Joe Beckwith (@jsbeckwith.bsky.social)

3D Super-Resolution Imaging of PSD95 Reveals an Abundance of Diffuse Protein Supercomplexes in the Mouse Brain PSD95 is an abundant scaffolding protein that assembles multiprotein complexes controlling synaptic physiology and behavior. Confocal microscopy has previously shown that PSD95 is enriched in the postsynaptic terminals of excitatory synapses and two-dimensional (2D) super-resolution microscopy further revealed that it forms nanoclusters. In this study, we utilized three-dimensional (3D) super-resolution microscopy to examine the nanoarchitecture of PSD95 in the mouse brain, characterizing the spatial arrangement of over 8 million molecules. While we were able to identify molecular arrangements that have been previously reported, imaging in 3D allowed us to classify these with higher accuracy. Furthermore, 3D super-resolution microscopy enabled the quantification of protein levels, revealing that an abundance of PSD95 molecules existed outside of synapses as a diffuse population of supercomplexes, containing multiple copies of PSD95. Further analysis of the supercomplexes containing two units identified two populations: one that had PSD95 molecules separated by 39 ± 2 nm, and a second with a separation of 94 ± 27 nm. The finding that there exists supercomplexes containing two PSD95 units outside of the synapse suggests that supercomplexes containing multiple protein copies assemble outside the synapse and then integrate into the synapse to form a supramolecular nanocluster architecture. https://pubs.acs.org/doi/10.1021/acschemneuro.4c00684#