Extended Data Fig. 6. How synCAM morphology is perturbed by small molecule inhibitors of specific actin regulators and loss of function mutations (linked to Fig. 2).

(a) Example microscopy images of L929 fibroblasts expressing αGFP JAM-B, ICAM-1, or Tether spreading on a GFP coated surface and stained with Phalloidin (scale bar = 10 µm). Spreading is shown in the presence of the indicated inhibitor of actin regulation. A minimum of 10 regions of interest were imaged on two separate days. (b) Maximum projection confocal images (scale bar = 10 µm), and calculated contact angles of synCAM interfaces containing the ICAM-1 ICD with mutations in the ERM binding domains (BD) (box = 25th to 75th percentile, whiskers = min to max, centre = median, n = 20 pairs)⁶⁰. (c) Maximum projection confocal images (scale bar = 10 µm), and calculated contact angles of synCAM interfaces containing the Intβ1 ICD with mutations in the two “NPxY” talin binding domain motifs (box = 25th to 75th percentile, whiskers = min to max, centre = median, n = 20 pairs)⁶¹. (d) Maximum projection confocal images (scale bar = 10 µm), and calculated contact angles of synCAM interfaces containing the Intβ2 ICD with mutations in the two “NPxF” talin binding domain motifs (box = 25th to 75th percentile, whiskers = min to max, centre = median, n = 20 pairs)⁶². (e) Maximum projection confocal images (scale bar = 10 µm), and calculated contact angles of synCAM interfaces containing the Ecad ICD with mutations in the β -catenin binding domain (box = 25th to 75th percentile, whiskers = min to max, centre = median, n = 20 pairs)⁶³. (f) Maximum projection confocal images (scale bar = 10 µm), and calculated contact angles and GFP enrichment of synCAM interfaces containing the MUC-4 ICD with mutations in Ser and Tyr phosphorylation sites (box = 25th to 75th percentile, whiskers = min to max, centre = median, n = 20 pairs). (g) Maximum projection confocal images (scale bar = 10 µm), and calculated contact angles and GFP enrichment of synCAM interfaces containing the JAM ICD with mutations in the PDZ binding domain (box = 25th to 75th percentile, whiskers = min to max, centre = median, n = 20 pairs). (h) Maximum projection confocal images (scale bar = 10 µm), and calculated contact angles and GFP enrichment of synCAM interfaces containing the NCAM-1 ICD with mutations in the Cys palmitoylation site (box = 25th to 75th percentile, whiskers = min to max, centre = median, n = 20 pairs)⁶⁴.

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