The small GTPase RAB-35 facilitates the initiation of phagosome maturation and acts as a robustness factor for apoptotic cell clearance.

We recently identified the novel function of the small GTPase RAB-35 in apoptotic cell clearance in Caenorhabditis elegans, a process in which dying cells are engulfed and degraded inside phagosomes. We have found that RAB-35 functions in two separate steps of cell corpse clearance, cell corpse recognition and the initiation of phagosome maturation. During the latter process, RAB-35 facilitates the removal of phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) from the membranes of nascent phagosomes and the simultaneous production of phosphatidylinositol-3-P (PI(3)P) on these same membranes, a process that we have coined the PI(4,5)P2 to PI(3)P shift. RAB-35 also promotes the recruitment of the small GTPase RAB-5 to the phagosomal surface. During these processes, the activity of RAB-35 is controlled by the candidate GTPase-activating protein (GAP) TBC-10 and the candidate guanine nucleotide exchange factor (GEF) FLCN-1. Overall, RAB-35 leads a third pathway during cell corpse clearance that functions in parallel to the two known pathways, one led by the phagocytic receptor CED-1 and the other led by the CED-10/Rac1 GTPase. Here, we further report that RAB-35 acts as a robustness factor that maintains the clearance activity and embryonic viability under conditions of heat stress. Moreover, we obtained additional evidence suggesting that RAB-35 acts upstream of RAB-5 and RAB-7. To establish a precise temporal pattern for its own dissociation from phagosomal surfaces, RAB-35 controls the removal of its own GAP. We propose that RAB-35 defines a largely unexplored initial phase of phagosome maturation.

Interplay between bacterial deubiquitinase and ubiquitin E3 ligase regulates ubiquitin dynamics on Legionella phagosomes.

Legionella pneumophila extensively modulates the host ubiquitin network to create the Legionella-containing vacuole (LCV) for its replication. Many of its virulence factors function as ubiquitin ligases or deubiquitinases (DUBs). Here, we identify Lem27 as a DUB that displays a preference for diubiquitin formed by K6, K11, or K48. Lem27 is associated with the LCV where it regulates Rab10 ubiquitination in concert with SidC and SdcA, two bacterial E3 ubiquitin ligases. Structural analysis of the complex formed by an active fragment of Lem27 and the substrate-based suicide inhibitor ubiquitin-propargylamide (PA) reveals that it harbors a fold resembling those in the OTU1 DUB subfamily with a Cys-His catalytic dyad and that it recognizes ubiquitin via extensive hydrogen bonding at six contact sites. Our results establish Lem27 as a DUB that functions to regulate protein ubiquitination on L. pneumophila phagosomes by counteracting the activity of bacterial ubiquitin E3 ligases.

The NADPH Oxidase and the Phagosome.

The key purpose of phagocytosis is the destruction of pathogenic microorganisms. The phagocytes exert a wide array of killing mechanisms that allow mastering the vast majority of pathogens. One of these mechanisms consists in the production of reactive oxygen species inside the phagosome by a specific enzyme, the phagocyte NADPH oxidase. This enzyme is composed of 6 proteins that need to assemble to form a complex on the phagosomal membrane. Multiple signaling pathways tightly regulate the assembly. We briefly summarize key features of the enzyme and its regulation. We then focus on several related topics that address the activity of the NADPH oxidase during phagocytosis. Novel fluorescence microscopy techniques combined with fluorescent protein labeling of NADPH oxidase subunits opened the view on the structure and dynamics of these proteins in living cells. This combination revealed details of the role of anionic phospholipids in the control of phagosomal ROS production. It also added critical information to propose a 3D model of the complex between the cytosolic subunits prior to activation, in complement to other structural data on the oxidase.

Post-translational modifications of Beclin 1 provide multiple strategies for autophagy regulation.

Autophagy is a conserved intracellular degradation pathway essential for protein homeostasis, survival and development. Defects in autophagic pathways have been connected to a variety of human diseases, including cancer and neurodegeneration. In the process of macroautophagy, cytoplasmic cargo is enclosed in a double-membrane structure and fused to the lysosome to allow for digestion and recycling of material. Autophagosome formation is primed by the ULK complex, which enables the downstream production of PI(3)P, a key lipid signalling molecule, on the phagophore membrane. The PI(3)P is generated by the PI3 kinase (PI3K) complex, consisting of the core components VPS34, VPS15 and Beclin 1. Beclin 1 is a central player in autophagy and constitutes a molecular platform for the regulation of autophagosome formation and maturation. Post-translational modifications of Beclin 1 affect its stability, interactions and ability to regulate PI3K activity, providing the cell with a plethora of strategies to fine-tune the levels of autophagy. Being such an important regulator, Beclin 1 is a potential target for therapeutic intervention and interfering with the post-translational regulation of Beclin 1 could be one way of manipulating the levels of autophagy. In this review, we provide an overview of the known post-translational modifications of Beclin 1 that govern its role in autophagy and how these modifications are maintained by input from several upstream signalling pathways. ▓.

Cryptococcus neoformans urease affects the outcome of intracellular pathogenesis by modulating phagolysosomal pH.

Cryptococcus neoformans is a facultative intracellular pathogen and its interaction with macrophages is a key event determining the outcome of infection. Urease is a major virulence factor in C. neoformans but its role during macrophage interaction has not been characterized. Consequently, we analyzed the effect of urease on fungal-macrophage interaction using wild-type, urease-deficient and urease-complemented strains of C. neoformans. The frequency of non-lytic exocytosis events was reduced in the absence of urease. Urease-positive C. neoformans manifested reduced and delayed intracellular replication with fewer macrophages displaying phagolysosomal membrane permeabilization. The production of urease was associated with increased phagolysosomal pH, which in turn reduced growth of urease-positive C. neoformans inside macrophages. Interestingly, the ure1 mutant strain grew slower in fungal growth medium which was buffered to neutral pH (pH 7.4). Mice inoculated with macrophages carrying urease-deficient C. neoformans had lower fungal burden in the brain than mice infected with macrophages carrying wild-type strain. In contrast, the absence of urease did not affect survival of yeast when interacting with amoebae. Because of the inability of the urease deletion mutant to grow on urea as a sole nitrogen source, we hypothesize urease plays a nutritional role involved in nitrogen acquisition in the environment. Taken together, our data demonstrate that urease affects fitness within the mammalian phagosome, promoting non-lytic exocytosis while delaying intracellular replication and thus reducing phagolysosomal membrane damage, events that could facilitate cryptococcal dissemination when transported inside macrophages. This system provides an example where an enzyme involved in nutrient acquisition modulates virulence during mammalian infection.

Independent trafficking of flavocytochrome b558 and myeloperoxidase to phagosomes during phagocytosis visualised by energy-filtering and energy-dispersive spectroscopy-scanning transmission electron microscopy.

When polymorphonuclear leukocytes (PMNs) phagocytose opsonised zymosan particles (OPZ), free radicals and reactive oxygen species (ROS) are formed in the phagosomes. ROS production is mediated by NADPH oxidase (Nox), which transfers electrons in converting oxygen to superoxide (O2- ). Nox-generated O2- is rapidly converted to other ROS. Free radical-forming secretory vesicles containing the Nox redox center flavocytochrome b558, a membrane protein, and azurophil granules with packaged myeloperoxidase (MPO) have been described. Presuming the probable fusion of these vesicular and granular organelles with phagosomes, the translation process of the enzymes was investigated using energy-filtering and energy-dispersive spectroscopy-scanning transmission electron microscopy. In this work, the primary method for imaging cerium (Ce) ions demonstrated the localisation of H2 O2 generated by phagocytosing PMNs. The MPO activity of the same PMNs was continuously monitored using 0.1% 3,3'-diaminobenzidine-tetrahydrochloride (DAB) and 0.01% H2 O2 . A detailed view of these vesicular and granular structures was created by overlaying each electron micrograph with pseudocolors: blue for Ce and green for nitrogen (N).

The Voltage-Dependent Anion Channels (VDAC) of Mycobacterium avium phagosome are associated with bacterial survival and lipid export in macrophages.

Mycobacterium avium subsp. hominissuis is associated with infection of immunocompromised individuals as well as patients with chronic lung disease. M. avium infects macrophages and actively interfere with the host killing machinery such as apoptosis and autophagy. Bacteria alter the normal endosomal trafficking, prevent the maturation of phagosomes and modify many signaling pathways inside of the macrophage by secreting effector molecules into the cytoplasm. To investigate whether M. avium needs to attach to the internal surface of the vacuole membrane before releasing efferent molecules, vacuole membrane proteins were purified and binding to the surface molecules present in intracellular bacteria was evaluated. The voltage-dependent anion channels (VDAC) were identified as components of M. avium vacuoles in macrophages. M. avium mmpL4 proteins were found to bind to VDAC-1 protein. The inactivation of VDAC-1 function either by pharmacological means or siRNA lead to significant decrease of M. avium survival. Although, we could not establish a role of VDAC channels in the transport of known secreted M. avium proteins, we demonstrated that the porin channels are associated with the export of bacterial cell wall lipids outside of vacuole. Suppression of the host phagosomal transport systems and the pathogen transporter may serve as therapeutic targets for infectious diseases.

A Rab20-Dependent Membrane Trafficking Pathway Controls M. tuberculosis Replication by Regulating Phagosome Spaciousness and Integrity.

The intracellular pathogen Mycobacterium tuberculosis (Mtb) lives within phagosomes and also disrupts these organelles to access the cytosol. The host pathways and mechanisms that contribute to maintaining Mtb phagosome integrity have not been investigated. Here, we examined the spatiotemporal dynamics of Mtb-containing phagosomes and identified an interferon-gamma-stimulated and Rab20-dependent membrane trafficking pathway in macrophages that maintains Mtb in spacious proteolytic phagolysosomes. This pathway functions to promote endosomal membrane influx in infected macrophages, and is required to preserve Mtb phagosome integrity and control Mtb replication. Rab20 is specifically and significantly upregulated in the sputum of human patients with active tuberculosis. Altogether, we uncover an immune-regulated cellular pathway of defense that promotes maintenance of Mtb within intact membrane-bound compartments for efficient elimination.

GOP-1 promotes apoptotic cell degradation by activating the small GTPase Rab2 in C. elegans.

Apoptotic cells generated by programmed cell death are engulfed by phagocytes and enclosed within plasma membrane-derived phagosomes. Maturation of phagosomes involves a series of membrane-remodeling events that are governed by the sequential actions of Rab GTPases and lead to formation of phagolysosomes, where cell corpses are degraded. Here we identified gop-1 as a novel regulator of apoptotic cell clearance in Caenorhabditis elegans Loss of gop-1 affects phagosome maturation through the RAB-5-positive stage, causing defects in phagosome acidification and phagolysosome formation, phenotypes identical to and unaffected by loss of unc-108, the C. elegans Rab2 GOP-1 transiently associates with cell corpse-containing phagosomes, and loss of its function abrogates phagosomal association of UNC-108. GOP-1 interacts with GDP-bound and nucleotide-free UNC-108/Rab2, disrupts GDI-UNC-108 complexes, and promotes activation and membrane recruitment of UNC-108/Rab2 in vitro. Loss of gop-1 also abolishes association of UNC-108 with endosomes, causing defects in endosome and dense core vesicle maturation. Thus, GOP-1 is an activator of UNC-108/Rab2 in multiple processes.

Endoplasmic reticulum-resident Rab8A GTPase is involved in phagocytosis in the protozoan parasite Entamoeba histolytica.

Phagocytosis is indispensable for the pathogenesis of the intestinal protozoan parasite Entamoeba histolytica. Here, we showed that in E. histolytica Rab8A, which is generally involved in trafficking from the trans-Golgi network to the plasma membrane in other organisms but was previously identified in phagosomes of the amoeba in the proteomic analysis, primarily resides in the endoplasmic reticulum (ER) and participates in phagocytosis. We demonstrated that down-regulation of EhRab8A by small antisense RNA-mediated transcriptional gene silencing remarkably reduced adherence and phagocytosis of erythrocytes, bacteria and carboxylated latex beads. Surface biotinylation followed by SDS-PAGE analysis revealed that the surface expression of several proteins presumably involved in target recognition was reduced in the EhRab8A gene-silenced strain. Further, overexpression of wild-type EhRab8A augmented phagocytosis, whereas expression of the dominant-negative form of EhRab8A resulted in reduced phagocytosis. These results indicated that EhRab8A regulates transport of surface receptor(s) for the prey from the ER to the plasma membrane. To our knowledge, this is the first report that the ER-resident Rab GTPase is involved in phagocytosis through the regulation of trafficking of a surface receptor, supporting a premise of direct involvement of the ER in phagocytosis.

IGF-I and IGFBP-2 Stimulate AMPK Activation and Autophagy, Which Are Required for Osteoblast Differentiation.

IGF-I/insulin-like growth factor binding protein 2 (IGFBP-2) coordinately stimulate osteoblast differentiation but the mechanisms by which they function have not been determined. AMP-activated protein kinase (AMPK) is induced during differentiation and AMPK knockout mice have reduced bone mass. IGF-I modulates AMPK in other cell types; therefore, these studies determined whether IGF-I/IGFBP-2 stimulate AMPK activation and the mechanism by which AMPK modulates differentiation. Calvarial osteoblasts and MC-3T3 cells expressed activated AMPK early in differentiation and AMPK inhibitors attenuated differentiation. However, expression of constitutively activated AMPK inhibited differentiation. To resolve this discrepancy we analyzed the time course of AMPK induction. AMPK activation was required early in differentiation (day 3-6) but down-regulation of AMPK after day 9 was also necessary. IGF-I/IGFBP-2 induced AMPK through their respective receptors and blocking-receptor activation blocked AMPK induction. To determine the mechanism by which AMPK functioned we analyzed components of the autophagosome. Activated AMPK stimulated ULK-1 S555 phosphorylation as well as beclin-1 and microtubule-associated protein 1A/1B light-chain phosphatidylethanolamine conjugate (LC3II) induction. Inhibition of AMPK attenuated these changes and direct inhibition of autophagy inhibited differentiation. Conversely, expression of activated AMPK was associated with persistence of these changes beyond day 9 and inhibited differentiation. Blocking AMPK activation after day 9 down-regulated these autophagosome components and rescued differentiation. This allowed induction of mechanistic target of rapamycin and AKT, which suppressed autophagy. The results show that early induction of AMPK in response to IGF-I/IGFBP-2 followed by suppression is required for osteoblast differentiation. AMPK functions through stimulation of autophagy. The findings suggest that these early catabolic changes are important for determining the energy source for osteoblast respiration and down-regulation of these components may be required for induction of glycolysis, which is required during the final anabolic stages of differentiation.

Molecular imaging analysis of Rab GTPases in the regulation of phagocytosis and macropinocytosis.

Phagocytosis and macropinocytosis, actin-dependent endocytic pathways that mediate the uptake of particles and fluid, respectively, are fundamental routes that enable cells to sample their environment, eliminate pathogens and endogenous cell debris, and contribute to immunoprotection and the maintenance of tissue homeostasis. These processes require a well-organized network of actin cytoskeletal remodeling and membrane transport, which are spatiotemporally regulated by small GTPases. The Rab family of small GTPases, which functions as molecular switches, plays central roles in intracellular membrane trafficking. Although multiple Rab proteins are localized to phagosomes and regulate phagosome maturation, the precise role of each Rab family member in Fcγ receptor (FcγR)-mediated phagocytosis is not fully characterized. Recently, we revealed that Rab35 and Rab20 are important regulators of phagosome formation and maturation, respectively. This review summarizes the functional implication of these Rab GTPases during FcγR-mediated phagocytosis in macrophages. Currently, compared with our knowledge of the regulatory mechanisms of receptor-mediated endocytosis including phagocytosis, the molecular components and signaling cascades of macropinocytosis remain poorly elucidated. Our time-lapse imaging showed that several Rab GTPases are sequentially recruited to the membrane of macropinosomes. Based on our observations, these findings regarding the spatiotemporal localization of Rab GTPases during macropinocytosis are introduced.

Heparanase Enhances Tumor Growth and Chemoresistance by Promoting Autophagy.

Heparanase is the only enzyme in mammals capable of cleaving heparan sulfate, an activity implicated in tumor inflammation, angiogenesis, and metastasis. Heparanase is secreted as a latent enzyme that is internalized and subjected to proteolytic processing and activation in lysosomes. Its role under normal conditions has yet to be understood. Here, we provide evidence that heparanase resides within autophagosomes, where studies in heparanase-deficient or transgenic mice established its contributions to autophagy. The protumorigenic properties of heparanase were found to be mediated, in part, by its proautophagic function, as demonstrated in tumor xenograft models of human cancer and through use of inhibitors of the lysosome (chloroquine) and heparanase (PG545), both alone and in combination. Notably, heparanase-overexpressing cells were more resistant to stress and chemotherapy in a manner associated with increased autophagy, effects that were reversed by chloroquine treatment. Collectively, our results establish a role for heparanase in modulating autophagy in normal and malignant cells, thereby conferring growth advantages under stress as well as resistance to chemotherapy. Cancer Res; 75(18); 3946-57. ©2015 AACR.

Identification of glucose-6-phosphate transporter as a key regulator functioning at the autophagy initiation step.

Autophagy is a catabolic process involving autophagosome formation via lysosome. However, the initiation step of autophagy is largely unknown. We found an interaction between ULK1 and ATG9 in mammalian cells and utilized the interaction to identify novel regulators of autophagy upstream of ULK1. We established a cell-based screening assay employing bimolecular fluorescence complementation. By performing gain-of-function screening, we identified G6PT as an autophagy activator. G6PT enhanced the interaction between N-terminal Venus-tagged ULK1 and C-terminal Venus-tagged ATG9, and increased autophagic flux independent of its transport activity. G6PT negatively regulated mTORC1 activity, demonstrating that G6PT functions upstream of mTORC1 in stimulating autophagy.

Visualization of Atg3 during autophagosome formation in Saccharomyces cerevisiae.

Macroautophagy (autophagy) is a highly conserved cellular recycling process involved in degradation of eukaryotic cellular components. During autophagy, macromolecules and organelles are sequestered into the double-membrane autophagosome and degraded in the vacuole/lysosome. Autophagy-related 8 (Atg8), a core Atg protein essential for autophagosome formation, is a marker of several autophagic structures: the pre-autophagosomal structure (PAS), isolation membrane (IM), and autophagosome. Atg8 is conjugated to phosphatidylethanolamine (PE) through a ubiquitin-like conjugation system to yield Atg8-PE; this reaction is called Atg8 lipidation. Although the mechanisms of Atg8 lipidation have been well studied in vitro, the cellular locale of Atg8 lipidation remains enigmatic. Atg3 is an E2-like enzyme that catalyzes the conjugation reaction between Atg8 and PE. Therefore, we hypothesized that the localization of Atg3 would provide insights about the site of the lipidation reaction. To explore this idea, we constructed functional GFP-tagged Atg3 (Atg3-GFP) by inserting the GFP portion immediately after the handle region of Atg3. During autophagy, Atg3-GFP transiently formed a single dot per cell on the vacuolar membrane. This Atg3-GFP dot colocalized with 2× mCherry-tagged Atg8, demonstrating that Atg3 is localized to autophagic structures. Furthermore, we found that Atg3-GFP is localized to the IM by fine-localization analysis. The localization of Atg3 suggests that Atg3 plays an important role in autophagosome formation at the IM.

ZMP: a master regulator of one-carbon metabolism.

In this issue, Kim et al. (2015) show that ZMP (5-aminoimidazole-4-carboxamide ribonucleotide) binds to and activates a conserved riboswitch to regulate expression of one-carbon metabolism genes.

mTORC1 phosphorylates UVRAG to negatively regulate autophagosome and endosome maturation.

mTORC1 plays a key role in autophagy as a negative regulator. The currently known targets of mTORC1 in the autophagy pathway mainly function at early stages of autophagosome formation. Here, we identify that mTORC1 inhibits later stages of autophagy by phosphorylating UVRAG. Under nutrient-enriched conditions, mTORC1 binds and phosphorylates UVRAG. The phosphorylation positively regulates the association of UVRAG with RUBICON, thereby enhancing the antagonizing effect of RUBICON on UVRAG-mediated autophagosome maturation. Upon dephosphorylation, UVRAG is released from RUBICON to interact with the HOPS complex, a component for the late endosome and lysosome fusion machinery, and enhances autophagosome and endosome maturation. Consequently, the dephosphorylation of UVRAG facilitates the lysosomal degradation of epidermal growth factor receptor (EGFR), reduces EGFR signaling, and suppresses cancer cell proliferation and tumor growth. These results demonstrate that mTORC1 engages in late stages of autophagy and endosome maturation, defining a broader range of mTORC1 functions in the membrane-associated processes.

An improved flow cytometry assay to monitor phagosome acidification.

Phago-lysosome formation is important for cell-autonomous immunity to intracellular pathogens, antigen presentation and metabolism. A hallmark feature of phago-lysosomal compartments is that they undergo progressive luminal acidification controlled by the activation of vacuolar V-ATPase. Acidification is required for many enzymatic processes taking place in phago-lysosomes, like proteolysis, and supports the microbicidal activity of macrophages. Here we present a new quantitative methodology to assess phagosome acidification by flow cytometry based on the use of bi-fluorescent particles. This method relies on the use of UV polystyrene beads labelled with the acid sensor pHrodo-succinimidyl ester (pHrodo(TM) SE red) and enables us to dissociate particle association with phagocytes from their engulfment in acidified compartments. This methodology is well suited to monitor the acidification of phagosomes formed in vivo after fluorescent bead administration.

Myeloperoxidase in human neutrophil host defence.

Human neutrophils represent the predominant leucocyte in circulation and the first responder to infection. Concurrent with ingestion of microorganisms, neutrophils activate and assemble the NADPH oxidase at the phagosome, thereby generating superoxide anion and hydrogen peroxide. Concomitantly, granules release their contents into the phagosome, where the antimicrobial proteins and enzymes synergize with oxidants to create an environment toxic to the captured microbe. The most rapid and complete antimicrobial action by human neutrophils against many organisms relies on the combined efforts of the azurophilic granule protein myeloperoxidase and hydrogen peroxide from the NADPH oxidase to oxidize chloride, thereby generating hypochlorous acid and a host of downstream reaction products. Although individual components of the neutrophil antimicrobial response exhibit specific activities in isolation, the situation in the environment of the phagosome is far more complicated, a consequence of multiple and complex interactions among oxidants, proteins and their by-products. In most cases, the cooperative interactions among the phagosomal contents, both from the host and the microbe, culminate in loss of viability of the ingested organism.

The lysosomal cathepsin protease CPL-1 plays a leading role in phagosomal degradation of apoptotic cells in Caenorhabditis elegans.

During programmed cell death, the clearance of apoptotic cells is achieved by their phagocytosis and delivery to lysosomes for destruction in engulfing cells. However, the role of lysosomal proteases in cell corpse destruction is not understood. Here we report the identification of the lysosomal cathepsin CPL-1 as an indispensable protease for apoptotic cell removal in Caenorhabditis elegans. We find that loss of cpl-1 function leads to strong accumulation of germ cell corpses, which results from a failure in degradation rather than engulfment. CPL-1 is expressed in a variety of cell types, including engulfment cells, and its mutation does not affect the maturation of cell corpse-containing phagosomes, including phagosomal recruitment of maturation effectors and phagosome acidification. Of importance, we find that phagosomal recruitment and incorporation of CPL-1 occurs before digestion of cell corpses, which depends on factors required for phagolysosome formation. Using RNA interference, we further examine the role of other candidate lysosomal proteases in cell corpse clearance but find that they do not obviously affect this process. Collectively, these findings establish CPL-1 as the leading lysosomal protease required for elimination of apoptotic cells in C. elegans.