A high affinity RIM-binding protein/Aplip1 interaction prevents the formation of ectopic axonal active zones

Synaptic vesicles (SVs) fuse at active zones (AZs) covered by a protein scaffold, at Drosophila synapses comprised of ELKS family member Bruchpilot (BRP) and RIM-binding protein (RBP). We here demonstrate axonal co-transport of BRP and RBP using intravital live imaging, with both proteins co- accumulating in axonal aggregates of several transport mutants. RBP, via its C-terminal Src-homology 3 (SH3) domains, binds Aplip1/JIP1, a transport adaptor involved in kinesin-dependent SV transport. We show in atomic detail that RBP C-terminal SH3 domains bind a proline-rich (PxxP) motif of Aplip1/JIP1 with submicromolar affinity. Pointmutating this PxxP motif provoked formation of ectopic AZ-like structures at axonal membranes. Direct interactions between AZ proteins and transport adaptors seem to provide complex avidity and shield synaptic interaction surfaces of pre-assembled scaffold protein transport complexes, thus, favouring physiological synaptic AZ assembly over premature assembly at axonal membranes. - See more at: http://elifesciences.org/content/4/e06935#sthash.oVGZ8cdi.dpu

Presynaptic spinophilin tunes neurexin signalling to control active zone architecture and function

Assembly and maturation of synapses at the Drosophila neuromuscular junction (NMJ) depend on trans-synaptic neurexin/neuroligin signalling, which is promoted by the scaffolding protein Syd-1 binding to neurexin. Here we report that the scaffold protein spinophilin binds to the C-terminal portion of neurexin and is needed to limit neurexin/neuroligin signalling by acting antagonistic to Syd-1. Loss of presynaptic spinophilin results in the formation of excess, but atypically small active zones. Neuroligin-1/neurexin-1/Syd-1 levels are increased at spinophilin mutant NMJs, and removal of single copies of the neurexin-1, Syd-1 or neuroligin-1 genes suppresses the spinophilin-active zone phenotype. Evoked transmission is strongly reduced at spinophilin terminals, owing to a severely reduced release probability at individual active zones. We conclude that presynaptic spinophilin fine-tunes neurexin/neuroligin signalling to control active zone number and functionality, thereby optimizing them for action potential-induced exocytosis

Syntaxin 1A forms larger clusters at active zones.

<p>(A) STED images of a Drosophila neuromuscular junction (NMJ), co-stained for Bruchpilot (left, green) andSyntaxin-1A (middle, red), and their overlay (right). Top row (15 μm x 15 μm): Syntaxin-1A is abundant over the entire NMJ and Bruchpilot forms ring-like structures. The middle row (1.75 μm x 1.75 μm): zoom showing seven active zones indicated by the Bruchpilot rings. Syntaxin-1A appears in patchy structures identifying clusters. Bottom row (0.5 μm x 0.5 μm): zoom showing one Bruchpilot ring. Syntaxin-1A micro-domains situated beneath or near the Bruchpilot ring structure. The active zone region as defined here is shown as shaded region. (B) Illustration of an active zone model showing the Bruchpilot and Syntaxin-1A cluster positions as observed in the STED images. (C) Analysis of the Syntaxin-1A cluster size with respect to their position towards the active zone. The cluster size distribution of identified Syntaxin-1A clusters, with cluster sizes defined by the diameter of the full width half maximum area. The distribution for whole NMJs (All) as well as the distributions in Syntaxin-1A clusters at (at AZ) and outside of active zones (Outside) are shown. (D) Syntaxin cluster size as a function of their distance to the nearest active zone (BRP ring structure). Boxplots show the median and distribution of cluster sizes for 8 distance ranges. Asterisks indicate degree of statistical significance and are inferred from the probability (P-Value) of the difference in means using a T-test, * = P<0.05, ** = P<0.01, *** = P<0.001. Asterisks are attached to bars which indicate the corresponding pair being compared in the T-test. Notches indicate 95% confidence interval for the median. The number of clusters within a specific range is shown inside the boxplot. (E) The fluorescence intensity of Syntaxin (red channel) for active zones (green channel intensity above zero) and outside active zones. (F) The density of clusters at the active zone compared with that outside of active zones. The number of clusters for a specific location is shown inside the boxplot.</p

The differences in Syntaxin-1A mobility and cluster dynamics explain Syntaxin cluster function at specific locations.

<p>(A) Lower degree of clustering allows faster membrane exploration and aggregation at target sites. i) Progress of membrane coverage (percentage of visited area). ii) Density plots (upper row) and step length plots (lower row) of one sample simulation run for four different degrees of clustering. iii) The aggregation time of clusters at a newly formed target site as a function of cluster size. (B) Higher degree of clustering enables the formation of more SNARE complexes leading to higher fusion probability. i) Histogram of docking candidates, i.e. the number of all Syntaxin particles in a vesicle-sized area. ii) Histogram of SNARE candidates, i.e. the number of free Syntaxin particles and Syntaxins on the cluster rim. Candidates are tracked in an area with the size of a synaptic vesicle. iii) Positions of SNARE candidates superimposed on density plots. Areas with many SNARE candidates are at the periphery of clusters.</p

Computational model of Syntaxin-1A reproduces experimental results.

<p>(A) Schematic presentation of the computational model of Syntaxin-1A cluster formation. The ReaDDy two-particle model of Syntaxin (top left) with the “membrane” potential profile (top right) and the SNARE-SNARE attraction/clustering potential (bottom left) and the starting topology of 500 Syntaxins on a circular area with 300 nm radius (bottom right). (B) Cluster size distributions for different potential well depths (i-iv) show strong differences in the clustering behavior. The simulated cluster size distributions for the two potential parameters E_a,Outside (ii) and E_a,AZ (iii) correspond well to the experimental cluster size distributions found outside (CSD-Outside) and at active zones (CSD-AZ) shown as dashed lines in ii and iii, respectively. (C) Line-plot showing the average cluster size and the fraction of “single” syntaxins with respect to the potential strength parameter Ea, also indicated are average cluster size of active zone and outside region from the experimental STED data (dashed lines). (D) Recovery curves of simulated FRAP experiments for the two selected potential strengths compared with experimentally derived FRAP curve from Fig 4B in Sieber <i>et al</i>.[<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004407#pcbi.1004407.ref006" target="_blank">6</a>].</p

Unc13A dynamically stabilizes vesicle priming at synaptic release sites for short-term facilitation and homeostatic potentiation

Summary: Presynaptic plasticity adjusts neurotransmitter (NT) liberation. Short-term facilitation (STF) tunes synapses to millisecond repetitive activation, while presynaptic homeostatic potentiation (PHP) of NT release stabilizes transmission over minutes. Despite different timescales of STF and PHP, our analysis of Drosophila neuromuscular junctions reveals functional overlap and shared molecular dependence on the release-site protein Unc13A. Mutating Unc13A’s calmodulin binding domain (CaM-domain) increases baseline transmission while blocking STF and PHP. Mathematical modeling suggests that Ca2+/calmodulin/Unc13A interaction plastically stabilizes vesicle priming at release sites and that CaM-domain mutation causes constitutive stabilization, thereby blocking plasticity. Labeling the functionally essential Unc13A MUN domain reveals higher STED microscopy signals closer to release sites following CaM-domain mutation. Acute phorbol ester treatment similarly enhances NT release and blocks STF/PHP in synapses expressing wild-type Unc13A, while CaM-domain mutation occludes this, indicating common downstream effects. Thus, Unc13A regulatory domains integrate signals across timescales to switch release-site participation for synaptic plasticity