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. 2023 May 15;34(6):ar51.
doi: 10.1091/mbc.E22-08-0372. Epub 2022 Dec 21.

An approach for quantitative mapping of synaptic periactive zone architecture and organization

Steven J Del Signore  1 , Margalit G Mitzner  1 , Anne M Silveira  1 , Thomas G Fai  2 , Avital A Rodal  1
Affiliations

An approach for quantitative mapping of synaptic periactive zone architecture and organization

Steven J Del Signore et al. Mol Biol Cell. .

Abstract

Following exocytosis at active zones, synaptic vesicle membranes and membrane-bound proteins must be recycled. The endocytic machinery that drives this recycling accumulates in the periactive zone (PAZ), a region of the synapse adjacent to active zones, but the organization of this machinery within the PAZ, and how PAZ composition relates to active zone release properties, remains unknown. The PAZ is also enriched for cell adhesion proteins, but their function at these sites is poorly understood. Here, using Airyscan and stimulated emission depletion imaging of Drosophila synapses, we develop a quantitative framework describing the organization and ultrastructure of the PAZ. Different endocytic proteins localize to distinct regions of the PAZ, suggesting that subdomains are specialized for distinct biochemical activities, stages of membrane remodeling, or synaptic functions. We find that the accumulation and distribution of endocytic but not adhesion PAZ proteins correlate with the abundance of the scaffolding protein Bruchpilot at active zones-a structural correlate of release probability. These data suggest that endocytic and exocytic activities are spatially correlated. Taken together, our results identify novel relationships between the exocytic and endocytic apparatus at the synapse and provide a new conceptual framework to quantify synaptic architecture.

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Figures

FIGURE 1:
FIGURE 1:
Endocytic proteins heterogeneously localize within the PAZ. (A) Schematic of the Drosophila NMJ prep. Left, cartoon illustration of the Drosophila larval fillet prep, with muscles highlighted in light red and the brain/motor neurons highlighted in green. Right, Example confocal image of a muscle 4 NMJ labeled with α-HRP. (B) Airyscan image of NMJ labeled with BRP (cyan), Nwk (magenta), and Dynamin (green). (C) Visualization of PAZ segmentation workflow. First, we generate maximum-intensity projections of boutons from the medial cross-section to the ventral surface of the neuron (i.e., the surface that is more deeply embedded in the muscle). To generate a composite segmentation, we normalize all channel intensities and add (PAZ) and subtract (active zone) channels to generate a single grayscale intensity map representing the NMJ. This image is then processed to enhance edge detection, and images are segmented by a watershed algorithm using local minima as seeds. For active zone object segmentation, local maxima are used as seeds followed by an additional distance transform watershed to separate touching objects. (D) The range of patterns of accumulation of endocytic proteins at the NMJ, as labeled. Note that Nwk and FasII form a more highly restricted mesh, Dynamin frequently intercalates into the core region, and Clc accumulates in a more punctate pattern. Right panels show example segmentations of the indicated channels into representative composite PAZ mesh. See Materials and Methods for more detail. (E) Schematic representation of a single PAZ mesh unit, divided into mesh and core regions. (F) Pearson R between the indicated pairwise comparisons within the mesh region indicates that colocalization of PAZ proteins is quite variable and ranges from low (∼0.2) to moderately high (∼0.6) but never approaches perfect or homogeneous colocalization. Letters above each group indicate which groups are significantly different from the given column at p < 0.05. Dots in F represent the average mesh value for a single image. Black dot and error bars indicate mean ± SD for all NMJs. For this and subsequent figures, see Supplemental Table S1 for a summary of number of animals, neurons, and PAZ units analyzed per experiment.
FIGURE 2:
FIGURE 2:
Endocytic proteins differentially distribute within the PAZ. (A) Cartoon illustrating potential ways that endocytic proteins might differentially accumulate—radially (from core to mesh), laterally (within the plane of the mesh region), or relatively more diffuse or punctate. (B) Example NMJ boutons labeled with BRP and single PAZ proteins to highlight differences in accumulation. (C, D) Radial profiles of BRP and PAZ proteins plotted against position from mesh edge (0) to core center (1), as depicted in the cartoon schematic. (C) Plots of mean normalized intensity by radial distance; BRP is inversely and more highly polarized compared with PAZ proteins. (D) Nwk and Dyn intensity (normalized min to max) plotted against radial position. Nwk is more highly polarized than Dyn, particularly at the core center. See Supplemental Figure S3 for all other pairwise profiles. (E) Quantification of the mesh ratio: the mean intensity in the mesh region divided by the mean intensity in the core, as depicted in the cartoon schematic. Nwk and FasII are significantly more enriched in the mesh than Dyn. (F) Quantification of ‘spottiness,” measured as the root mean squared of each image processed with a Laplacian of Gaussian filter. Higher values correspond to more punctate signal. (G) Cartoon summarizing radial distributions of PAZ and active zone proteins. Lines in C and D indicate the mean ± SD mesh profile from three to nine independent experiments per channel (see Supplemental Table S1 for details). $ in E indicates that BRP is significantly different from all other groups (p < 0.001). Dots in E and F represent the average mesh value for a single image; spot color indicates independent experimental replicates and does not correspond to colors in other figure legends. Black dot and error bars indicate mean ± SD for all NMJs.
FIGURE 3:
FIGURE 3:
Active zone position and pattern only partially define PAZ architecture. (A, B) Example observed and model patterning of BRP and composite mesh centroids. (A) Left, Example NMJ with composite segmentation labeled in cyan. Right, Modeled clustered, regularly spaced, and random spatial point distributions for the example NMJ compared with the observed composite mesh centroid pattern. (B) Quantification of Ripley’s g function shows that spatial patterning of BRP and composite segmentations are indistinguishable and best resemble a regularly spaced distribution. (C, D) Comparison of active zone and composite PAZ segmentations. Both Jaccard index (C) and centroid error (D) indicate that composite segmentations match active zone and individual PAZ meshes similarly well and significantly better than active zone–based segmentations. (E) Probability density estimates of mesh areas from composite-based segmentations, broken down by the number of active zones in the mesh. Active zone number correlates with mesh size. (F) Quantification of mesh area of composite meshes containing 0–3 active zones. The area of meshes significantly increases as active zone number increases. Dots in C and D represent the average value for a single image; black dot and error bars indicate mean ± SD for all images. Lines in B and E represent the mean ± SD per image (B) or experiment (E). Dots in F represent the average value for a single image; colors represent measurements made from independent experiments. Black dot and error bars indicate mean ± SD for all NMJs.
FIGURE 4:
FIGURE 4:
Synaptic endocytic protein architecture correlates with active zone structural properties. (A) Schematic of comparisons between active zone intensity and PAZ protein distributions. (B) Table summarizing the Pearson correlation between active zone integrated density and the indicated PAZ metrics. Notably, accumulations of all PAZ proteins except FasII in core and mesh regions generally correlate with active zone intensity. Mesh ratio exhibits a variable but generally negative correlation to active zone intensity. Additional metrics are shown in Supplemental File 1. (C–E) Comparison of core mean intensity (C), mesh mean intensity (D), and mesh ratio (E) for PAZ and active zone proteins binned by active zone intensity quantile. (C, D) Consistent with Pearson values, in most cases PAZ proteins exhibit significantly higher core means in the high active zone intensity bin, with FasII a notable exception. (E) Clc, Dap160, and Nwk show a clear decrease in mesh enrichment as active zone integrated density increases. Values in B represent the average Pearson R per experiment, calculated from individual meshes of all images. Dots in C–E represent the average value per image; color indicates the active zone integrated density bin. Black dot and error bars indicate mean ± SD of all NMJs.
FIGURE 5:
FIGURE 5:
Live imaging and STED microscopy validate measurements of PAZ architecture. (A, B) Maximum-intensity projection (A) and mid–bouton plane of a muscle 4 bouton labeled with endogenously tagged NwkHALO (JF549) and BRPMiMIC. (C, D) Quantifications of distribution of Nwk and BRP by radial profiling (C) and mesh ratio (D) show strong polarization of Nwk in the PAZ mesh and BRP in the PAZ core, consistent with observations in fixed tissues. (E, F) Comparison of Nwk and BRP intensity. Nwk accumulation and distribution correlate with BRP intensity, similar to results in fixed tissues. Correlations between BRP and Nwk CoV and Entropy differ slightly, suggesting that features of the Nwk distribution may be sensitive to fixation. (G–K) Imaging and analysis of PAZ architecture by STED microscopy. Nwk-BRP and Dyn-BRP comparisons were made in independent experiments. (G) Single plane 2D STED microscopy images of Nwk and BRP (top) or Dyn and BRP (bottom). (H, I) Radial distribution by radial profile (H) or mesh ratio (I) demonstrates that Nwk and Dyn are enriched in the PAZ mesh, while BRP is strongly enriched in the PAZ core, consistent with Airyscan microscopy. (J, K) Quantification of active zone intensity—PAZ relationships. Similar to Airyscan, Nwk and Dyn accumulation in both mesh and core correlates with active zone intensity.
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