3D Super-Resolution\nImaging of PSD95 Reveals an Abundance\nof Diffuse Protein Supercomplexes in the Mouse Brain

PSD95 is an abundant\nscaffolding protein that assembles multiprotein\ncomplexes controlling synaptic physiology and behavior. Confocal microscopy\nhas previously shown that PSD95 is enriched in the postsynaptic terminals\nof excitatory synapses and two-dimensional (2D) super-resolution microscopy\nfurther revealed that it forms nanoclusters. In this study, we utilized\nthree-dimensional (3D) super-resolution microscopy to examine the\nnanoarchitecture of PSD95 in the mouse brain, characterizing the spatial\narrangement of over 8 million molecules. While we were able to identify\nmolecular arrangements that have been previously reported, imaging\nin 3D allowed us to classify these with higher accuracy. Furthermore,\n3D super-resolution microscopy enabled the quantification of protein\nlevels, revealing that an abundance of PSD95 molecules existed outside\nof synapses as a diffuse population of supercomplexes, containing\nmultiple copies of PSD95. Further analysis of the supercomplexes containing\ntwo units identified two populations: one that had PSD95 molecules\nseparated by 39 ± 2 nm, and a second with a separation of 94\n± 27 nm. The finding that there exists supercomplexes containing\ntwo PSD95 units outside of the synapse suggests that supercomplexes\ncontaining multiple protein copies assemble outside the synapse and\nthen integrate into the synapse to form a supramolecular nanocluster\narchitecture.