Figure 3.

Hemis ynaptogenesis assay reveals distinct functional codes for neurexins acting on LRRTM2 or neuroligin-1. The coculture assay was modified such that YFP-LRRTM2 or YFP-NLG1(+B) were expressed in hippocampal neurons, and these were cocultured with COS7 cells expressing CFP-tagged neurexins 1β, 2β′, 3β′, 1α, 2α, and 3α, (−S4) or (+S4). Cocultures were immunostained for PSD-95 and synapsin I. A, Illustration of the assay showing YFP-LRRTM2 expressed in neurons clustering at contacts with COS7 cells expressing Nrx1β(−S4)-CFP. PSD-95 coclusters at these sites. The absence of synapsin I at these clusters indicates the absence of native synapses. B, Nrx1β(−S4) on COS7 cells clusters both YFP-LRRTM2 and YFP-NLG1 in neurons, Nrx-1β(+S4) clusters only YFP-NLG1, Nrx1α(−S4) clusters only YFP-LRRTM2, and Nrx 1α(+S4) does not cluster either of them. C, Table showing the clustering code for YFP-LRRTM2 and YFP-NLG1 with all 12 neurexin variants studied. Whereas YFP-LRRTM2 in neurons clustered at contacts with COS7 cells expressing all (−S4) neurexin variants, YFP-NLG1 clustered neurexin β variants and not α variants. (+++++, 90–95% contact sites were positive; +++, 60–80% contact sites were positive; −, 0–5% contact sites were positive; n > 30 contact sites). Scale bars: 10 μm.