MPP2 and GABA_AR α1 form highly overlapping nanoclusters.

<p>Mature (DIV21) primary rat hippocampal neurons were immunostained for endogenous MPP2 (cyan, second column), GABA_AR α1 (magenta, third column), and the PSD marker Homer1 (yellow, fourth column) and subjected to triple-colour <i>d</i>STORM imaging. <b>(a)</b> Overview of a dendritic segment (upper row). White box indicates location of detail image below. Zooming onto an individual synapse expressing Homer1, MPP2, and GABA_AR α1 (lower row) reveals a close association between clusters of MPP2 and GABA_AR α1 and the bracelet-like arrangements of MPP2 surrounding a centrally positioned Homer1 cluster. Scale bars: overview 1 μm; detail 250 nm. <b>(b)</b> Histograms illustrating the distribution of nano-cluster sizes (longest axis) for MPP2 (cyan) and GABA_AR α1 (magenta). Clusters were detected via DBSCAN. Mean ± SEM; <i>n</i> = 26 images from <i>N</i> = 3 independent experiments. <b>(c)</b> NN analysis of MPP2 and GABA_AR α1 protein clusters after DBSCAN. NN distances were calculated from the cluster centres. Closest GABA_AR α1 to MPP2 were analysed (grey bars). Dashed lines represent the random control by toroidal shift. Note the close association of both clusters (approximately 10 nm distance between both centres), which is well below the cluster sizes (20 to 40 and 30 to 60 nm, see b) and shifted after randomisation. Mean ± SEM; <i>n</i> = 26 images from <i>N</i> = 3 independent experiments. Please see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.3001503#pbio.3001503.s008" target="_blank">S8 Fig</a> for related analysis in the reverse direction. Underlying data can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.3001503#pbio.3001503.s016" target="_blank">S2 Data</a>. DIV, days in vitro; <i>d</i>STORM, direct stochastic optical reconstruction microscopy; GABA, γ-aminobutyric acid; MPP2, membrane protein palmitoylated 2; NN, nearest neighbour; PSD, postsynaptic density.</p>