Roberto J. Botelho

Phagosome maturation: aging gracefully

Foreign particles and apoptotic bodies are eliminated from the body by phagocytic leucocytes. The initial stage of the elimination process is the internalization of the particles into a plasma membrane-derived vacuole known as the phagosome. Such nascent phagosomes, however, lack the ability to kill pathogens or to degrade the ingested targets. These properties are acquired during the course of phagosomal maturation, a complex sequence of reactions that result in drastic remodelling of the phagosomal membrane and contents. The determinants and consequences of the fusion and fission reactions that underlie phagosomal maturation are the topic of this review.

Distinct roles of class I and class III phosphatidylinositol 3-kinases in phagosome formation and maturation

Phagosomes acquire their microbicidal properties by fusion with lysosomes. Products of phosphatidylinositol 3-kinase (PI 3-kinase) are required for phagosome formation, but their role in maturation is unknown. Using chimeric fluorescent proteins encoding tandem FYVE domains, we found that phosphatidylinositol 3-phosphate (PI[3]P) accumulates greatly but transiently on the phagosomal membrane. Unlike the 3′-phosphoinositides generated by class I PI 3-kinases which are evident in the nascent phagosomal cup, PI(3)P is only detectable after the phagosome has sealed. The class III PI 3-kinase VPS34 was found to be responsible for PI(3)P synthesis and essential for phagolysosome formation. In contrast, selective ablation of class I PI 3-kinase revealed that optimal phagocytosis, but not maturation, requires this type of enzyme. These results highlight the differential functional role of the two families of kinases, and raise the possibility that PI(3)P production by VPS34 may be targeted during the maturation arrest induced by some intracellular parasites.

Phosphatidylinositol-4,5-bisphosphate hydrolysis directs actin remodeling during phagocytosis

The Rho GTPases play a critical role in initiating actin polymerization during phagocytosis. In contrast, the factors directing the disassembly of F-actin required for fission of the phagocytic vacuole are ill defined. We used fluorescent chimeric proteins to monitor the dynamics of association of actin and active Cdc42 and Rac1 with the forming phagosome. Although actin was found to disappear from the base of the forming phagosome before sealing was complete, Rac1/Cdc42 activity persisted, suggesting that termination of GTPase activity is not the main determinant of actin disassembly. Furthermore, fully internalized phagosomes engineered to associate constitutively with active Rac1 showed little associated F-actin. The disappearance of phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) from the phagosomal membrane closely paralleled the course of actin disassembly. Furthermore, inhibition of PI(4,5)P2 hydrolysis or increased PI(4,5)P2 generation by overexpression of phosphatidylinositol phosphate kinase I prevented the actin disassembly necessary for the completion of phagocytosis. These observations suggest that hydrolysis of PI(4,5)P2 dictates the remodeling of actin necessary for completion of phagocytosis.

Phagosome Maturation: A Few Bugs in the System

Cells of the innate immune system ingest and destroy invading microorganisms by initially engulfing them into a specialized vacuole, known as the phagosome. The membrane of the forming phagosome is similar to the plasmalemma and its contents resemble the extracellular milieu. As such, the nascent phagosome is not competent to kill and eliminate the ingested microorganisms. However, shortly after sealing, the phagosome undergoes a series of rapid and extensive changes in its composition, the result of a sophisticated sequence of membrane fusion and fission reactions. Understanding the molecular basis of these events is of particular importance, since they are often the target of disruption by intracellular parasites such as Mycobacterium, Salmonella and Legionella. The objective of this review is to summarize the current knowledge of the molecular mechanisms underlying phagosomal maturation and its subversion by parasitic microorganisms.

Critical role for scaffolding adapter Gab2 in FcγR-mediated phagocytosis

Grb2-associated binder 2 (Gab2), a member of the Dos/Gab subfamily scaffolding molecules, plays important roles in regulating the growth, differentiation, and function of many hematopoietic cell types. In this paper, we reveal a novel function of Gab2 in Fcγ receptor (FcγR)–initiated phagocytosis in macrophages. Upon FcγR activation, Gab2 becomes tyrosyl phosphorylated and associated with p85, the regulatory subunit of phosphoinositide 3-kinase (PI3K), and the protein–tyrosine phosphatidylinositol Shp-2. FcγR-mediated phagocytosis is severely impaired in bone marrow–derived macrophages from Gab2−/− mice. The defect in phagocytosis correlates with decreased FcγR-evoked activation of Akt, a downstream target of PI3K. Using confocal fluorescence microscopy, we find that Gab2 is recruited to the nascent phagosome, where de novo PI3K lipid production occurs. Gab2 recruitment requires the pleckstrin homology domain of Gab2 and is sensitive to treatment with the PI3K inhibitor wortmannin. The Grb2 binding site on Gab2 also plays an auxiliary role in recruitment to the phagosome. Because PI3K activity is required for FcγR-mediated phagocytosis, our results indicate that Gab2 acts as a key component of FcγR-mediated phagocytosis, most likely by amplifying PI3K signaling in the nascent phagosome.

Phagosomal Maturation, Acidification, and Inhibition of Bacterial Growth in Nonphagocytic Cells Transfected with FcγRIIA Receptors

Phagocytosis and killing of microbial pathogens by professional phagocytes is an essential component of the innate immune response. Recently, heterologous transfection of individual receptors into nonmyeloid cells has been used successfully to elucidate the early steps that signal phagosome formation. It is unclear, however, whether the vacuoles formed by such transfected cells are bona fide phagosomes, capable of fusion with endomembranes, of luminal acidification, and of controlling the growth of microorganisms. The aim of the current study was to determine whether COS-1 and Chinese hamster ovary cells, rendered phagocytic by expression of human FcγRIIA receptors, express the cellular machinery required to support phagosomal maturation. Immunolocalization studies demonstrated that early endosomes, as well as late endosomes and/or lysosomes, fuse sequentially with phagosomes in the transfectants. Microfluorescence ratio imaging of particles labeled with pH-sensitive dyes revealed that maturation of the phagosome was accompanied by luminal acidification. The drop in pH, which attained levels comparable to those reported in professional phagocytes, was prevented by inhibitors of vacuolar-type H+-ATPases. Optimal phagosomal acidification required elevation of cytosolic [Ca2+], suggesting that it results from fusion of endomembranes bearing proton pumps. Moreover, the transfected cells effectively internalized live bacteria. Opsonization was essential for bacterial internalization, implying that it occurred by FcγRIIA-mediated phagocytosis, as opposed to invasion. Uptake into phagolysosomes was associated with inhibition of bacterial growth, due at least in part to the low intraphagosomal pH. These studies indicate that the biochemical events that follow receptor-mediated particle internalization in cells transfected with FcγRIIA receptors closely resemble the process of phagosomal maturation in neutrophils and macrophages. FcγRIIA-transfected cells can, therefore, be used as a model for the study of additional aspects of phagocyte biology.

Assembly of a Fab1 Phosphoinositide Kinase Signaling Complex Requires the Fig4 Phosphoinositide Phosphatase

Phosphatidylinositol-3,5-bisphosphate [PtdIns(3,5)P(2)] regulates several vacuolar functions, including acidification, morphology, and membrane traffic. The lipid kinase Fab1 converts phosphatidylinositol-3-phosphate [PtdIns(3)P] to PtdIns(3,5)P(2). PtdIns(3,5)P(2) levels are controlled by the adaptor-like protein Vac14 and the Fig4 PtdIns(3,5)P(2)-specific 5-phosphatase. Interestingly, Vac14 and Fig4 serve a dual function: they are both implicated in the synthesis and turnover of PtdIns(3,5)P(2) by an unknown mechanism. We now show that Fab1, through its chaperonin-like domain, binds to Vac14 and Fig4 and forms a vacuole-associated signaling complex. The Fab1 complex is tethered to the vacuole via an interaction between the FYVE domain in Fab1 and PtdIns(3)P on the vacuole. Moreover, Vac14 and Fig4 bind to each other directly and are mutually dependent for interaction with the Fab1 kinase. Our observations identify a protein complex that incorporates the antagonizing Fab1 lipid kinase and Fig4 lipid phosphatase into a common functional unit. We propose a model explaining the dual roles of Vac14 and Fig4 in the synthesis and turnover of PtdIns(3,5)P(2).

Phosphatidylinositol‐3,5‐Bisphosphate: No Longer the Poor PIP2

Phosphoinositides play an important role in organelle identity by recruiting effector proteins to the host membrane organelle, thus decorating that organelle with molecular identity. Phosphatidylinositol‐3,5‐bisphos‐ phate [PtdIns(3,5)P2] is a low‐abundance phosphoinositide that predominates in endolysosomes in higher eukaryotes and in the yeast vacuole. Compared to other phosphoinositides such as PtdIns(4,5)P2, our understanding of the regulation and function of PtdIns(3,5)P2 remained rudimentary until more recently. Here, we review many of the recent developments in PtdIns(3,5)P2 function and regulation. PtdIns(3,5)P2 is now known to espouse functions, not only in the regulation of endolysosome morphology, trafficking and acidification, but also in autophagy, signaling mediation in response to stresses and hormonal cues and control of membrane and ion transport. In fact, PtdIns(3,5)P2 misregulation is now linked with several human neuropathologies including Charcot‐Marie‐Tooth disease and amyotrophic lateral sclerosis. Given the functional versatility of PtdIns(3,5)P2, it is not surprising that regulation of PtdIns(3,5)P2 metabolism is proving rather elaborate. PtdIns(3,5)P2 synthesis and turnover are tightly coupled via a protein complex that includes the Fab1/PIKfyve lipid kinase and its antagonistic Fig4/Sac3 lipid phosphatase. Most interestingly, many PtdIns(3,5)P2 regulators play simultaneous roles in its synthesis and turnover.

An electrostatic switch displaces phosphatidylinositol phosphate kinases from the membrane during phagocytosis

PIP5K is held at the membrane of forming phagosomes by a conserved, positively charged patch. During particle engulfment, the surface charge of the phagosome decreases, releasing PIP5K and enabling phagocytosis to proceed.

Rab7 and Arl8 GTPases are Necessary for Lysosome Tubulation in Macrophages

Lysosomes provide a niche for molecular digestion and are a convergence point for endocytic trafficking, phagosome maturation and autophagy. Typically, lysosomes are small, globular organelles that appear punctate under the fluorescence microscope. However, activating agents like phorbol esters transform macrophage lysosomes into tubular lysosomes (TLs), which have been implicated in retention of pinocytic uptake and phagosome maturation. Moreover, dendritic cells exposed to lipopolysaccharides (LPSs) convert their punctate class II major histocompatibility complex compartment, a lysosome‐related organelle, into a tubular network that is thought to be involved in antigen presentation. Other than a requirement for microtubules and kinesin, little is known about the molecular mechanisms that drive lysosome tubulation. Here, we show that macrophage cell lines readily form TLs after LPS exposure, with a requirement for the Rab7 GTPase and its effectors RILP (Rab7‐interacting lysosomal protein) and FYCO1 (coiled‐coil domain‐containing protein 1), which respectively modulate the dynein and kinesin microtubule motor proteins. We also show that Arl8B, a recently identified lysosomal GTPase, and its effector SKIP, are also important for TL biogenesis. Finally, we reveal that TLs are significantly more motile than punctate lysosomes within the same LPS‐treated cells. Therefore, we identify the first molecular regulators of lysosome tubulation and we show that TLs represent a more dynamic lysosome population.