Cbln family proteins promote synapse formation by regulating distinct neurexin signaling pathways in various brain regions
Article first published online: 17 MAR 2011
© 2011 The Authors. European Journal of Neuroscience © 2011 Federation of European Neuroscience Societies and Blackwell Publishing Ltd
European Journal of Neuroscience
Volume 33, Issue 8, pages 1447–1461, April 2011
How to Cite
Matsuda, K. and Yuzaki, M. (2011), Cbln family proteins promote synapse formation by regulating distinct neurexin signaling pathways in various brain regions. European Journal of Neuroscience, 33: 1447–1461. doi: 10.1111/j.1460-9568.2011.07638.x
- Issue published online: 17 APR 2011
- Article first published online: 17 MAR 2011
- Received 2 November 2010, revised 9 January 2011, accepted 26 January 2011
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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