide independent evidence that Unc13A promotes a fast, transient component of SV release at the ORN-to-PN synapse.…”

Subsynaptic spatial organization as a regulator of synaptic strength and plasticity

“…Combined pHuse and live-cell, single-molecule imaging of expressed RIM1 found that AZ subregions with high RIM1 density were the preferred sites of vesicle fusion after an action potential [2**]. In the glutamatergic Drosophila neuromuscular junction, (M)Unc13 distribution measured by STED aligned with functional synaptic vesicle release sites as measured by postsynaptic Ca 2+ sensor GCaMP [8**]. In particular, the isoform Unc13-A appeared critical for evoked release, as deletion of the isoform resulted in drastic reduction in evoked release stemming from a reduced number of vesicles docked close to Ca 2+ channels [9].…”

Regulation of synaptic release‐site Ca2+ channel coupling as a mechanism to control release probability and short‐term plasticity

“…Additionally, the essential release factors Sec1/ (M)Unc18 and (M)Unc13 cooperate with the SNAREs, and are essential for transmitter release across species (UNC13 and UNC18 are the gene names, in the cases of the proteins, 'M' stands for 'mammalian' while the proteins are calledlike the genes -Unc13/Unc18 (Uncoordinated 13/18) in D. melanogaster and C. elegans; for an overview of AZs in different species and differences in protein nomenclature see [2,14]) [7,[15][16][17][18][19][20][21]. It was recently shown that the precise localization of (M)Unc13 proteins at and within the AZ is particularly important, because they mark the exact SV release site [22][23][24].…”

“…3), the number, and position of release sites, pV r , and STP [43,98,99,[109][110][111][112][113][114][115][116]. A likely function of this core scaffold is the AZ localization of (M)Unc13s to generate SV-release sites [22,51,53,117]. Indeed, loss of RIM and either RBP or ELKS in mammals, or of BRP and RBP in Drosophila resulted in loss of (M)Unc13 isoforms, loss of membrane docked SVs (Fig.…”