Phagosome formation relies on profound reorganization of actin and membranes, but the mechanism of phagosome closure remains poorly understood. We used an original experimental setup to monitor phagosome formation and closure in three dimensions in living macrophages using Total Internal Reflection Fluorescence (TIRF) Microscopy. We reveal that a cross talk between actin and dynamin-2 takes place for phagosome formation and closure, and that dynamin-2 plays a critical role in the effective scission of phagosomes from the plasma membrane.
Phagocytosis is a mechanism used by macrophages to internalize and eliminate microorganisms or cellular debris. It relies on profound rearrangements of the actin cytoskeleton that is the driving force allowing plasma membrane extension around the particle. The closure step of phagocytosis, however, remains poorly defined. We used a dedicated experimental setup with Total Internal Reflection Fluorescence Microscopy (TIRFM) to monitor phagosome formation and closure in three dimensions in living cells. We show that dynamin-2, which mediates the scission of endocytic vesicles, was recruited early and concomitantly with actin during phagosome formation. Dynamin-2 accumulated at the site of phagosome closure in living macrophages. Inhibition of its activity with dominant negative mutants or drugs demonstrated that dynamin-2 is implicated in actin dynamics and pseudopod extension. Depolymerization of actin led to impaired dynamin-2 recruitment or activity. Finally, we show that dynamin-2 plays a critical role in the effective scission of the phagosome from the plasma membrane. Thus, we establish that a cross talk between actin and dynamin takes place for phagosome formation and closure before dynamin functions for scission.