Phagosome maturation: going through the acid test

Abstract

Phagosome maturation is the process by which internalized particles (such as bacteria and apoptotic cells) are trafficked into a series of increasingly acidified membrane-bound structures, leading to particle degradation. The characterization of the phagosomal proteome and studies in model organisms and mammals have led to the identification of numerous candidate proteins that cooperate to control the maturation of phagosomes containing different particles. A subset of these candidate proteins makes up the first pathway to be identified for the maturation of apoptotic cell-containing phagosomes. This suggests that a machinery that is distinct from receptor-mediated endocytosis is used in phagosome maturation.

Figure 1. Endocytosis as a paradigm for phagocytosis

Phagocytosis and receptor mediated endocytosis are similar processes, but also have distinct features. (a) During receptor-mediated endocytosis, activated receptor sinks into the cell, in this case into a clathrin-coated pit. Following scission of the vesicle from the membrane mediated by dynamin, clathrin is removed; receptors are then trafficked to the `recycling endosome,' where they are eventually sorted back to the cell surface. Activated receptors are also removed from phagosomes and presumably are also targeted to recycling endosomes. Endocytosed vesicles acquire the GTPase Rab5, which is activated by GEFs (such as RabEx-5 and GapEx-5) which allows homotypic fusion with other Rab5-positive structures, , as well as heterotypic fusion with Rab4-positive recycling endosomes. Rab5 is then exchanged for Rab7 via the HOPS complex, which contains the Rab7 GEF Vps39, resulting in recruitment of the Rab7 effector RILP and ultimately fusion with acidic lysosomes and acquisition of the lysosomal markers LAMP-1 and LAMP-2. (b) In contrast, during phagocytosis the plasma membrane is extended around the apoptotic cell, forming the phagosome, . Phagosomes also `mature' through Rab5(+) and Rab7(+) stages, ultimately resulting in fusion with lysosome structures, , , , , , however phagosomes appear to utilize a distinct mechanism for GTPase regulation. In the nematode, known RAB-5 GEFs are not required for phagosome maturation), and the HOPS complex is not required for the recruitment of Rab7 to the phagosome. This may reflect the observations that phagosomes rarely fuse together and tend to mature individually. Other events, such as trafficking of proteins from the Golgi to the phagosome have been described, although whether this occurs during endocytosis remains to be determined. A number of GTPases have also been found associated with the phagosome during phagosome maturation (see Box 3), many of which have not been described to play a role on the endosome.

Figure 2. Activation of Rab5 during phagosome maturation

During internalization of apoptotic cells Dynamin and Vps34 are recruited to the phagosome by an unknown mechanism. Vps34 can interact with inactive (GDP-bound) or active (GTP-bound) Rab5; the recruitment of Dynamin to the forming phagosome results in the recruitment of Vps34 and RAB-5^GDP to the phagosome. Dynamin (and DYN-1) has also been shown to play a role in the maintenance of the actin cytoskeleton, which has implications for phagocytosis as well, . Once on the phagosome, an unidentified GEF likely activates RAB-5 (converting it to the GTP-bound state), activating Vps34 kinase activity, which generates PtdIns(3)P (indicated as yellow dots) on the phagosome. It is noteworthy that GapEx-5 has been identified as a possible GEF for Rab5 in one mammalian cell context. Effector proteins bind to active Rab5 in the context of PtdIns(3)P on the phagosome; the assembly of effectors on the phagosome (which have yet to be identified) results in the exchange of Rab5 for Rab7, leading to the continued maturation of the phagosome.

Figure 3. Pathways for phagosome maturation versus endocytosis

Pathways for phagosome maturation and endosome maturation derived from publications describing the requirements of each protein in the different endocytic processes. (a) In the nematode, recruitment of RAB-5 to the phagosome (via interaction with DYN-1 and VPS-3441) begins the process of acidification, leading to VPS-34 activation and recruitment of as-yet unidentified effectors. Rab7 is then recruited to the phagosome, resulting in recruitment of the HOPS complex, which activates Rab7, promoting further maturation events and eventual fusion with lysosome structures. (b) Activation of Rab5 on endocytic vesicles (by the GEFs RabEx-5, GapEx-5, or RIN1) begins a concentration step, where vesicles merge and ligand is concentrated, potentially through the formation of a fusion pore by the Rab5 effector EEA1, which binds PtdIns(3)P on the phagosome generated by Vps34. Another Rab5 effector, Rabenosyn, binds Vps45, a component of the Rab7 GEF/effector HOPS complex, potentially mediating Rab5 recruitment to the maturing vesicle, . The HOPS complex member Vps39 activated Rab7, resulting in recruitment of the Rab7 effector RILP, potentially resulting in lysosome fusion. While some of the players have been shown to play a role in mammalian engulfment, a step-wise pathway for apoptotic cell-containing phagosome maturation in mammalian systems is just beginning to be defined.