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. Author manuscript; available in PMC: 2014 Oct 3.
Published in final edited form as: Neuron. 2013 Oct 2;80(1):10.1016/j.neuron.2013.07.016. doi: 10.1016/j.neuron.2013.07.016

Figure 3. Full-Length Calsyntenin-3 is Synaptogenic but Cleaved Calsyntenin-3 Inhibits the Synaptogenic Activity of α-Neurexin Partners.

Figure 3

(A) An antibody raised against an N-terminal region of calsyntenin-3 recognizes Myc-calsyntenin-3 (Myc-C3) and shows little or no signal for Myc-calsyntenin-1 or -2 (Myc-C1, Myc-C2) expressed in COS cells. In mouse brain homogenate, the two major bands correspond to full-length (F. L.) calsyntenin-3 and the cleaved extracellular fragment. The COS cell lysate analyzed here contains mainly full-length calsyntenin-3; the cleaved extracellular fragment is found mainly in COS cell media.

(B) Domain structure of Myc-calsyntenin-3-CFP and variants: CAD, cadherin domain; LNS, laminin neurexin sex hormone binding protein domain; TM, transmembrane domain; Intra, intracellular domain; CD8TM, the calsyntenin-3 transmembrane domain was replaced with the CD8 lymphocyte protein transmembrane domain.

(C) Western blot with anti-Myc antibody of lysate and media from COS cells expressing the indicated constructs confirms secretion of Myc-C3secreted and indicates that Myc-C3EX-CD8-CFP was not cleaved whereas all other constructs assayed were partially cleaved generating large fragments in the media.

(D) Representative images of calsyntenin-3 variants in the co-culture assay. All membrane-associated forms with the calsyntenin-3 extracellular domain are synaptogenic including the non-cleavable Myc-C3EX-CD8-CFP.

(E) Quantitation of the percentage of COS cells expressing the indicated construct that induced clusters of synapsin lacking MAP2 in the co-culture assay. ANOVA, p < 0.0001, n≥3 experiments counting≥100 cells per experiment, ◆p < 0.001 compared to negative control N-cadherin-CFP by post-hoc Bonferroni multiple comparison test, * p < 0.001 compared to Myc-C3-CFP by post-hoc Bonferroni multiple comparison test.

(F) Representative co-culture images showing suppression of synaptogenic activity of CFP-tagged α-neurexin partners neuroligin 2 (Nlg2), calsyntenin-3 (Clstn3) and LRRTM2 by co-expression of Myc-C3secreted mimicking the shed calsyntenin-3 ectodomain as compared with co-expression of alkaline phosphatase (AP) control. Myc-C3secreted had no effect on Slitrk2 which acts via a non-neurexin presynaptic partner.

(G) Quantification of suppression of synaptogenic activity of α-neurexin partners by Myc-C3secreted. Clusters of synapsin associated with CFP-positive COS cells and lacking MAP2 were measured. ANOVA, p<0.0001, n= >30 cells each, *** p < 0.001 and * p < 0.05 compared to AP control by post-hoc Bonferroni multiple comparison test.

Scale bars: 20 μm. Data are presented as mean ± SEM.

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