Structural insights into the in situ assembly of clustered protocadherin γB4

Clustered protocadherins (cPcdhs) belong to the cadherin superfamily and play important roles in neural development. cPcdhs can mediate homophilic adhesion and lead to self-avoidance and tiling by giving neurons specific identities in vertebrates. Structures and functions of cPcdhs have been studied extensively in the past decades, but the mechanisms behind the functions have not been fully understood. Here we investigate the in situ assembly of cPcdh-γB4, a member in the γ subfamily of cPcdhs, by electron tomography and find that the full length cPcdh-γB4 does not show regular organization at the adhesion interfaces. By contrast, cPcdh-γB4 lacking the intracellular domain can generate an ordered zigzag pattern between cells and the cis interacting mode is different from the crystal packing of the ectodomain. We also identify the residues on the ectodomain that might be important for the zigzag pattern formation by mutagenesis. Furthermore, truncation mutants of the intracellular domain of cPcdh-γB4 reveal different assembly patterns between cell membranes, suggesting that the intracellular domain plays a crucial role in the intermembrane organization of cPcdh-γB4. Taken together, these results suggest both ectodomain and intracellular domain regulate the in situ assembly of cPcdh-γB4 at the adhesion interfaces, thereby providing mechanistic insights into the functional roles of cPcdhs during neuronal wiring.