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. 2020 Oct 28;40(44):8438–8462. doi: 10.1523/JNEUROSCI.1091-20.2020

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Figure 3. — Distinct sets of intracellular proteins are involved in LAR-RPTP- and Nrxn-mediated presynaptic assembly. A, B, Representative images of heterologous synapse-formation activities of Nlgn1 and Slitrk6. Cultured hippocampal neurons were infected with KD lentiviruses against the indicated scaffold proteins at DIV4 and cocultured with Nlgn1- or Slitrk6-expressing HEK293T cells for 12 h (Nlgn1) or 24 h (Slitrk6). Hemi-synapse induction was analyzed by double-immunostaining for HA (blue) and VGLUT1 (red, A) or GAD67 (red, B). Scale bar, 10 μm. β-Catn, β-Catenin; N-Cad, N-cadherin. For semiquantitative immunoblot data to analyze KD efficacies of a subset of presynaptic proteins, see Extended Data Figure 3-1. C, Quantification of synapse-formation activity in A and B by measuring the ratio of red staining intensity to blue staining intensity. Data are mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; Mann–Whitney U test. Green-colored proteins represent liprin-α-binding proteins. Blue-colored proteins represent PTP substrates. Red-colored proteins represent active-zone proteins. n indicates the number of cells as follows: Control/Control/VGLUT1, n = 52; sh-liprin-α/Control/VGLUT1, n = 10; sh-Trio/Control/VGLUT1, n = 10; sh-MIM-B/Control/VGLUT1, n = 12; sh-Caskins/Control/VGLUT1, n = 10; sh-β-Catn/Control/VGLUT1, n = 13; sh-Abl/Control/VGLUT1, n = 17; sh-Ena/Control/VGLUT1, n = 16; sh-p250GAP/Control/VGLUT1, n = 12; sh-N-Cad/Control/VGLUT1, n = 10; sh-Piccolo/Control/VGLUT1, n = 10; sh-ELKS/Control/VGLUT1, n = 11; sh-SYD1A/Control/VGLUT1, n = 9; sh-CASK/Control/VGLUT1, n = 11; sh-RIM1/Control/VGLUT1, n = 9; sh-RIM-BPs/Control/VGLUT1, n = 11; sh-Control/Slitrk6/VGLUT1, n = 20; sh-liprin-α/Slitrk6/VGLUT1, n = 24; sh-Trio/Slitrk6/VGLUT1, n = 18; sh-MIM-B/Slitrk6/VGLUT1, n = 21; sh-Caskins/Slitrk6/VGLUT1, n = 33; sh-β-Catn/Slitrk6/VGLUT1, n = 17; sh-Abl/Slitrk6/VGLUT1, n = 19; sh-Ena/Slitrk6/VGLUT1, n = 20; sh-p250GAP/Slitrk6/VGLUT1, n = 18; sh-N-Cad/Slitrk6/VGLUT1, n = 28; sh-Piccolo/Slitrk6/VGLUT1, n = 21; sh-ELKS/Slitrk6/VGLUT1, n = 19; sh-SYD1A/Slitrk6/VGLUT1, n = 21; sh-CASK/Slitrk6/VGLUT1, n = 20; sh-RIM1/Slitrk6/VGLUT1, n = 19; sh-RIM-BPs/Slitrk6/VGLUT1, n = 23; sh-Control/Nlgn1/VGLUT1, n = 35; sh-liprin-α/Nlgn1/VGLUT1, n = 18; sh-Trio/Nlgn1/VGLUT1, n = 18; sh-MIM-B/Nlgn1/VGLUT1, n = 15; sh-Caskins/Nlgn1/VGLUT1, n = 16; sh-β-Catn/Nlgn1/VGLUT1, n = 16; sh-Abl/Nlgn1/VGLUT1, n = 17; sh-Ena/Nlgn1/VGLUT1, n = 18; sh-p250GAP/Nlgn1/VGLUT1, n = 17; sh-N-Cad/Nlgn1/VGLUT1, n = 17; sh-Piccolo/Nlgn1/VGLUT1, n = 16; sh-ELKS/Nlgn1/VGLUT1, n = 16; sh-SYD1A/Nlgn1/VGLUT1, n = 17; sh-CASK/Nlgn1/VGLUT1, n = 17; sh-RIM1/Nlgn1/VGLUT1, n = 16; sh-RIM-BPs/Nlgn1/VGLUT1, n = 18; sh-Control/Control/GAD67, n = 41; sh-liprin-α/Control/GAD67, n = 10; sh-Trio/Control/GAD67, n = 13; sh-MIM-B/Control/GAD67, n = 11; sh- Caskins/Control/GAD67, n = 9; sh-β-Catn/Control/GAD67, n = 13; sh-Abl/Control/GAD67, n = 12; sh-Ena/Control/GAD67, n = 13; sh-p250GAP/Control/GAD67, n = 12; sh-N-Cad/Control/GAD67, n = 10; sh-Piccolo/Control/GAD67, n = 9; sh-ELKS/Control/GAD67, n = 9; sh-SYD1A/Control/GAD67, n = 9; sh-CASK/Control/GAD67, n = 11; sh-RIM1/Control/GAD67, n = 10; sh-RIM-BPs/Control/GAD67, n = 12; sh-Control/Slitrk6/GAD67, n = 23; sh-liprin-α/Slitrk6/GAD67, n = 21; sh-Trio/Slitrk6/VGLUT1, n = 19; sh-MIM-B/Slitrk6/GAD67, n = 26; sh-Caskins/Slitrk6/GAD67, n = 27; sh-β-Catn/Slitrk6/GAD67, n = 16; sh-Abl/Slitrk6/GAD67, n = 17; sh-Ena/Slitrk6/GAD67, n = 16; sh-p250GAP/Slitrk6/GAD67, n = 16; sh-N-Cad/Slitrk6/GAD67, n = 26; sh-Piccolo/Slitrk6/GAD67, n = 17; sh-ELKS/Slitrk6/GAD67, n = 20; sh-SYD1A/Slitrk6/GAD67, n = 10; sh-CASK/Slitrk6/GAD67, n = 20; sh-RIM1/Slitrk6/GAD67, n = 26; sh-RIM-BPs/Slitrk6/GAD67, n = 26; sh-Control/Nlgn1/GAD67, n = 35; sh-liprin-α/Nlgn1/GAD67, n = 18; sh-Trio/Nlgn1/GAD67, n = 18; sh-MIM-B/Nlgn1/GAD67, n = 18; sh-Caskins/Nlgn1/GAD67, n = 17; sh-β-Catn/Nlgn1/GAD67, n = 23; sh-Abl/Nlgn1/GAD67, n = 22; sh-Ena/Nlgn1/GAD67, n = 19; sh-p250GAP/Nlgn1/GAD67, n = 21; sh-N-Cad/Nlgn1/GAD67, n = 17; sh-Piccolo/Nlgn1/GAD67, n = 16; sh-ELKS/Nlgn1/GAD67, n = 16; sh-SYD1A/Nlgn1/GAD67, n = 18; sh-CASK/Nlgn1/GAD67, n = 19; sh-RIM1/Nlgn1/GAD67, n = 21; and sh-RIM-BPs/Nlgn1/GAD67, n = 19. p values for each comparison are as follows: sh-Control versus sh-liprin-α/Slitrk6/VGLUT1, p < 0.0001; sh-Control versus sh-Trio/Slitrk6/VGLUT1, p = 0.8228; sh-Control versus sh-MIM-B/Slitrk6/VGLUT1, p = 0.0943; sh-Control versus sh-Caskins/Slitrk6/VGLUT1, p = 0.0078; sh-Control versus sh-β-Catn/Slitrk6/VGLUT1, p = 0.4586; sh-Control versus sh-Abl/Slitrk6/VGLUT1, p = 0.0503; sh-Control versus sh-Ena/Slitrk6/VGLUT1, p = 0.2515; sh-Control versus sh-p250GAP/Slitrk6/VGLUT1, p < 0.0001; sh-Control versus sh-N-Cad/Slitrk6/VGLUT1, p = 0.0034; sh-Control versus sh-Piccolo/Slitrk6/VGLUT1, p = 0.0098; sh-Control versus sh-ELKS/Slitrk6/VGLUT1, p = 0.9197; sh-Control versus sh-SYD1A/Slitrk6/VGLUT1, p = 0.0059; sh-Control versus sh-CASK/Slitrk6/VGLUT1, p = 0.4047; sh-Control versus sh-RIM1/Slitrk6/VGLUT1, p = 0.7157; sh-Control versus sh-RIM-BPs/Slitrk6/VGLUT1, p < 0.0001; sh-Control versus sh-liprin-α/Nlgn1/VGLUT1, p = 0.0005; sh-Control versus sh-Trio/Nlgn1/VGLUT1, p = 0.3909; sh-Control versus sh-MIM-B/Nlgn1/VGLUT1, p = 0.0039; sh-Control versus sh-Caskins/Nlgn1/VGLUT1, p = 0.4677; sh-Control versus sh-β-Catn/Nlgn1/VGLUT1, p = 0.0026; sh-Control versus sh-Abl/Nlgn1/VGLUT1, p < 0.0001; sh-Control versus sh-Ena/Nlgn1/VGLUT1, p = 0.0242; sh-Control versus sh-p250GAP/Nlgn1/VGLUT1, p < 0.0001; sh-Control versus sh-N-Cad/Nlgn1/VGLUT1, p = 0.0017; sh-Control versus sh-Piccolo/Nlgn1/VGLUT1, p = 0.2858; sh-Control versus sh-ELKS/Nlgn1/VGLUT1, p = 0.2703; sh-Control versus sh-SYD1A/Nlgn1/VGLUT1, p = 0.0223; sh-Control versus sh-CASK/Nlgn1/VGLUT1, p = 0.7299; sh-Control versus sh-RIM1/Nlgn1/VGLUT1, p = 0.6353; sh-Control versus sh-RIM-BPs/Nlgn1/VGLUT1, p = 0.0042; sh-Control versus sh-liprin-α/Slitrk6/GAD67, p < 0.0001; sh-Control versus sh-Trio/Slitrk6/GAD67, p =0.1079; sh-Control versus sh-MIM-B/Slitrk6/GAD67, p = 0.0008; sh-Control versus sh-Caskins/Slitrk6/GAD67, p < 0.0001; sh-Control versus sh-β-Catn/Slitrk6/GAD67, p = 0.0022; sh-Control versus sh-Abl/Slitrk6/GAD67, p = 0.0200; sh-Control versus sh-Ena/Slitrk6/GAD67, p = 0.0002; sh-Control versus sh-p250GAP/Slitrk6/GAD67, p < 0.0001; sh-Control versus sh-N-Cad/Slitrk6/GAD67, p < 0.0001; sh-Control versus sh-Piccolo/Slitrk6/GAD67, p < 0.0001; sh-Control versus sh-ELKS/Slitrk6/GAD67, p = 0.9177; sh-Control versus sh-SYD1A/Slitrk6/GAD67, p = 0.0044; sh-Control versus sh-CASK/Slitrk6/GAD67, p = 0.0633; sh-Control versus sh-RIM1/Slitrk6/GAD67, p = 0.0384; sh-Control versus sh-RIM-BPs/Slitrk6/GAD67, p < 0.0001; sh-Control versus sh-liprin-α/Nlgn1/GAD67, p < 0.0001; sh-Control versus sh-Trio/Nlgn1/GAD67, p = 0.079; sh-Control versus sh-MIM-B/Nlgn1/GAD67, p = 0.0016; sh-Control versus sh-Caskins/Nlgn1/GAD67, p = 0.0006; sh-Control versus sh-β-Catn/Nlgn1/GAD67, p < 0.0001; sh-Control versus sh-Abl/Nlgn1/GAD67, p < 0.0001; sh-Control versus sh-Ena/Nlgn1/GAD67, p = 0.0861; sh-Control versus sh-p250GAP/Nlgn1/GAD67, p = 0.1483; sh-Control versus sh-N-Cad/Nlgn1/GAD67, p < 0.0001; sh-Control versus sh-Piccolo/Nlgn1/GAD67, p = 0.0079; sh-Control versus sh-ELKS/Nlgn1/GAD67, p = 0.0087; sh-Control versus sh-SYD1A/Nlgn1/GAD67, p < 0.0001; sh-Control versus sh-CASK/Nlgn1/GAD67, p = 0.0068; sh-Control versus sh-RIM1/Nlgn1/GAD67, p = 0.0004; and sh-Control versus sh-RIM-BPs/Nlgn1/GAD67, p > 0.9999.