Sequential replacement of PSD95 subunits in postsynaptic supercomplexes is slowest in the cortex
- EaStCHEM School of Chemistry, University of Edinburgh, United Kingdom
- Genes to Cognition Program, Centre for Clinical Brain Sciences, University of Edinburgh, United Kingdom
- EMBL Imaging Centre, European Molecular Biology Laboratory, Germany
- IRR Chemistry Hub, Institute for Regeneration and Repair, University of Edinburgh, United Kingdom
- Simons Initiative for the Developing Brain (SIDB), Centre for Discovery Brain Sciences, University of Edinburgh, United Kingdom
- The Patrick Wild Centre for Research into Autism, Fragile X Syndrome & Intellectual Disabilities, Centre for Discovery Brain Sciences, University of Edinburgh, United Kingdom
- Centre for Inflammation Research, University of Edinburgh, United Kingdom
Figures
Figure 1 with 2 supplements
Stoichiometry and spatial arrangement of PSD95 within individual supercomplexes.
(a) Brain containing either endogenously tagged PSD95-eGFP or PSD95-mEoS2 was extracted from the genetically modified mouse, and the forebrain was homogenized to solubilize PSD95-containing …
Figure 1—figure supplement 1
Stoichiometry of eGFP determined using photobleaching step count analysis.
(a) Stoichiometries of eGFP determined using the same approach as that for eGFP-tagged PSD95. (b) Example intensity trace with fitting.
Figure 1—figure supplement 2
Multimers of PSD95 in wild-type supercomplexes.
Homogenate extracted from wild-type mice was processed using the same procedure as that for PSD95-eGFP mice. It was subsequently diluted 1:1000 and 100 µl of this solution was incubated on a …
Figure 2 with 1 supplement
PSD95 turnover within supercomplexes in mouse brain homogenate.
(a) PSD95-HaloTag homozygous mice were injected with SiR-Halo ligand and culled 6 hr (day-0) or 7 days (day-7) post injection. The forebrains were homogenized and post hoc stained with AF488-Halo …
Figure 2—figure supplement 1
Absolute number graphs for Figure 2c.
Number of labeled supercomplexes at day-0 and day-7 containing only SiR-labeled ‘old’ protein (SIR), AF488-labeled ‘new’ protein (AF488), or both ‘mixed’. In total, 13,710 supercomplexes were …
Figure 3
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. Seven days later, the forebrain was extracted and synaptosomes isolated. AF488-HaloTag ligand …
Figure 4 with 2 supplements
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 …
Figure 4—figure supplement 1
Characterization of PSD95 turnover in 3-week-old mice.
(a) Key showing the five dissected brain regions (i). Percentage of total PSD95 proteins in old-only (ii), new-only (iii), and mixed (iv) supercomplexes (mean ± SD, n = 3). (b) Mouse brain region …
Figure 4—figure supplement 2
Comparison between the density-based PSD95 puncta half-life and old, new, and mixed proteins in supercomplexes.
The percentage of complexes that contain (a) old-only, (b) new-only, or (c) mixed old and new PSD95 (mean ± SD for % PSD95, and ± 95% CI for half-life, n = 3). There is no statistically significant …
Tables
Table 1
MINFLUX iterations.
| 1st | 2nd | 3rd | 4th | 5th | |
|---|---|---|---|---|---|
| L size [nm] | 300 | 300 | 150 | 75 | 40 |
| TCP – pattern | Hexagon | Hexagon | Hexagon | Hexagon | Hexagon |
| Minimum number of collected photons | 100 | 150 | 100 | 100 | 150 |
| Laser power factor | 1x | 1x | 2x | 4x | 6x |
| TCP dwell time [ms] | 1 | 1 | 1 | 1 | 1 |
| CFR | x | 0.5 | x | 0.8 | x |
| Background threshold [kHz] | 15 | 15 | 15 | 15 | 15 |
Table 2
Post hoc t-tests to locate the source of significance between the means of the mixed supercomplexes in the five brain regions analyzed.
Only p-values less than 0.05 are shown. There are high levels of significance between the means of the isocortex and all other regions.
| Isocortex | Hippocampus | Olfactory bulb | Cerebellum | Subcortex | |
|---|---|---|---|---|---|
| Isocortex | 6.4 × 10−4 | 3.8 × 10−6 | 1.5 × 10−5 | 3.1 × 10−3 | |
| Hippocampus | 3.8 × 10−3 | 1.0 × 10−2 | N.S. | ||
| Olfactory bulb | N.S. | 9.3 × 10−3 | |||
| Cerebellum | N.S. | ||||
| Subcortex |
