Fig. 6. The formation of phagocytic F-actin structures by CEACAM3 is triggered by receptor aggregation and critically depends on the tyrosine residues of the cytoplasmic receptor domain. (A) Confocal sections through COS-7 cells transiently transfected to express either CEACAM3 or the double mutant Y230/241F and infected with N313 (Opa₅₇) for 20 min. Phase contrast shows cells and adhering bacteria. Immunofluorescence labelling of CEACAM receptors (green) reveals strong aggregation of both receptors at sites of bacterial adherence. Phagocytic F-actin structures as visualized by TRITC–phalloidin (red) are present in cells expressing wild-type CEACAM3 (arrows) but absent from cells expressing the Y230/241F double mutant (arrowheads). (B) Magnetic beads coated with the D14HD11 mouse monoclonal antibody against CEACAM receptors were incubated with HeLa cell lines for 20 min at 37°C at a ratio of five beads per cell. Receptor aggregation by anti-CEACAM beads is sufficient to induce phagocytic actin rearrangements in cells expressing CEACAM3 (arrows) but not in cells producing the Y230/241F double mutant. Beads were visualized in fixed and permeabilized samples by a dichlorotriazinylamino fluorescein (DTAF)-conjugated secondary antibody against the coating mouse monoclonal antibody.