Phagosomes Fuse with Late Endosomes and/or Lysosomes by Extension of Membrane Protrusions along Microtubules: Role of Rab7 and RILP

Abstract

Nascent phagosomes must undergo a series of fusion and fission reactions to acquire the microbicidal properties required for the innate immune response. Here we demonstrate that this maturation process involves the GTPase Rab7. Rab7 recruitment to phagosomes was found to precede and to be essential for their fusion with late endosomes and/or lysosomes. Active Rab7 on the phagosomal membrane associates with the effector protein RILP (Rab7-interacting lysosomal protein), which in turn bridges phagosomes with dyneindynactin, a microtubule-associated motor complex. The motors not only displace phagosomes in the centripetal direction but, strikingly, promote the extension of phagosomal tubules toward late endocytic compartments. Fusion of tubules with these organelles was documented by fluorescence and electron microscopy. Tubule extension and fusion with late endosomes and/or lysosomes were prevented by expression of a truncated form of RILP lacking the dynein-dynactin-recruiting domain. We conclude that full maturation of phagosomes requires the retrograde emission of tubular extensions, which are generated by activation of Rab7, recruitment of RILP, and consequent association of phagosomes with microtubule-associated motors.Leukocytes eliminate pathogens and apoptotic cells by initially engulfing them into a phagocytic vacuole. The vacuole, which is derived from the plasmalemma, needs to undergo extensive remodeling to acquire microbicidal and lytic capabilities (28). Such remodeling, also known as maturation, involves sequential fusion with various components of the endolysosomal pathway and concomitant fission events that maintain the vacuolar size nearly constant (1, 28).The molecular machinery underlying maturation, particularly the process of phagolysosome formation, is poorly understood. It is generally thought that phagosomal maturation shares some features with the progression of endosomes to lysosomes, a complex process that is orchestrated by Rab GTPases (33). Accordingly, Rab5 and Rab7 have been detected on the membranes of early and intermediate phagosomes, respectively (12, 13, 23). Moreover, using an in vitro reconstitution system, Funato and colleagues (15) found that inhibition of Rab function by addition of excess Rab-GDP dissociation inhibitor impaired phagosome maturation. Inhibition of Rab5 may be at least partly responsible for this effect, since immunodepletion of this protein precluded the fusion of phagosomes with endosomes (2, 3). The mode of action of Rab5 and the possible role of other Rabs, particularly Rab7, in phagosomal maturation remain obscure.The objective of the present studies was to analyze the involvement of Rab7 and its only known effector, RILP (Rab7-interacting lysosomal protein), in phagolysosome formation by mammalian macrophages. To this end, we transfected cells of the murine monocyte/macrophage line RAW 264.7 with chimeric constructs of enhanced green fluorescent protein (EGFP) and either normal or mutated forms of Rab7 and RILP, and we monitored their distribution, dy...

CASP4/caspase-11 promotes autophagosome formation in response to bacterial infection

“…RAB7 has been implicated in lysosome biogenesis [46] and controls the interaction and fusion of late endosomes with lysosomes [47]. Active GTP-bound RAB7 on the surface of phagosomes promotes membrane motility leading to phagolysosome formation [26]. In addition, RAB7 regulates the final maturation of late autophagic vacuoles during starvation-induced autophagy [48].…”

“…RAB7 on the surface of phagosomes was further reported to promote membrane motility leading to phagolysosome formation via recruitment of the effector protein RILP, which mediates the contact of phagosomes with microtubule-associated motor proteins [26]. Microtubules organize the formation of autophagosomes, their transport, and subsequent fusion events [54–56] by electrostatic interactions originating from the N-terminal domain of LC3 thereby allowing the attachment of autophagosomes to the microtubule network [57,58].…”

“…CASP4 deficiency leads to increased replication of B. cenocepacia in murine macrophages in vitro and infected lungs accompanied by elevated bacterial dissemination to the spleen in vivo . Furthermore, transport of B. cenocepacia to phagophores, the precursors to autophagosomes, and subsequent delivery to lysosomes is compromised in casp4 −/- macrophages resulting in the accumulation of RAB7, a small GTPase that is involved in phagosome maturation and acidification [26]. Our findings provide new insights into non-pyroptotic functions of CASP4 by facilitating autophagosome biogenesis and trafficking in response to intracellular pathogens.…”

Powering membrane traffic in endocytosis and recycling

“…RAB proteins might be involved in cargo binding as well. For example, at the Golgi apparatus, RAB6, its interactor Bicaudal-D and Golgi-specific β-III spectrin interact with distinct dynactin subunits and/or directly with dynein 141 , whereas at phagosomes, RAB7 and its interactor RAB7-interacting lysosomal protein (RILP) recruit the dynein-dynactin complex 68 . affect organelle distribution and organism survival, arguing against a role for +TIP-associated dynactin in vesicle trafficking 58 .…”

“…Its two effectors, RAB7-interacting lysosomal protein (RILP) and oxysterol-binding protein (OSBP)-related protein-1L (ORP1L), recruit the dynein -dynactin complex to these compartments, promoting dyneinmediated tubulation 68 and transport [69][70][71] . A potential site in RILP that is implicated in motor recruitment has been identified 72 , although it is still unclear whether RILP interacts directly with dynein.…”