Figures and data
![](https://prod--epp.elifesciences.org/iiif/2/99303%2Fv1%2Fcontent%2F548866v2_fig1.tif/full/max/0/default.jpg)
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.
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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.
![](https://prod--epp.elifesciences.org/iiif/2/99303%2Fv1%2Fcontent%2F548866v2_fig4.tif/full/max/0/default.jpg)
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).
![](https://prod--epp.elifesciences.org/iiif/2/99303%2Fv1%2Fcontent%2F548866v2_fig5.tif/full/max/0/default.jpg)
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.