Phagocytes engulf unwanted particles into phagosomes that then fuse with lysosomes to degrade the enclosed particles. Ultimately, phagosomes must be recycled to help recover membrane resources that were consumed during phagocytosis and phagosome maturation, a process referred to as “phagosome resolution.” Little is known about phagosome resolution, which may proceed through exocytosis or membrane fission. Here, we show that bacteria-containing phagolysosomes in macrophages undergo fragmentation through vesicle budding, tubulation, and constriction. Phagosome fragmentation requires cargo degradation, the actin and microtubule cytoskeletons, and clathrin. We provide evidence that lysosome reformation occurs during phagosome resolution since the majority of phagosome-derived vesicles displayed lysosomal properties. Importantly, we show that clathrin-dependent phagosome resolution is important to maintain the degradative capacity of macrophages challenged with two waves of phagocytosis. Overall, our work suggests that phagosome resolution contributes to lysosome recovery and to maintaining the degradative power of macrophages to handle multiple waves of phagocytosis.

Abbreviations: Arl 8: Arf-like GTPase 8, CLC-GFP: GFP-fusion of the clathrin-light chain; ConA: concanamycin A; DQ-BSA: dye-quenched bovine serum albumin; IKA: ikarugamycin; LAMP1: lysosomal-associated membrane protein 1; LAMP2: lysosomal-associated membrane protein 2; LB: Luria-Bertani medium; Lp: Legionella pneumophila; mTORC1: mechanistic target of rapamycin complex 1; PDV: phagosome-derived vesicle; PtdIns(3)P: phosphatidylinositol 3-phosphate; PtdIns(3,5)P 2 : phosphatidylinositol 3,5-biphosphate; PtdIns(4)P: phosphatidylinositol 4-phosphate; TFEB: transcription factor EB Summary Phagocytes engulf particles into phagolysosomes for degradation. However, the ultimate fate of phagolysosomes is undefined. Lancaster, Fountain et al. show that phagosomes undergo fragmentation to reform lysosomes in a clathrin-dependent manner. This process is necessary to maintain the degradative capacity of phagocytes during subsequent phagocytosis. AbstractDuring phagocytosis, phagocytes like macrophages engulf and sequester unwanted particles like bacteria into phagosomes. Phagosomes then fuse with lysosomes to mature into phagolysosomes, resulting in the degradation of the enclosed particle. Ultimately, phagosomes must be recycled to help recover membrane resources like lysosomes consumed during phagocytosis, a process referred to as phagosome resolution. Little is known about phagosome resolution, which may proceed through exocytosis or membrane fission. Here, we show that bacteria-containing phagolysosomes in macrophages undergo fragmentation through vesicle budding, tubulation, and constriction. Phagosome fragmentation required cargo degradation, the actin and microtubule 3 cytoskeletons, and clathrin. We provide evidence that lysosome reformation occurs during phagosome resolution since the majority of phagosome-derived vesicles displayed lysosomal properties. Importantly, we showed that the clathrin-dependent phagosome resolution is important to maintain the degradative capacity of macrophages challenged with two waves of phagocytosis. Overall, our work suggests that phagosome resolution contributes to lysosome recovery and to maintain the degradative power of macrophages to handle multiple waves of phagocytosis.