Fig. 1.

Binding of β-neurexin-coated beads and recruitment of postsynaptic proteins is neuroligin-specific. (A) Rat hippocampal neurons at 7 or 8 DIV were incubated with 4-μm microspheres coated with Nrx1β-Fc, N-cadherin-Fc (not shown), or human Fc for 1 h, then rinsed and fixed at given time intervals. Some cultures were co-transfected with PSD-95-GFP and WT Nlg1 (B) or an Nlg1 construct unable to interact with neurexin (Nlg1-SWAP). (C) Number of beads bound per cell in each condition. Data are presented as mean ± SEM and the number of beads examined is given in italics. (D) Example of recruitment of Nlg1-mCherry (arrows) at a Nrx1β-Fc bead. The enrichment factor (bead vs. neurite fluorescence levels) for Nlg1-mCherry was 1.71 ± 0.08 (n = 18 beads). (E) Example of recruitment of PSD-mCherry; position of the bead is represented by a dashed circle. Immuno-stained synaptotagmin puncta co-localize with spontaneous PSD-95 clusters (arrows), but not with PSD-95-mCherry accumulated at Nrx1β-Fc beads. Neuronal areas covered by synapses were 7.6% ± 1.0% (n = 13) outside beads and only 4.5% ± 1.3% (n = 48) at Nrx1β-Fc beads, quantitatively showing that Nrx1β-Fc beads do not recruit native synapses. (F) Time course of PSD-95-GFP accumulation at Nrx1β-Fc beads, the fit being a first-order exponential function.