Stoichiometry and spatial arrangement of PSD95 within individual supercomplexes.

a) Brain containing either endogenously-tagged PSD95-GFP or PSD95-mEoS was extracted from the genetically-modified mouse, and the forebrain was homogenized to solubilize PSD95-containing supercomplexes. PSD95 supercomplexes were immobilized on glass coverslips and imaged using single-molecule and super-resolution approaches. bi) Individual PSD95-GFP supercomplexes (boxed) were imaged using TIRF microscopy. Scale bar = 5 µm, inset scale bar = 500 nm. Photobleaching step counting revealed a distribution of PSD95 stoichiometries (bii) (10,178 PSD95-GFP photobleaching steps were counted across 6,461 supercomplexes). Representative intensity traces with fits shown in biii. ci) Example PALM images of individual PSD95 supercomplexes. Scale bar = 500 nm, inset scale bar = 50 nm. cii) Subsequent analysis revealed that PSD95 exists at a range of stoichiometries within the supercomplexes (132,929 PSD95-mEoS molecules were detected in 82,501 individual supercomplexes). Plots show mean ± SD, n = 3 biological repeats. ciii) Class averaging of the dimer population shows a distinct separation between the PSD95 proteins within the supercomplexes (class average of 9,743 supercomplexes). di) Example MINFLUX images of PSD95 supercomplexes. Scale bar = 100 nm, inset scale bar = 10 nm. dii) Analysis of the supercomplexes containing two PSD95 molecules (1011 supercomplexes) showed a distribution of PSD95 separation distances. diii) Class averaging of this population revealed two peaks separated by 12.7 nm.

Protein turnover in synaptic and total forebrain PSD95 supercomplexes.

a) SiR-Halo ligand was injected into the tail vein of three 3-month-old PSD95-HaloTag knock-in mice. 7 days later, the forebrain was extracted and synaptosomes isolated. AF488-HaloTag ligand was incorporated during the homogenization process. DOC, deoxycholate. b) Comparison of populations of PSD95 supercomplexes in the total forebrain and synaptic fraction.

Differences in PSD95 turnover between mouse brain regions.

a) Mouse brains were dissected into five broad regions. b) The percentage of total PSD95 imaged contained in SiR-labeled (i), AF488-labeled (ii) and mixed (iii) supercomplexes. Error bars show the SD of 6 biological repeats. c) The percentage of SiR-labeled (i), AF488-labeled (ii) and mixed (iii) PSD95 supercomplexes in each region of the brain. For statistical significance, see Supplementary Methods and Materials (Table S2).

PSD95 turnover within supercomplexes in mouse brain homogenate.

a) PSD95-HaloTag homozygous mice were injected with SiR-Halo ligand and culled 6 hours (day-0) or 7 days (day-7) post injection. The forebrains were homogenized and post-hoc stained with AF488-Halo ligand to saturate remaining binding sites. At day-0, the vast majority of PSD95 is labeled with SiR-Halo only. After 7 days of protein turnover, three populations of supercomplex are possible: SiR-Halo only, AF488-Halo only, both fluorophores. b) Images of supercomplexes from homogenate at day-0 and day-7, showing increased AF488-Halo:SiR-Halo ratios at day-7, with increased coincidence. Scale bar is 5 μm and 500 nm in the zoom. c) Quantified percentages of supercomplexes labeled only with SiR-Halo, AF488-Halo, or both. At day-0, 96% of supercomplexes were labeled with SiR-Halo only, indicating saturation of PSD95-HaloTag binding sites by injection. At day-7, this had decreased to 56%, with expansion of the AF488-Halo and co-labeled populations, indicating that PSD95 protein turnover had occurred over the 7 days.