
Synaptic scaffold proteins, such as PSD95, are organized into molecular supercomplexes that are maintained by the sequential replacement of individual subunits over time, a process linked to the protein's lifetime in different brain regions.


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The concept that dimeric protein complexes in synapses can sequentially replace their subunits has been a cornerstone of Francis Crick’s 1984 hypothesis, explaining how long-term memories could be maintained in the face of short protein lifetimes. However, it is unknown whether the subunits of protein complexes that mediate memory are sequentially replaced in the brain and if this process is linked to protein lifetime. We address these issues by focusing on supercomplexes assembled by the abundant postsynaptic scaffolding protein PSD95, which plays a crucial role in memory. We used single-molecule detection, super-resolution microscopy and MINFLUX to probe the molecular composition of PSD95 supercomplexes in mice carrying genetically encoded HaloTags, eGFP and mEos2. We found a major population of PSD95-containing supercomplexes comprised of two copies of PSD95, with a dominant 12.7 nm separation. Time-stamping of PSD95 subunits in vivo revealed that each PSD95 subunit was sequentially replaced over days and weeks. Comparison of brain regions showed subunit replacement was slowest in the cortex, where PSD95 protein lifetime is longest. Our findings reveal that protein supercomplexes within the postsynaptic density can be maintained by gradual replacement of individual subunits providing a mechanism for stable maintenance of their organization. Moreover, we extend Crick’s model by suggesting that synapses with slow subunit replacement of protein supercomplexes and long protein lifetimes are specialized for long-term memory storage and that these synapses are highly enriched in superficial layers of the cortex where long-term memories are stored.

### Competing Interest Statement

The authors have declared no competing interest.

*   SR
    :   super-resolution
    PALM
    :   photoactivation localization microscopy
    MINFLUX
    :   MINimal photon FLUXes
    TIRF
    :   total internal reflection fluorescence
    GFP
    :   green fluorescent protein
    LPL
    :   long protein lifetime
    SPL
    :   short protein lifetime

Sequential replacement of PSD95 subunits in postsynaptic supercomplexes is slowest in the cortex

sequential-replacement-of-synaptic-scaffold-proteins-maintains-the-stability-of-molecular-supercomplexes-in-the-brain


Sequential Replacement of Synaptic Scaffold Proteins Maintains the Stability of Molecular Supercomplexes in the Brain
