Visualization of clustered protocadherin neuronal self-recognition complexes

Neurite self-recognition and avoidance are fundamental properties of all nervous systems(1). These processes facilitate dendritic arborization(2,3), prevent formation of autapses(4) and allow free interaction among non-self neurons(1,2,3,4,5). Avoidance among self neurites is mediated by stochastic cell-surface expression of combinations of about 60 isoforms of alpha-, beta- and gamma-clustered protocadherin that provide mammalian neurons with single-cell identities(1,2,4-13). Avoidance is observed between neurons that express identical protocadherin repertoires(2,5), and single-isoform differences are sufficient to prevent self-recognition(10). Protocadherins form isoform-promiscuous cis dimers and isoform-specific homophilic trans dimersi (10,14-20) Although these interactions have previously been characterized in isolation(15,17-20), structures of full-length protocadherin ectodomains have not been determined, and how these two interfaces engage in self-recognition between neuronal surfaces remains unknown. Here we determine the molecular arrangement of full-length clustered protocadherin ectodomains in single-isoform self-recognition complexes, using X-ray crystallography and cryo-electron tomography. We determine the crystal structure of the clustered protocadherin , gamma B4 ectodomain, which reveals a zipper-like lattice that is formed by alternating cis and trans interactions. Using cryo-electron tomography, we show that clustered protocadherin gamma B6 ectodomains tethered to liposomes spontaneously assemble into linear arrays at membrane contact sites, in a configuration that is consistent with the assembly observed in the crystal structure. These linear assemblies pack against each other as parallel arrays to form larger two-dimensional structures between membranes. Our results suggest that the formation of ordered linear assemblies by clustered protocadherins represents the initial self-recognition step in neuronal avoidance, and thus provide support for the isoform-mismatch chain-termination model of protocadherin-mediated self-recognition, which depends on these linear chains(11).