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. 2015 Sep 14;11(9):e1004407.
doi: 10.1371/journal.pcbi.1004407. eCollection 2015 Sep.

Dynamical Organization of Syntaxin-1A at the Presynaptic Active Zone

Free PMC article

Dynamical Organization of Syntaxin-1A at the Presynaptic Active Zone

Alexander Ullrich et al. PLoS Comput Biol. .
Free PMC article


Synaptic vesicle fusion is mediated by SNARE proteins forming in between synaptic vesicle (v-SNARE) and plasma membrane (t-SNARE), one of which is Syntaxin-1A. Although exocytosis mainly occurs at active zones, Syntaxin-1A appears to cover the entire neuronal membrane. By using STED super-resolution light microscopy and image analysis of Drosophila neuro-muscular junctions, we show that Syntaxin-1A clusters are more abundant and have an increased size at active zones. A computational particle-based model of syntaxin cluster formation and dynamics is developed. The model is parametrized to reproduce Syntaxin cluster-size distributions found by STED analysis, and successfully reproduces existing FRAP results. The model shows that the neuronal membrane is adjusted in a way to strike a balance between having most syntaxins stored in large clusters, while still keeping a mobile fraction of syntaxins free or in small clusters that can efficiently search the membrane or be traded between clusters. This balance is subtle and can be shifted toward almost no clustering and almost complete clustering by modifying the syntaxin interaction energy on the order of only 1 kBT. This capability appears to be exploited at active zones. The larger active-zone syntaxin clusters are more stable and provide regions of high docking and fusion capability, whereas the smaller clusters outside may serve as flexible reserve pool or sites of spontaneous ectopic release.

Conflict of interest statement

The authors have declared that no competing interests exist.


Fig 1
Fig 1. Syntaxin 1A forms larger clusters at active zones.
(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.
Fig 2
Fig 2. Computational model of Syntaxin-1A reproduces experimental results.
(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 Ea,Outside (ii) and Ea,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 et al.[6].
Fig 3
Fig 3. Degree of clustering influences Syntaxin-1A mobility and cluster dynamics.
(A) Influence of clustering on Syntaxin-1A mobility. i) The diffusion constant of the cluster center is inversely proportional to the number of particles in the cluster. ii) The distribution of step lengths of single particles in a time period of 200 μs shows a wide variety of diffusive behaviors. Slow diffusion (small step length) is likely caused by particles in a clustered state, while faster diffusion (large step lengths) indicate to freely diffusing particles. (B) Cluster dynamics in relation to clustering degree. i) The rate of particles dissociating from a cluster as a function of the respective cluster size. ii) The distribution of residence times of particles in a cluster. (C) i) A sample trajectory of one Syntaxin-1A particle over the course of one simulation run. ii) Step length, number of neighbors and the size of the cluster in which the particle resides tracked for the sample trajectory.
Fig 4
Fig 4. The differences in Syntaxin-1A mobility and cluster dynamics explain Syntaxin cluster function at specific locations.
(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.

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Deutsche Forschungsgemeinschaft (DFG) through Sonderforschungsbereich (SFB) 958, sponsored AU and MAB. European Commission through ERC starting grant "pcCell", sponsored FN. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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