3I). These results indicate that Cbln1 bound to NRXs in a manner distinct from NLs or LRRTMs. As

Cbln1 binds to GluD2 at the postsynaptic site, we next examined whether the binding between Cbln1 and GluD2 was affected by extracellular Ca2+ concentrations. Immunocytochemical analyses of the surface HA-Cbln1 revealed that HA-Cbln1 bound to HEK293 expressing GluD2 under low extracellular Ca2+ concentrations (Fig. 3J). Together, these results indicate that, unlike NRX/NL- or NRX/LRRTM-based cell adhesion, trans-synaptic cell adhesion mediated by NRX1β(S4+)/Cbln1/GluD2 is resistant to low extracellular Ca2+ concentrations. Cbln1, which accumulates at the synaptic junction by binding to GluD2, serves as a presynaptic organizer (Matsuda et al., 2010). As NRX is known to recruit selleck synaptic vesicles (Dean et al., 2003), it probably mediates the presynaptic organizing function of Cbln1. To examine this hypothesis, we first examined whether Cbln1 and GluD2 formed a tripartite complex HCS assay with NRXs. Immunocytochemical analyses showed that NRX1β(S4+)-Fc but not NRX1β(S4−)-Fc specifically bound to HEK293 cells expressing GluD2 only when HA-Cbln1 was

added to the culture medium (Fig. 4A). Similarly, when NRX1β(S4+) and GFP were coexpressed in cbln1-null cerebellar granule cells, NRX1β(S4+) accumulated in GFP-positive axons around the beads coated with HA-Cbln1 but not around uncoated beads (Fig. 4B). We expressed NRX1β(S4+)-Flag, in which the region necessary for binding to presynaptic organizing proteins such as calcium/calmodulin-dependent serine protein kinase (CASK) (Hata et al., 1996; Dean et al., 2003) was disrupted by attaching the Flag tag at the extreme Interleukin-3 receptor C-terminus of NRX1β(S4+) (Fairless et al., 2008) in wild-type hippocampal neurons. Importantly, NRX1β(S4+)-Flag also accumulated in axons contacting the beads coated with HA-Cbln1 without recruiting the presynaptic marker synapsin I (Supporting

information Fig. S2A), indicating that accumulation of NRX1β(S4+) was directly caused by HA-Cbln1 and not by other presynaptic molecules that bound to the C-terminus of NRX1β(S4+). In addition, not only overexpressed NRX1β(S4+), but also endogenous NRXs in cbln1-null granule cells preferentially accumulated in axons contacting the beads coated with HA-Cbln1 (Supporting Information Fig. S2B). Furthermore, NRX1β(S4+)-Flag expressed in cbln1-null granule cells accumulated in axons that crossed Purkinje cells only when HA-Cbln1 was added to the culture medium (Supporting Information Fig. S2C), indicating that Cbln1, which was bound to GluD2 on Purkinje cell dendrites, induced clustering of NRX1β(S4+) at presynaptic terminals. Although beads coated with Cbln1 accumulated synapsin I-positive synaptic vesicles in cbln1-null granule cell axons (Matsuda et al., 2010), addition of NRX1β(S4+)-Fc and not NRX1β(S4−)-Fc to the culture medium significantly inhibited Cbln1 presynaptic organizing function (Fig. 4C).