(A) Aggregation assay using N-cadherin deficient HEK-293 cells transiently transfected with wild-type (left panel) and N22R mutant (middle panel) Sdk1-RFP. Cytoplasmic RFP transfection was used as a negative control (right panel). (B) Quantification of the aggregation assay shown in A for wild-type (WT) Sdk1 and several Sdk1 dimer interface mutants (n = 15, mean ± S.E.). (C) Immunolabeling of Sdk1 (red) with a monoclonal antibody to Sdk1 in interacting L cells shows wild-type Sdk1 (left panel) localizes to the cell-cell junctions whereas the Sdk1 N22R mutant is diffusely localized (right panel). Counterstaining with wheat germ agglutinin (WGA, green) was used to visualize the cell surface. (D) Immunolabeled Sdk1 (red, stained with anti-Sdk1 cytoplasmic domain) and Sdk1YFP (green, Sdk1’s cytoplasmic domain was replaced with YFP) co-localize at cell-cell junctions between Sdk1 and Sdk1YFP expressing L cells (top panels). Arrows indicate co-localization of red and green fluorescence. By contrast, Sdk1 N22R does not localize to cell-cell junctions between Sdk1 N22R and Sdk1YFP expressing cells (bottom panels). (E) Immunolabeling of Sdk1 with a monoclonal antibody to Sdk1 (red) in solitary L cells shows that wild-type Sdk1 localizes in puncta on the cell surface (left) whereas the Sdk1 N22R mutant is diffusely localized (right). (F) HEK-293 cells transiently transfected with both Sdk1RFP and Sdk1YFP express both proteins, which co-localize to cell-cell junctions (top row). Co-transfection of a Rapsyn::RFPnanobody induces clustering of Sdk1RFP away from cell-cell junctions (second and third rows). Sdk2YFP does not co-cluster with Sdk1RFP (second row), but Sdk1YFP does co-cluster (third row). However Sdk1YFP does not co-cluster with Sdk1 N22R-RFP/Rapsyn::RFPnanobody clusters (bottom row). (G) Our data suggest Sdk dimerizes using the crystallographically-determined interface, both between molecules emanating from opposing cell surfaces (in trans)—mediating cell-cell interactions—and between molecules emanating from the same cell surface (in cis)—mediating Sdk clustering. These interactions are shown schematically, using the Sdk1Ig1–5 crystal structure to illustrate the dimer interaction. The remaining 1 Ig and 13 FNIII domains that constitute the rest of the Sdk extracellular domain are abbreviated to a dashed line, with the transmembrane and intracellular domains shown as solid lines. Scale bars in A, C, D, E and F, 5 μm.