PMC

N-cadherin is required for both the induction of presynaptic vesicle clusters by neuroligin-1 and the postsynaptic clustering of neuroligin. (A) Images of dendrites of N-cad^+/− and N-cad^−/− neurons (6-7 DIV) cotransfected (at 4–5 DIV) with DsRed2-VAMP2 and either neuroligin-1-EGFP (Nlg-GFP) or EGFP. (Left) EGFP-labeled dendrites. (Center) DsRed2-VAMP2-labeled vesicle clusters (Center Left, original images; Center Right, thresholded DsRed2-VAMP2 puncta). (Right) Overlay of EGFP-labeled dendrites (green) and DsRed2-VAMP2 puncta (red). (Scale bars: 5 μm.) (B and C) Quantification of the induction of presynaptic vesicle clusters by overexpression of neuroligin-1. Coexpression of either neuroligin-1-EGFP or EGFP with DsRed2-VAMP2 is indicated on bars. N-cad^+/−: GFP, n = 45; Nlg-GFP, n = 31. N-cad^−/−: GFP, n = 38; Nlg-GFP, n = 36. (D) Immunostainings for endogenous neuroligin (Nlg) and VAMP2 in N-cad^+/− and N-cad^−/− neurons at 7 DIV. (D Left) Original images of endogenous neuroligin and corresponding thresholded neuroligin puncta. (Center) Thresholded VAMP2 puncta. (Right) Overlay of thresholded neuroligin (green) and VAMP2 puncta (red) visualizing colocalization. (Scale bars: 2 μm.) (E) Density of endogenous neuroligin puncta in dendrites. N-cad^+/−, n = 19; N-cad^−/−, n = 21. (F) Colocalization of neuroligin puncta with VAMP2 puncta. N-cad^+/−, n = 22; N-cad^−/−, n = 15. (G) Density of endogeneous PSD95 puncta in dendrites. N-cad^+/−, n = 21; N-cad^−/−, n = 21. Mean ± SEM; *P < 0.05; ***P < 0.001, unpaired t test.

Neuroligin-1-induced increase in both miniature EPSC frequency and release probability requires N-cadherin function. (A) Example traces of whole-cell recordings of AMPA mEPSCs in N-cad^−/− and control (N-cad^+/−) neurons at 12–14 DIV expressing either neuroligin-1-EGFP (Nlg-GFP) or EGFP (transfected at 10–12 DIV). Holding potential: −60 mV. Frequencies (B) and amplitudes (C) of AMPA mEPSCs upon neuroligin-1-EGFP overexpression. N-cad^+/−: GFP, n = 35; Nlg-GFP, n = 24. N-cad^−/−: GFP, n = 28; Nlg-GFP, n = 25. Mean ± SEM; *P < 0.05, unpaired t test. (D) Neuroligin-1-EGFP overexpression (n = 6) accelerated the MK801 block of evoked NMDA EPSCs in cultured cortical neurons at 12–14 DIV (transfected at 10–12 DIV) as compared with control EGFP expression (n = 5), indicating an increased release probability. (E) Upon coexpression of N-cadΔE the enhancing effect of neuroligin-1 on release probability was blocked (n = 5; controls n = 5). Mean ± SEM. Biexponential fits are shown.

Cooperation of N-cadherin and neuroligin-1 in CA3 pyramidal neurons. (A) Three-dimensional reconstruction of an EGFP expressing CA3 pyramidal neuron in a rat hippocampal slice culture (transfected by single cell electroporation). (Scale bar, 10 μm.) (B–D) Neuroligin-1-EGFP (Nlg-GFP) or EGFP were coelectroporated with dominant-negative N-cadΔE (ectodomain deleted) into single cells at 5 DIV and presynaptic vesicle clusters were immunostained for VAMP2 2 d later at 7 DIV. (B) Three-dimensional images of proximal dendrites. VAMP2 puncta on EGFP expressing dendrites are indicated by arrows. (Scale bar, 3 μm.) (C) Density of VAMP2 puncta on dendrites. (D) Change in VAMP2 puncta density upon neuroligin-1-EGFP expression depends on N-cadherin function. (E and F) Neuroligin-1-EGFP clustering depends on N-cadherin function. (E) (Upper) Original images of neuroligin-1-EGFP-expressing dendrites. (Lower) Thresholded neuroligin-1-EGFP puncta. (Scale bars: 2 μm.) (F) Dendritic density of neuroligin-1-EGFP puncta. Mean ± SEM; *P < 0.05; **P < 0.01, unpaired t test.

Cooperation of N-cadherin and neuroligin-1 is mediated by the scaffolding protein S-SCAM in cultured cortical neurons. (A and B) Inhibition of S-SCAM function by expression of truncated S-SCAM proteins blocks the induction of presynaptic vesicle clusters by neuroligin-1-EGFP (transfected at 4–5 DIV, analyzed at 6–7 DIV). (A) DsRed2-VAMP2 was coexpressed with either EGFP or neuroligin-1-EGFP (Nlg-GFP) and either full-length S-SCAM (S-SCAM full) or S-SCAM with PDZ5 deleted (S-SCAM-PDZ5 del) or S-SCAM with WW and PDZ1 deleted (S-SCAM-PDZ1 del). (Left) EGFP-labeled dendrites. (Center) Thresholded DsRed2-VAMP2 puncta. (Right) Overlay of EGFP fluorescence (green) and DsRed2-VAMP2 puncta (red) to visualize vesicle clusters on dendrites. (Scale bars: 2 μm.) (B) Changes in vesicle cluster density upon neuroligin-1-EGFP expression. Type of coexpressed S-SCAM is indicated below bars. S-SCAM-full, n = 16; S-SCAM-PDZ5del, n = 20; S-SCAM-PDZ1del, n = 17. (C–E) Clustering of neuroligin-1-EGFP and colocalization with vesicle clusters depends on S-SCAM function. (C) Overlay images of thresholded neuroligin-1-EGFP puncta (green) and DsRed2-VAMP2 puncta (red) to visualize colocalization. Type of coexpressed S-SCAM is indicated. (Scale bar, 2 μm.) Density of neuroligin-1-EGFP puncta on dendrites (D), and colocalization of neuroligin-1-EGFP puncta with DsRed2-VAMP2 puncta (E). No S-SCAM, n = 27; S-SCAM full, n = 16; S-SCAM PDZ5del, n = 20; S-SCAM PDZ1del, n = 17. (F) Colocalization of immunoctochemically stained endogeneous N-cadherin puncta with endogeneous neuroligin puncta depends on S-SCAM function. No S-SCAM, n = 13; S-SCAM full, n = 11; S-SCAM PDZ5del, n = 12; S-SCAM PDZ1del, n = 13. (G and H) RNAi-mediated S-SCAM knockdown in low-density cultures of cortical neurons (transfected at 2 DIV, analyzed at 8 DIV). Quantification of immunocytochemically stained neuroligin puncta (G) and VAMP2 puncta (H) at 8 DIV. Control, no transfection; Mismatch, mismatch control; KD, knockdown. Mean ± SEM; *P < 0.05; **P < 0.01; ***P < 0.001, one-way ANOVA; unpaired t test in G and H.

Presynaptic vesicle accumulation depends on N-cadherin in immature neurons. (A) EGFP expressing mouse ES cell-derived neurons at 5 DIV (transfected at 3 DIV). N-cad^−/−, N-cadherin knockout neurons; N-cad^+/−, control neurons. (Scale bars: 10 μm.) (B and C) No change in axon length or in the number of dendrites was observed in N-cad^−/− neurons. N-cad^+/−, n = 30; N-cad^−/−, n = 30. (D and H) Immunocytochemical stainings of vesicle clusters (VAMP2) and Bassoon clusters (Bsn) on dendrites at 6–7 DIV. (Right) Original images. (Center) Thresholded VAMP2 or Bsn puncta. (Left) Overlay image of VAMP2 or Bsn puncta and MAP2 staining of dendrites. (Scale bars: 2 μm.) (E–G and I) Dendritic density (E), mean intensity (F), and area (G) of VAMP2 puncta are reduced in the absence of N-cadherin, whereas clusters of the active zone protein Bassoon are unaltered (I). VAMP2 puncta: N-cad^+/−, n = 20; N-cad^−/−, n = 19; Bsn puncta: N-cad^+/−, n = 21; N-cad^−/−, n = 16. Mean ± SEM; *P < 0.05; **P < 0.01, unpaired t test. (J) Examples of FRAP experiments in EGFP-VAMP2 expressing N-cad^+/− and N-cad^−/− neurons at 6–7 DIV (transfected at 4–5 DIV). Individual EGFP-VAMP2 puncta on dendrites were photobleached (arrows) and fluorescence recovery was imaged at indicated time points. Fluorescence intensity is color-coded. BB, before photobleaching; AB, after photobleaching (t = 0 min). (Scale bars: 5 μm.) (K) Normalized fluorescence intensity of photobleached vesicle clusters shown in J versus observation time. (L) Mean recovery of fluorescence intensity. N-cad^+/−, n = 13; N-cad^−/−, n = 11. Mean ± SEM.