Clusters of MPP2 and SynCAM 1 form bracelet-like arrangements at the edge of the PSD.

Mature (DIV21) primary rat hippocampal neurons immunostained for endogenous PSD-95 (blue, second column), MPP2 (green, third column), and SynCAM 1 (red, fourth column) and subjected to 3D SIM. (a) More than 90% of the imaged dendritic spines express all 3 proteins of interest (overview maximum projection, first row). A single synapse detail (second row) depicts the bracelet-like arrangements of MPP2 and SynCAM 1 surrounding central PSD-95 puncta. A 3D rendering of that particular synapse in top (third row) and side view (fourth row) reveals that SynCAM 1 and MPP2 clusters are arranged in an interlocked, bracelet-like form, surrounding a central cluster of PSD-95. Scale bars: overview 1 μm; detail 1 μm. Three-dimensional rendering box size: 2.8 μm. (b) Histograms illustrating the distribution of protein cluster sizes for PSD-95 (top, blue), MPP2 (middle, green), and SynCAM 1 (bottom, red). Indicated radii were calculated based on extracted cluster volumes, assuming a spherical shape. The final bin in each histogram contains summarised data for cluster sizes greater than 400 nm. Histograms reflect clusters associated with approximately 40,000 synapses (in 50 images from N = 3 independent experiments). (c) Three-dimensional radial intensity profiles of PSD-95, MPP2, and SynCAM 1 signals in relation to the centres of PSD-95 clusters. Plot shows averaged normalised mean ± SEM from 3 independent experiments (approximately 40,000 synapses from 50 images). For details on the analysis, please see the Materials and methods. (d) NN analysis of MPP2 and SynCAM 1 protein clusters after 3D segmentation. NN distances from MPP2 to the nearest SynCAM 1 cluster were calculated between cluster centres (upper panel, grey bars) and cluster surfaces (lower panel). Dashed lines represent the upper and lower envelopes of CSR. CSR was calculated by randomly distributing MPP2 within the volume and SynCAM 1 on the surface of spheres of 0.8 μm diameter as indicated by the grey dotted line (mean ± SEM, 95% confidence interval, 10 simulations per synapse, N = 3 independent experiments, approximately 40,000 synapses from 50 images). See S6 Fig for NN analysis in the reverse direction. Underlying data are provided in S2 Data. CSR, complete spatial randomness; DIV, days in vitro; MPP2, membrane protein palmitoylated 2; NN, nearest neighbour; PSD, postsynaptic density; SIM, structured illumination microscopy.