We found that cadherin-9-Fc but not control Fc binds the surface of 293T cells expressing cadherin-9
in a calcium-dependent manner indicating that, like other classic cadherins, cadherin-9 undergoes homophilic binding (Figures 5B and 5C) (Shimoyama et al., 2000). One consequence of cadherin binding is recruitment and activation of β-catenin (Arikkath and Reichardt, 2008 and Stepniak et al., 2009). To determine if cadherin-9-mediated adhesion recruits β-catenin, we expressed cadherin-9 in 293T cells and found that β-catenin is recruited to the interface between cadherin-9 cells (Figure 5D). Additionally, epitope-tagged cadherin-9 colocalizes with β-catenin in neurons (Figure 5E). These results suggest that cadherin-9 is capable of intracellular signaling via catenin proteins. We next analyzed the location of cadherin-9 protein in neurons. Consistent with in situ hybridization results selleck chemicals showing that cadherin-9 is expressed selectively Apoptosis Compound Library high throughput by DG and CA3 neurons, we found that endogenous cadherin-9 protein is found in puncta along axons and dendrites in a subset of cultured hippocampal neurons (Figures 5F and 5G). Cadherin-9 puncta do not directly overlap with synaptic vesicles or the postsynaptic density but are frequently found adjacent to synaptic active zones (Figure 5H), similar to what has been observed for other
cadherins (Fannon and Colman, 1996 and Uchida et al., 1996). Antibody incompatibility prevented us from determining precisely which cell types are cadherin-9 positive in culture and from analyzing endogenous cadherin-9 protein
in vivo. However, we were able to examine the location of epitope-tagged cadherin-9 in DG neurons in the brain. DG neurons of P5 rats were infected with a lentivirus expressing cadherin-9 and GFP using a 2A peptide to drive expression of both genes from a single promoter (Figure S3A). Because the 2A peptide remains attached to cadherin-9, it was used as an epitope tag to visualize cadherin-9 with anti-2A these antibodies. We found that at P14 cadherin-9-2A localizes to dendrites and axons of DG neurons (Figures S3B–S3E). Although cadherin-9-2A was found diffusely throughout the dendrites, it localized to discrete bands at mossy fiber terminals adjacent to but not overlapping presynaptic vesicles marked by VGlut1 (Figures 5I and S3B–S3E), consistent with a role as a synaptic organizer. To examine the role of cadherin-9 in synapse formation, we generated a cadherin-9 shRNA that markedly reduces expression of a tagged cadherin-9 construct in 293T cells (Figure 6A). The construct does not significantly affect expression of several other cadherins expressed in the hippocampus, including cadherin-2 (N-cadherin), cadherin-8, and cadherin-10, based on western blot and quantitative PCR analysis (Figures 6B and 6C, and S4).