Distribution of AmBRP and Synapsin in the honeybee central brain.

<p>Optical sections of a honeybee central brain incubated with the anti-BRP^last200 and anti-SYNORF1 to visualize the presynaptic proteins AmBRP and Synapsin. <b>a</b> Longitudinal section through a schematic honeybee brain showing ventrally located regions (nomenclature after Ito et al. (2014) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0175894#pone.0175894.ref034" target="_blank">34</a>]). <b>b-d</b> Distribution of BRP^last200 signals (b) and anti-SYNORF1 signals (c) in ventrally located brain regions of a 29-day-old bee. Both antibodies show staining in all brain regions with almost similar distribution (d). Prominent stained regions are the vertical lobes and the antennal lobes. <b>e</b> Longitudinal section through a schematic honeybee brain showing dorsally located regions (nomenclature after Ito et al. (2014) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0175894#pone.0175894.ref034" target="_blank">34</a>]).<b>f-h</b> Distribution of BRP^last200 signals (f) and anti-SYNORF1 signals (g) in dorsally located brain regions of a 29-day-old bee. Both antibodies show staining in all brain regions with similar distribution (h) Prominent stained regions are the peduncles, especially in the AmBRP staining. LCA, lateral calyx; MCA, medial calyx; VL, vertical lobe; PED, peduncle; ML, medial lobe; CB, central body; AL, antennal lobe; NA, neuraxis anterior; M, medial; NP, neuraxis posterior; L, lateral. Scale bars: 200 μm for b-d and f-h.</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

Primary antibodies used in this study.

<p>Primary antibodies used in this study.</p

AmBRP variants detected in the honeybee brain.

<p><b>a</b> Map of the BRP^last200 and the BRP^D2 antibodies’ epitopes in the large <i>Drosophila</i> BRP isoforms D (NP_724796). <b>b</b> Immunoblot of honeybee central brain and fruit fly head homogenate. The BRP^last200 antibody and the BRP^D2 antibody recognize two bands around 220 kDa in honeybee central brains (lane 2, 4), and two major bands and several light bands between 120 and 220 kDa in fruit fly heads (lane 3, 5).</p

The median number of anti-BRP^last200- and anti-SYNORF1-positive pixels per ROI varies with age in lip and collar.

<p><b>a</b> The median number of anti-BRP^last200-positive (anti-BRP) pixels per ROI in the collar is higher in 43-day-old bees compared with 1-, 8- and 15-day-old bees. <b>b</b> The median number of anti-SYNORF1-positive (anti-SYNORF1) pixels per ROI in the collar is lower in 43-day-old bees compared with 1- and 15-day-old bees. <b>c</b> The ratio of anti-BRP^last200-positive pixels to anti-SYNORF1-positive pixels per ROI in the collar is higher in 43-day-old bees compared with 1-, 8- and 15-day-old bees. <b>d</b>. 1-day- and 43-day-old bees differ in their median number of anti-BRP^last200-positive pixels per ROI in the collar. <b>e</b> The median number of anti-SYNORF1-positive pixels did not change with age in the lip. <b>f</b> The ratio of anti-BRP^last200-positive pixels to anti-SYNORF1-positive pixels per ROI in the lip is higher in 8-day-old bees compared with 1-day-old bees and in 43-day old bees compared with 15- and 29-day-old bees. Box blots show median, 25% and 75% quartiles and value range (min-max). (*) Significant differences (p < 0.05) detected with Mann-Whitney U test after Kruskal Wallis ANOVA.</p

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

AmBRP is predominantly located in the vicinity of the membrane of presynaptic boutons within microglomeruli in the mushroom body calyces.

<p><b>a-c</b> Confocal images of the medial calyx showing BRP^last200 staining (AmBRP, green, b) in combination with Phalloidin staining (F-actin, magenta, a) and an anti-SYNORF1 counterstaining (Synapsin, blue, a) to visualize pre- and postsynaptic structures in a 8-day-old bee. The calyx can be subdivided into three regions, lip, collar and basal ring. Experiments focused on the lip and the dense region of the collar (dCO). <b>d</b> Schematic representation of a microglomerulus (MG) (modified after [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0175894#pone.0175894.ref035" target="_blank">35</a>] showing already established pre- and postsynaptic marker (Synapsin, blue; F-actin, magenta). The bouton of a projection neuron is surrounded by spines from Kenyon cell dendrites. Anti-SYNORF1 labels the vesicle-associated protein Synapsin (blue) whereas Alexa Fluor 546 Phalloidin binds to F-actin located in dendritic spines (magenta). <b>e-k</b> Confocal images of MG in the dense collar region with labeled Synapsin (e), F-actin (f) and AmBRP (h). The F-actin signals form circles around Synapsin signals (g). AmBRP is located predominantly at the outer rim of the Synapsin-labeled signals and at the inner rim of the F-actin signals (i-k). The insets show a single, magnified MG from the corresponding image. LI, lip; CO, collar; BR, basal ring; dCO, dense collar; PN, projection neuron; KC, Kenyon cell.Scale bars: 20 μm for a-c, 2 μm for e-k, 1 μm for insets.</p