Distinct SAP102 and PSD-95 Nano-organization Defines Multiple Types of Synaptic Scaffold Protein Domains at Single Synapses

Abstract

MAGUK scaffold proteins play a central role in maintaining and modulating synaptic signaling, providing a framework to retain and position receptors, signaling molecules, and other synaptic components. In particular, the MAGUKs SAP102 and PSD-95 are essential for synaptic function at distinct developmental timepoints and perform both overlapping and unique roles. While their similar structures allow for common binding partners, SAP102 is expressed earlier in synapse development and is required for synaptogenesis, whereas PSD-95 expression peaks later and is associated with synapse maturation. PSD-95 and other key synaptic proteins organize into subsynaptic nanodomains that have a significant impact on synaptic transmission, but the nanoscale organization of SAP102 is unknown. How SAP102 is organized within the synapse, and how it relates spatially to PSD-95 on a nanometer scale, could underlie its unique functions and impact how SAP102 scaffolds synaptic proteins. Here we used DNA-PAINT super-resolution microscopy to measure SAP102 nano-organization and its spatial relationship to PSD-95 at individual synapses in mixed-sex rat cultured neurons. We found that like PSD-95, SAP102 accumulates in high-density subsynaptic nanoclusters. However, SAP102 nanoclusters were smaller and denser than PSD-95 nanoclusters across development. Additionally, only a subset of SAP102 nanoclusters co-organized with PSD-95, revealing MAGUK nanodomains within individual synapses containing either one or both proteins. These MAGUK nanodomain types had distinct nanocluster properties and were differentially enriched with the presynaptic release protein Munc13-1. This organization into both shared and distinct subsynaptic nanodomains may underlie the ability of SAP102 and PSD-95 to perform both common and unique synaptic functions.Significance statementSAP102 and PSD-95 are two key members of the membrane-associated guanylate kinase (MAGUK) family of synaptic scaffold proteins that are critical for synapse development, maintenance, and plasticity. Because PSD-95 has a highly complex subsynaptic nanostructure that impacts synaptic function, we asked if SAP102 is similarly organized into nanoclusters and how it relates to PSD-95 synaptic organization. We found that SAP102 forms subsynaptic nanoclusters with unique properties from PSD-95. Within individual synapses, these proteins form both MAGUK-specific and overlapping nanodomains with unique properties and trans-synaptic enrichments with the vesicle priming protein Munc13-1. Thus, organization of synaptic proteins into nanoclusters is likely maintained within the MAGUK family and reveals potential mechanisms for specializing functions within individual synapses based on scaffold protein nanodomains.