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Fig. S1. Effects of soluble NRX1β(S4+) or (S4−) on artificial synapse formation induced by NL1(−) or LRRTM2. HEK293 cells expressing GFP-NL1(−) or LRRTM2 plus GFP were cocultured with cbln1-null cerebellar neurons in the presence of NRX1β(S4+) or (S4−)-Fc (100 μg/mL) for 3 days. Representative confocal images of cells immunostained for synaptophysin (Syn; red or white) and GFP (green) are shown. Scale bar, 25 μm. Mean intensities of synaptophysin immunoreactivity in the GFP-positive area in the presence of NRX1β (S4+) or (S4−)-Fc are normalized to those in cultures untreated with NRXs-Fc and summarized in the lower graph. Error bars represent SEMs. At least n = 270 cells were analyzed in two independent experiments. **P = 5.52 × 10−21 for NL1(−). **P = 2.8 × 10−6 for LRRTM2.

Fig. S2. Exogenous HA-Cbln1 accumulate axonal NRX1β(S4+) in transfected neurons. (A) Accumulation of axonal NRX1β(S4+) on HA-Cbln1-coated beads in hippocampal neurons. Wild-type hippocampal neurons expressing NRX1β(S4+)-Flag, in which the region necessary for presynaptic differentiation was disrupted by attaching the Flag tag at the extreme C-terminus of NRX1β(S4+), were cocultured with HA-Cbln1-coated beads. Confocal images of NRX1β(S4+) (red or white) and synapsin I (green or white) are shown. Red and white arrowheads indicate accumulated NRX1β(S4+) and synaptophysin around the beads, respectively. Scale bar, 20 μm. (B) Accumulation of endogenous NRXs in cerebellar neurons on HA-Cbln1-coated beads. cbln1-null cerebellar neurons were cocultured with beads coated or uncoated (control) with HA-Cbln1 from 9 to 11 DIV. HA-Cbln1-conjugated beads but not control caused clustering of endogenous NRXs (green). Scale bar, 20 μm. (C) NRX1β(S4+) in granule cell axons accumulates on Purkinje cells. Purkinje cells were cocultured with cbln1-null granule cells transfected with NRX1β(S4+)-Flag in the presence or absence of HA-Cbln1 (40 μg/mL) from 10 to 13 DIV. Confocal images of neurons immunostained for calbindin (blue) and NRX1β(S4+) (red or white) are shown. Red arrowheads indicate accumulated NRX1β(S4+) in granule cell axons in the proximity of Purkinje cells and open arrowheads indicate NRX1β(S4+) in other axonal regions. Cotransfected GFP diffusely stained axons (bottom panel).

Fig. S3. Cbln1 or Cbln2 directly causes clustering of NRX1β(S4+). Beads coated with HA-Cbln1, Cbln2, Cbln4, or CS-Cbln1 were incubated with HEK293 cells expressing NRX1β(S4+) for 2 days. Confocal images of HEK293 cells immunostained against NRX1β(S4+) (red or white) and beads (green) are shown. Scale bar, 25 μm.

Fig. S4. Cbln1 serves as a direct presynaptic organizer in hippocampal neurons. (A) Accumulation of functional presynaptic sites labeled with FM4-64 (red) around HA-Cbln1-coated beads (green). Scale bar, 20 μm. () Presynaptic sites were directly induced by HA-Cbln1-coated beads. Synapsin I-immunopositive terminals (red) were induced around HA-Cbln1-coated beads (arrowheads), which were located at extrasynaptic sites lacking endogenous AMPA receptors (detected by anti-pan AMPA receptor antibody; green). Scale bar, 20 μm.

Fig. S5. Cbln1 and Cbln2 but not Cbln4 induced presynaptic differentiation of cortical neurons. Beads coated with HA-Cbln1, Cbln2, Cbln4, or CS-Cbln1 were cocultured with cortical neurons. Confocal images of neurons immunostained for synapsin I (red or white) and beads (green) are shown. Scale bar, 20 μm.

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