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...

Phagosomal biogenesis is central for microbial killing and antigen presentation by leukocytes. However, the molecular mechanisms governing phagosome maturation are poorly understood. We analyzed the role and site of action of phosphatidylinositol 3-kinases (PI3K) and of Rab GTPases in maturation using both professional and engineered phagocytes. Rab5, which is recruited rapidly and transiently to the phagosome, was found to be essential for the recruitment of Rab7 and for progression to phagolysosomes. Similarly, functional PI3K is required for successful maturation. Remarkably, inhibition of PI3K did not preclude Rab5 recruitment to phagosomes but instead enhanced and prolonged it. Moreover, in the presence of PI3K inhibitors Rab5 was found to be active, as deduced from measurements of early endosome antigen 1 binding and by photobleaching recovery determinations. Though their ability to fuse with late endosomes and lysosomes was virtually eliminated by wortmannin, phagosomes nevertheless recruited a sizable amount of Rab7. Moreover, Rab7 recruited to phagosomes in the presence of PI3K antagonists retained the ability to bind its effector, Rab7-interacting lysosomal protein, suggesting that it is functionally active. These findings imply that (i) dissociation of Rab5 from phagosomes requires products of PI3K, (ii) PI3K-dependent effectors of Rab5 are not essential for the recruitment of Rab7 by phagosomes, and (iii) recruitment and activation of Rab7 are insufficient to induce fusion of phagosomes with late endosomes and lysosomes. Accordingly, transfection of constitutively active Rab7 did not bypass the block of phagolysosome formation exerted by wortmannin. We propose that Rab5 activates both PI3K-dependent and PI3K-independent effectors that act in parallel to promote phagosome maturation.

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