Abstract

Phagocytosis is initiated by lateral clustering of receptors, which in turn activates Src-family kinases (SFKs). Activation of SFKs requires exclusion of tyrosine phosphatases from the area of particle engagement. We investigated how the major phosphatase, CD45 , is excluded from sites of contact with the target using single-molecule tracking. A frustrated phagocytosis model was implemented to stabilize the focal plane. The mobility of CD45 , which was largely confined in unstimulated macrophages, increased markedly upon engagement of phagocytic (Fcγ) receptors. While individual CD45 molecules moved randomly in the plane of the membrane, they were displaced from the advancing phagocytic cup by an expanding diffusional barrier. Micropatterning of IgG, the ligand of Fcγ receptors, was used to better define the relationship between engaged receptors and the progressive diffusional barrier. Remarkably, the barrier extended well beyond the perimeter of the receptor-ligand engagement zone. Second messengers generated by Fcγ receptor activation were found to activate integrins, which were shown to form the diffusion barrier that excluded CD45 by a kinetic segregation process akin to that described in the immunological synapse. The expanding integrin wave facilitates the “zippering” of Fcγ receptors onto the target and integrates the information from sparse receptor-ligand complexes, coordinating the progression and ultimate closure of the phagocytic cup.