PHAGOSOME RESOLUTION: REDUCE, REUSE, RECYCLE

Candidate: Aaron Fountain

Supervisor: Dr. Roberto Botelho

ABSTRACT

To maintain tissue health, the body must be able to remove unwanted particles from the tissue. The removal of unwanted particles are typically carried out by phagocytes such as macrophages, that engulf and internalize unwanted particles into compartments called phagosomes. The phagosome itself will then undergo phagosome maturation to fuse with lysosomes to form phagolysosomes and degrade the unwanted particles within. The process beyond phagosome-lysosome fusion is not well understood in mammalian phagocytes such as macrophages.

We first determined the fate of phagosomes by allowing macrophages to ingest and fluorescently labeled bacteria. When allowing macrophages to ingest fluorescently labeled bacteria, we observed that phagosomes containing such bacteria fragment into smaller vesicles called Phagosome-Derived Vesicles (PDV). We also found that phagosome fragmentation requires cargo breakdown, cytoskeletal dynamics, and clathrin. The novel involvement of clathrin in phagosome fragmentation may play a role in lysosome reformation as it does in autophagosomes.

We next tested if phagosome fragmentation is able to reform lysosomes. We assessed PDVs for lysosome-like properties such as acidity and proteolytic activity and found that PDVs indeed possessed such lysosome-like properties. We also found that following phagocytosis decreases in lysosome levels with some lysosomes restored during phagosome fragmentation. Finally, we found that phagosome fragmentation is required to restore proteolytic activity. Thus phagosome fragmentation enables lysosome reformation from phagosomes after particle degradation.

As phagosomes absorb endomembranes such as lysosomes and render them inaccessible, we also tested to see if phagosome resolution can help macrophages maintain phagocytic activity. We observed that macrophages have a limit to phagocytic uptake, and that approaching that limit prevented macrophages from internalizing more particles. We also observed that clearance of phagosomes via fragmentation restored the ability to internalize particles in macrophages. When assessing lipid membrane contribution to phagosomes, we found that endomembrane, but not plasma membrane, levels decrease after phagocytosis. This suggests that endomembranes availability are one of the limiting factors to phagocytic activity, and that phagosome fragmentation may restore phagocytic activity by maintaining endomembrane levels.

Together, we show that phagosomes are cleared in mammalian phagocytes by fragmentation, which maintain phagocytic and degradative activity by reforming lysosomes.