Distinct SAP102 and PSD-95 nano-organization defines multiple types of synaptic scaffold protein domains at single synapses

Abstract

The MAGUK family of scaffold proteins plays a central role in maintaining and modulating synaptic signaling, providing a framework to retain and position receptors, signaling molecules, and other synaptic components. Of these scaffold proteins, SAP102 and PSD-95 are essential for synaptic function at distinct developmental timepoints and perform overlapping as well as unique roles. While their similar structures allow for common binding partners, SAP102 is expressed earlier in synapse development and is required for synaptogenesis, whereas PSD-95 expression peaks later in development and is associated with synapse maturation. PSD-95 and other key synaptic proteins organize into subsynaptic nanodomains that have a significant impact on synaptic transmission, but the nanoscale organization of SAP102 is unknown. How SAP102 is organized within the synapse, and how it relates spatially to PSD-95 on a nanometer scale, could impact how SAP102 clusters synaptic proteins and underlie its ability to perform its unique functions. Here we used DNA-PAINT super-resolution microscopy to measure SAP102 nano-organization and its spatial relationship to PSD-95 at individual synapses. We found that like PSD-95, SAP102 accumulates in high-density subsynaptic nanoclusters. However, SAP102 nanoclusters were smaller and denser than PSD-95 nanoclusters across development. Additionally, only a subset of SAP102 nanoclusters co-organized with PSD-95, revealing that within individual synapses there are nanodomains that contain either one or both proteins. This organization into both shared and distinct subsynaptic nanodomains may underlie the ability of SAP102 and PSD-95 to perform both common and unique synaptic functions.

Figure 1:. SAP102 is organized into high-density subsynaptic nanoclusters with distinct properties.

Outliers removed to aid visualization for violin plots, full data set is used for statistics A. (Left) Example stretch of DNA-PAINT super-resolved dendrite with both SAP102 (magenta) and PSD-95 (green). Scale bar: 2.5 mm (Right) Magnified views of selected synaptic puncta indicated by white boxes. Synapses 1–3 contain both SAP102 (magenta) and PSD-95 (green) while synapse 4 contains only PSD-95 and synapse 5 contains only SAP102. Scale bar: 200 nm. B. Synaptic puncta area for SAP102 and PSD-95. (SAP102: 0.044 ± 0.001 mm², n=1057 synapses; PSD-95: 0.071 ± 0.001 mm², n=1293 synapses; p<0.0001, unpaired t-test) C. Overall synaptic puncta localization density for SAP102 and PSD-95. (SAP102: 0.009 ± 0.0001 locs/nm², n=1057 synapses; PSD-95: 0.007 ± 0.0001 locs/nm², n=1293 synapses; p<0.0001, unpaired t-test) D. Autocorrelation of SAP102 and PSD-95 distributions. E. Example localization maps for SAP102 (left) and PSD-95 (right). Colormap indicates local density around each localization. Red circles indicate high density areas that were identified as NCs based on the NC detection algorithm used. F. Number of NCs per synapse for SAP102 and PSD-95. (SAP102: 1.856 ± 0.0366, n=1057 synapses; PSD-95: 1.837 ± 0.0309, n=1293 synapses; p=0.684, unpaired t-test) G. Relationship between number of NCs and synaptic puncta area for SAP102 and PSD-95 (SAP102: slope=24.82, PSD-95: slope=15.39, p<0.0001). H. Autoenrichment analysis for both SAP102 and PSD-95. I. NC area based on the NC detection algorithm for SAP102 NCs and PSD-95 NCs. (SAP102: 1953 ± 62.95 nm², n=1962 NCs; PSD-95: 3197 ± 79.54 nm², n=2375 NCs; p<0.0001, unpaired t-test) J. Fraction of the synaptic puncta occupied by NCs for SAP102 and PSD-95. (SAP102: 0.081 ± 0.002, n=1057 synapses; PSD-95: 0.08 ± 0.001, n=1293 synapses; p<0.0001, unpaired t test) K. Internal NC localization density for SAP102 and PSD-95. (SAP102: 0.058 ± 0.001 locs/nm², n=1962 NCs; PSD-95: 0.042 ± 0.0006 locs/nm², n=2375 NCs; p<0.0001, unpaired t-test) L. The difference between the overall synaptic density and NC internal density (Ddensity) for SAP102 and PSD-95. (SAP102: 0.033 ± 0.001 locs/nm², n=1057 synapses; PSD-95: 0.022 ± 0.0004 locs/nm², n=1293 synapses; p<0.0001, unpaired t-test) M. Schematic representing the key features in the subsynaptic organization of SAP102 (magenta) and PSD-95 (green). Circles represent molecules of each protein, the overall synaptic puncta area is defined by the solid line, and NCs boundaries are indicated by dashed lines.

Figure 2:. SAP102 and PSD-95 nanostructure is impacted by the presence of the other protein

A. Synaptic puncta area for SAP102 (magenta) and PSD-95 (green) at synapses with both proteins present (paired) or just one of the proteins (alone) (See Table 2-1 for statistics). B. Synaptic puncta density for SAP102 and PSD-95 at paired and alone synapses (See Table 2-1 for statistics). C. Left: autocorrelation analysis for SAP102 at paired (light magenta) and alone (dark magenta) synapses. Right: PSD-95 autocorrelation analysis at paired (light green) and alone (dark green) synapses. D. Autoenrichment analysis for SAP102 at paired and alone synapses (left) and PSD-95 at paired and alone synapses (right). E. Number of NCs per synapse for SAP102 and PSD-95 at paired and alone synapses (See Table 2-1 for statistics). F. NC area for SAP102 and PSD-95 at paired and alone synapses (See Table 2-1 for statistics). G. Relationship between number of NCs and synaptic puncta area for SAP102 (left; SAP102 paired: slope=24.62, SAP102 alone: slope=26.82, p=0.417) and PSD-95 (right; PSD-95 paired: slope=15.00, PSD-95 alone: slope=12.24, p=0.0106) at both paired and alone synapses. H. Linear regression lines describing the relationship between NC number and synaptic puncta area for SAP102 and PSD-95, reproduced from 2G for clarity. I. Area of the synapse incorporated into NCs for SAP102 and PSD-95 at paired and alone synapses (See Table 2-1 for statistics). J. NC internal density for SAP102 and PSD-95 at paired and alone synapses (See Table 2-1 for statistics). K. Ddensity for SAP102 and PSD-95 at paired and alone synapses (See Table 2-1 for statistics). L. Schematic representing the differences in subsynaptic organization between SAP102 (magenta) alone vs paired and PSD-95 (green) alone vs paired. Circles represent molecules of each protein, the overall synaptic puncta area is defined by the solid line, and NC boundaries are indicated by dashed lines.

Figure 3:. SAP102 and PSD-95 occupy both distinct and overlapping subsynaptic nanodomains.

A. Example synapse containing both PSD-95 (green) and SAP102 (magenta). Gray area is the intersection area where the proteins’ synaptic puncta overlap. Blue line indicates the total synapse area, defined by overlaying and combining the SAP102 and PSD-95 synaptic puncta. Scale bar is 100 nm. B. Percentage of the total combined synaptic area occupied by SAP102 and PSD-95. Outliers removed to aid visualization for violin plots, full data set is used for statistics. (SAP102: 52.84 ± 0.783 % of total area, n=602 synapses; PSD-95: 83.33 ± 0.616 % of total area, n=602 synapses; p<0.0001, unpaired t-test) C. Percent of SAP102, PSD-95, and total synaptic areas that contain both SAP102 and PSD-95 (intersection area). (SAP102: 79.53 ± 0.716 % overlap, n=602 synapses; PSD-95: 49.99 ± 0.785 % overlap, n=602 synapses; total: 41.36 ± 0.593 % overlap, n=602 synapses; p<0.0001, unpaired t-test) D. Example synapses with heat map indicating local density for each protein’s localizations (PSD-95: left and SAP102: middle) each with detected NCs for the other protein indicated (left: SAP102 NC in magenta; middle: PSD-95 NCs in green). Right: SAP102 (magenta) and PSD-95 (green) localizations with NCs for each protein overlaid. E. SAP102 and PSD-95 cross-enrichment with respect to NC centers of the other protein. Right: schematic illustrating the cross-enrichment analysis. F. Distribution of cross-enrichment indices scaled to NC area for each protein’s NCs. G. Schematic representing the distribution of SAP102 (magenta) and PSD-95 (green) within individual synapses. Circles represent molecules of each protein; the overall synaptic puncta area is defined by the solid line, and NCs boundaries are indicated by dashed lines.

Figure 4:. SAP102 and PSD-95 nanostructure each change cross development, yet their nanoscale spatial relationship remains largely consistent.

*Throughout this figure- the 2 week in vitro dataset is the same dataset as in the previous figures. A. Example rendered synapses from 1, 2, and 3 weeks in vitro. Scalebar is 100 nm. B. Percentage of synapses with SAP102 alone (magenta), PSD-95 alone (green), or both proteins (blue) across development. C. Synaptic puncta area at both paired and alone synapses across development for SAP102 (left) and PSD-95 (right). D. Fraction of total synaptic puncta area occupied by SAP102 (left) or PSD-95 (right) across development. E. Fraction of each protein’s puncta area that overlaps with the other protein (intersection area) for SAP102 (left) or PSD-95 (right) across development. F. NC number at both paired and alone synapses across development for SAP102 (left) and PSD-95 (right). G. NC area at both paired and alone synapses across development for SAP102 (left) and PSD-95 (right). H. Ddensity at both paired and alone synapses across development for SAP102 (left) and PSD-95 (right). I. SAP102 (left) and PSD-95 (right) cross-enrichment with respect to NC centers of the other protein for both paired and alone synapses across development. J. Distribution of cross-enrichment indices scaled to NC area for each protein’s NCs for SAP102 (left) and PSD-95 (right) across development.

Figure 1–1

A. Example super-resolved synapses demonstrating co-localization between bassoon (left) and SAP102 (middle). B. Distribution of overall SAP102 puncta area at puncta that co-localized with bassoon and those that did not. Dashed line indicated cutoff value. C. Percentage of SAP102 puncta that overlapped with bassoon without the area cutoff applied and with the area cutoff applied. D. Example super-resolved synapses demonstrating co-localization between bassoon (left) and PSD-95 (middle). E. Distribution of overall PSD-95 puncta area at puncta that co-localized with bassoon and those that did not. Dashed line indicated cutoff value. F. Percentage of PSD-95 puncta that overlapped with bassoon without the area cutoff applied and with the area cutoff applied.