Extended Data Figure 9. Enrichment of other scaffolding proteins within nanocolumns.

a, Enrichment of Homer1 with PSD-95 NCs, n = 118 NCs from 48 synapses, scale 100 nm. b, Enrichment of RIM1/2 to Shank NCs, n = 80 NCs from 32 synapses, scale 200 nm. *p<0.05, ANOVA on ranks with pairwise comparison procedures (Dunn’s method) in a and b. c, GKAP2 and Shank3 densities (determined with STORM, n = 6 and 12, respectively) within PSD-95 NCs (determined with PALM of transfected knockdown-replacement-PSD-95-mEos2) normalized to total PSD densities. Both proteins showed significant enrichment in PSD-95 NCs, *p<0.05, paired t-tests. d, Three-color STORM imaging of RIM1/2, GKAP1 and PSD-95 on the same synapses example (left) and protein enrichment profiles of RIM1/2 and GKAP1 with respect to PSD-95 NCs (right), n = 32 NCs from 17 synapses, scale 200 nm. e, Enrichment indices of RIM1/2 and GKAP1 relative to PSD-95 NCs. Color-coded bars represent the same set of randomizations as performed in Fig. 3c: orange denotes randomization of only out-of-NC localizations, cyan denotes randomization of NC positions within synaptic clusters and grey denotes randomization of all localizations. f, The percentage of PSD-95 NCs that were enriched with GKAP1, RIM1/2 or both with color-coded randomizations. *p<0.05, **p<0.01, ANOVA on ranks with pairwise comparison procedures (Dunn’s method), n = 32 NCs from 17 synapses in 7 different cultures. g, Schematic summary of the distribution of synaptic proteins within nanocolumns. The distributions of color-coded proteins are based on our results and the proteins in grey are hypothetical, some, such as Ca²⁺ channels, have been suggested previously to be clustered^49,50.