The coordination of signaling by 3' phosphoinositide concentration thresholds during phagocytosis.

摘要

Phagocytosis plays important roles in immune responses by ingesting and destroying pathogens. This thesis addresses the overall question of how signaling during Fcgamma receptor (FcR)-mediated phagocytosis is coordinated. The magnitude of signal molecule recruitment into forming phagosomes was measured as a function of ligand density, using a newly developed recruitment measurement method based on ratiometric fluorescence microscopy. Although early FcR-dependent signals correlated with IgG density on beads, later signals showed a threshold response which was regulated by the concentrations of 3' phosphoinositides in phagocytic cups. The suprathreshold concentrations of 3' phosphoinositides were required for the commitment to later stages of phagosome formation. Rho-family GTPases displayed an activity transition during phagocytosis. In particular, Rac1 was active throughout phagosome formation, Cdc42 activity was restricted to early stages after receptor ligation but Rac2 activity increased later during phagosome closure. Inhibition of PI3-kinases, which generate 3' phosphoinositides following FcR ligation, resulted in persistently active Cdc42 and Rac1, but not Rac2, indicating a role for 3' phosphoinositides in the Rho GTPase activity transition. Expression of constitutively active Cdc42 inhibited phagocytosis by a mechanism that was additive with PI3-Kinase inhibition, indicating that Rho GTPase activity transition was also required for the commitment to later stages of phagosome formation. In addition, 3' phosphoinositide concentration thresholds correlated with the phenomenon that the shape of target particles affected phagocytosis. Target geometry modulated the generation of intracellular 3' phosphoinositides, whose concentrations can affect the late stage signals and outcome of phagocytosis. In summary, this thesis establishes that 3' phosphoinositide concentration thresholds regulate commitment to phagocytosis by regulating late stage signal protein recruitment and GTPase activity transitions.