Figure 2. ADAM8 interacts with the actin-based motor protein Myo1f via SH3 domains and modulates cell motility.

(A) Domain structure of ADAM8 and Myo1f as interaction partner identified by a yeast-2-hybrid screen using a leukocyte library. ADAM8 CD (aa 678–824) was used as prey and the reporter gene indicated interaction (“+”) via the SH3 domain of Myo1f (clone BP19, aa 1032–1098). (B) Co-IP of activated human PMN (hPMN) and (C) HL-60 cells using polyclonal ADAM8 (A8) antibody coupled to magnetic beads. Negative controls, beads bound to control IgG (IgG); unconjugated beads (beads). (D) Immunofluorescence staining against Myo1f (green) and ADAM8 (red) of hPMN and HL-60 cells. Overlay (yellow) and colocalized pixel map (Coloc Map) indicate colocalization in cell protrusions. Scale bar, 10 μm. (E) Co-IP of hPMNs + BK-1361 or CP during activation. (F) Relative pixel intensity of Myo1f coprecipitated with ADAM8 normalized to input using ImageJ software (NIH). *, P < 0.05, Student’s t test. (G) Colocalization map of hPMNs + BK-1361 or CP as described in D. Scale bar, 10 μm. (H) Effect of BK-1361 or SH3 domain blockade (SH3-Inh) on fMLP-induced transendothelial migration of hPMNs (n = 4 donors). (I) Migration across a 3D gel matrix, respectively (n = 3 donors). (H and I) Data are mean ± SD, **, P < 0.01; 2-way ANOVA followed by Holm-Šídák multiple-comparison test. (J) 3D chemotactic migration of hPMNs preincubated with BK-1361 or CP toward an IL-8 (1 μg/mL) gradient using chemotaxis μ-Slides. Representative trajectory plots. Triangles indicate orientation of the gradient. Violin plots of (K) migration velocity and (L) mean Euclidian and (M) accumulated distance; BK-1361 (white), SH3 inhibitor (gray), control (black). (K–M) Three independent experiments, 30 cells per experiment were analyzed. *, P < 0.05; **, P < 0.01; ***, P < 0.001; 2-way ANOVA followed by Holm-Šídák multiple-comparison test. fMLP, N-formyl-methionyl-leucyl-phenylalanine.