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.