Topological studies on the hydrolases bound to the intestinal brush border membrane. I. Solubilization by papain and triton X-100

Papain digestion of closed, right side out vesicles from pig, rat and rabbit jejunum brush border induces the release of the hydrolases bound to the membrane without grossly affecting the lipid bilayer limiting the vesicles. This observation definitely proves that intestinal hydrolases are surface components attached to the external side of the membrane. All proteins released by papain could be identified by electrophoresis and immunoelectrophoresis to already known intestinal hydrolases, with the exception of an unidentified substance strongly stained by the Schiff's reagent.The early observation that the aminopeptidase form released from pig brush border by Triton X-100 is different from that released by papain was extended to other hydrolases from pig, rat and rabbit. In some cases, the Triton-released form could be converted by further proteolytic digestion into a new form similar to that liberated by papain. These facts may be related to the existence of hydrophobic anchors retaining the intestinal hydrolases to the membrane surface.     

Fusion of newly formed phagosomes with endosomes in intact cells and in a cell-free system

Phagosomes are membrane-bound vesicles, formed by the receptor-mediated internalization of particulate ligands, which exchange soluble and membrane proteins with other endocytic compartments as a part of their maturation process. This exchange of material is undoubtedly mediated by fusion of phagosomes with other membrane-bound compartments of the endocytic pathway. By using a particulate probe (fixed Staphylococcus aureus coated with mouse anti-dinitrophenol monoclonal antibody) localized in phagosomes and a soluble probe (dinitrophenol-derivitized beta-glucuronidase) internalized by receptor-mediated endocytosis, we have studied phagosome-endosome and phagosome-lysosome fusion in intact cells and in a cell-free system. Vesicle fusion was assessed by measuring beta-glucuronidase activity associated with S. aureus particles after lysis of the membranes. In intact macrophages, newly formed phagosomes fused with early endosomes and with lysosomes. Fusion with lysosomes was observed to commence after a short lag period of about 5 min. In broken-cell preparations, phagosomes were able to fuse with early endosomes. It was not possible to reconstitute phagosome-lysosome fusion in vitro. In vitro phagosome-endosome fusion required energy and cytosolic- and membrane-associated proteins. A nonhydrolyzable analog of GTP stimulated fusion at low cytosol concentrations and inhibited fusion at high cytosol concentrations. These observations indicate that the mechanisms mediating phagosome-endosome fusion are similar to those described for endosome-endosome fusion. Our results suggest that exchange of material with endosomes is an important step in the process of phagosome maturation.     

Membrane shuttle between plasma membrane, phagosomes, and pinosomes in Dictyostelium discoideum amoeboid cells

The intracellular redistribution of membrane internalized during endocytosis was studied quantitatively by a biochemical approach and by a morphometric analysis of autoradiographs in electron microscopy. Plasma membrane glycoconjugates, enzymatically labelled with radioactive galactose, were used as a membrane marker. In cells labelled at their surface either before or after the phagocytotic uptake of latex beads, subsequent endocytosis led to a redistribution of label between the plasma membrane and endosomal membranes until a steady-state was reached after about 1 h with 43% of the label on the plasma membrane. The steady-state resulted when all participating membranes carried the same surface density of label. During phagocytosis or pinocytosis the equivalent of the plasma membrane was internalized and recycled once every 20 min or 40 min, respectively. Compared to this rate a very rapid and complete mixing of membranes was observed between newly formed phagosomes and preexisting digestive vacuoles or between newly formed pinosomes and preexisting phagosomes. Due to this rapid mixing, the membranes enclosing undigestible latex beads remained fully linked to the shuttle of membrane to and from the cell surface.     

Rab20 regulates phagosome maturation in RAW264 macrophages during Fc gamma receptor-mediated phagocytosis

Rab20, a member of the Rab GTPase family, is known to be involved in membrane trafficking, however its implication in FcγR-mediated phagocytosis is unclear. We examined the spatiotemporal localization of Rab20 during phagocytosis of IgG-opsonized erythrocytes (IgG-Es) in RAW264 macrophages. By the live-cell imaging of fluorescent protein-fused Rab20, it was shown that Rab20 was transiently associated with the phagosomal membranes. During the early stage of phagosome formation, Rab20 was not localized on the membranes of phagocytic cups, but was gradually recruited to the newly formed phagosomes. Although Rab20 was colocalized with Rab5 to some extent, the association of Rab20 with the phagosomes persisted even after the loss of Rab5 from the phagosomal membranes. Then, Rab20 was colocalized with Rab7 and Lamp1, late endosomal/lysosomal markers, on the internalized phagosomes. Moreover, our analysis of Rab20 mutant expression revealed that the maturation of phagosomes was significantly delayed in cells expressing the GDP-bound mutant Rab20-T19N. These data suggest that Rab20 is an important component of phagosome and regulates the phagosome maturation during FcγR-mediated phagocytosis.     

Phagosomes are fully competent antigen-processing organelles that mediate the formation of peptide:class II MHC complexes

During the processing of particulate Ags, it is unclear whether peptide:class II MHC (MHC-II) complexes are formed within phagosomes or within endocytic compartments that receive Ag fragments from phagosomes. Murine macrophages were pulsed with latex beads conjugated with OVA. Flow or Western blot analysis of isolated phagosomes showed extensive acquisition of MHC-II, H-2M, and invariant chain within 30 min, with concurrent degradation of OVA. T hybridoma responses to isolated subcellular fractions demonstrated OVA (323-339):I-Ad complexes in phagosomes and plasma membrane but not within dense late endocytic compartments. Furthermore, when two physically separable sets of phagosomes were present within the same cells, OVA(323-339):I-Ad complexes were demonstrated in latex-OVA phagosomes but not in phagosomes containing latex beads conjugated with another protein. This implies that these complexes were formed specifically within phagosomes and were not formed elsewhere and subsequently transported to phagosomes. In addition, peptide:MHC-II complexes were shown to traffic from phagosomes to the cell surface. In conclusion, phagosomes are fully competent to process Ags and generate peptide:MHC-II complexes that are transported to the cell surface and presented to T cells.     

Regulation of the macrophage vacuolar ATPase and phagosome-lysosome fusion by Histoplasma capsulatum

Histoplasma capsulatum (Hc) maintains a phagosomal pH of about 6.5. This strategy allows Hc to obtain iron from transferrin, and minimize the activity of macrophage (Mo) lysosomal hydrolases. To determine the mechanism of pH regulation, we evaluated the function of the vacuolar ATPase (V-ATPase) in RAW264.7 Mo infected with Hc yeast or the nonpathogenic yeast Saccharomyces cerevisae (Sc). Incubation of Hc-infected Mo with bafilomycin, an inhibitor of the V-ATPase, did not affect the intracellular growth of Hc, nor did it affect the intraphagosomal pH. In contrast, upon addition of bafilomycin, phagosomes containing Sc rapidly changed their pH from 5 to 7. Hc-containing phagosomes had 5-fold less V-ATPase than Sc-containing phagosomes as quantified by immunoelectron microscopy. Furthermore, Hc-containing phagosomes inhibited phagolysosomal fusion as quantified by the presence of acid phosphatase, accumulation of LAMP2, and fusion with rhodamine B-isothiocyanate-labeled dextran-loaded lysosomes. Finally, in Hc-containing phagosomes, uptake of ferritin was equivalent to phagosomes containing Sc, indicating that Hc-containing phagosomes have full access to the early "bulk flow" endocytic pathway. Thus, Hc yeasts inhibit phagolysosomal fusion, inhibit accumulation of the V-ATPase in the phagosome, and actively acidify the phagosomal pH to 6.5 as part of their strategy to survive in Mo phagosomes.     

WASH is required for lysosomal recycling and efficient autophagic and phagocytic digestion

Wiskott-Aldrich syndrome protein and SCAR homologue (WASH) is an important regulator of vesicle trafficking. By generating actin on the surface of intracellular vesicles, WASH is able to directly regulate endosomal sorting and maturation. We report that, in Dictyostelium, WASH is also required for the lysosomal digestion of both phagocytic and autophagic cargo. Consequently, Dictyostelium cells lacking WASH are unable to grow on many bacteria or to digest their own cytoplasm to survive starvation. WASH is required for efficient phagosomal proteolysis, and proteomic analysis demonstrates that this is due to reduced delivery of lysosomal hydrolases. Both protease and lipase delivery are disrupted, and lipid catabolism is also perturbed. Starvation-induced autophagy therefore leads to phospholipid accumulation within WASH-null lysosomes. This causes the formation of multilamellar bodies typical of many lysosomal storage diseases. Mechanistically, we show that, in cells lacking WASH, cathepsin D becomes trapped in a late endosomal compartment, unable to be recycled to nascent phagosomes and autophagosomes. WASH is therefore required for the maturation of lysosomes to a stage at which hydrolases can be retrieved and reused.     

An Electron Microscopic Study of Erythrophagocytosis

The present study describes a submicroscopic surface fragmentation of erythrocytes which occurs in the ascitic fluid of rats bearing the Novikoff ascites hepatoma. The resulting fragments attach to the surface of macrophages and are phagocytized by pseudopod formation. Plasma membrane in the region of these phagocytosis vacuoles appears to condense into electron-opaque material, suggesting an alteration in its physicochemical state. Stages in intracellular digestion of intact erythrocytes or small fragments within the phagocytosis vacuoles are illustrated; no particles resembling ferritin are observed. The phagocytosis vacuoles possess high levels of acid phosphatase activity. They may be called phagosomes, a type of lysosome. There is no indication of a connection between phagosomes and other formed cytoplasmic organelles. Small vacuoles of the order of 80 mµ in diameter, which may represent pinocytosis vacuoles, are present in the cytoplasm and some appear to be in contact with the phagosome membrane, reminiscent of observations of Rose with HeLa cells.     

Chloroquine inhibits lysosomal enzyme pinocytosis and enhances lysosomal enzyme secretion by impairing receptor recycling

Adsorptive pinocytosis of acid hydrolases by fibroblasts depends on phosphomannosyl recognition markers on the enzymes and high-affinity pinocytosis receptors on the cell surface. In this study, beta- glucuronidase binding to the cell surface of attached fibroblasts was found to be saturable and inhibitable by mannose-6-phosphate (Man-6-P). Dissociation of cell-bound beta-glucuronidase occurred very slowly at neutral pH, but was greatly accelerated by lowering the pH below 6.0, or by exposure to Man-6-P. Comparison of the maximal cell surface binding and the observed rate of enzyme pinocytosis suggests that the pinocytosis receptors are replaced or reused about every 5 min. Enzyme pinocytosis was not affected by inhibition of new protein synthesis for several hours, suggesting a large pool of internal receptors and/or reuse of internalized receptors. Chloroquine treatment of normal human fibroblasts had three effects: (a) greatly enhanced secretion of newly synthesized acid hydrolases bearing the recognition marker for uptake, (b) depletion of enzyme-binding sites from the cell surface, and (c) inhibition of pinocytosis of exogenous enzyme. Only the third effect was seen in I-cell disease fibroblasts, which were also less sensitive than control cells to this effect. These observations are consistent with a model for transport of acid hydrolases that proposes that delivery of newly synthesized acid hydrolases to lysosomes requires the phosphomannosyl recognition marker on the enzymes, and intracellular receptors that segregate receptor-bound enzymes into vesicles for transport to lysosomes. This model explains how chloroquine, which raises intralysosomal pH, can disrupt both the intracellular pathway for newly synthesized acid hydrolases, and the one for uptake of exogenous enzyme by cell surface pinocytosis receptors.     

Heterogeneous modes of uptake for latex beads revealed through live cell imaging of phagocytes expressing a probe for phosphatidylinositol-(3,4,5)-trisphosphate and phosphatidylinositol-(3,4)-bisphosphate

Latex beads are the preferred phagocytic substrate in biochemical studies of phagosome composition and maturation. Using living Dictyostelium cells and fluorescent probes, we compared the properties of phagosomes formed to ingest latex beads or digestible prey. Significant differences were found during the initial steps of phagocytosis. During uptake of bacteria or yeast, PHcrac-GFP, a probe that binds to membranes enriched in PI(3,4,5)P(3) and PI(3,4)P(2), always labeled the nascent phagosome and faded shortly after it sealed. However, labeling of bead-containing phagosomes was highly variable. Beads were engulfed by phagosomes either lacking or displaying the PHcrac-GFP label, and that label, if present, often persisted for many minutes, revealing that early trafficking steps for bead-containing phagosomes are quite heterogeneous. Later stages of the endocytic pathway appeared more similar for phagosomes containing prey and latex beads. Both types of phagosomes fused with acidic endosomes while undergoing transport along microtubules, both acquired the V-ATPase and lost it prior to exocytosis, and both bound the late endosome marker vacuolin B, which was transferred to the plasma membrane upon exocytosis. We conclude that caution is needed in extrapolating results from latex bead phagosomes to phagosomes containing physiological substances, especially in early stages of the endocytic pathway.     

Biogenesis of phagolysosomes proceeds through a sequential series of interactions with the endocytic apparatus

We have examined the modifications occurring during the transformation of phagosomes into phagolysosomes in J-774 macrophages. The use of low density latex beads as markers of phagosomes (latex bead compartments, LBC) allowed the isolation of these organelles by flotation on a simple sucrose gradient. Two-dimensional gel electrophoresis, immunocytochemistry, and biochemical assays have been used to characterize the composition of LBC at different time points after their formation, as well as their interactions with the organelles of the endocytic pathway. Our results show that LBC acquire and lose various markers during their transformation into phagolysosomes. Among these are members of the rab family of small GTPases as well as proteins of the lamp family. The transfer of the LBC of lamp 2, a membrane protein associated with late endocytic structures, was shown to be microtubule dependent. Video-microscopy showed that newly formed phagosomes were involved in rapid multiple contacts with late components of the endocytic pathway. Collectively, these observations suggest that phagolysosome formation is a highly dynamic process that involves the gradual and regulated acquisition of markers from endocytic organelles.     

Uptake and utilization of human polymorphonuclear leukocyte granule myeloperoxidase by mouse peritoneal macrophages

Summary Functional myeloperoxidase contained in granules of polymorphonuclear neutrophil leukocytes or in fixed whole cells can be endocytosed by mouse peritoneal macrophages. Acquired myeloperoxidase was distributed in what we considered to be the secondary lysosomal system and, following a phagocytic stimulation, was delivered to newly formed phagosomes containing the targets.     

Quantitative and dynamic assessment of the contribution of the ER to phagosome formation

Phagosomes were traditionally thought to originate from an invagination and scission of the plasma membrane to form a distinct intracellular vacuole. An alternative model implicating the endoplasmic reticulum (ER) as a major component of nascent and maturing phagosomes was recently proposed (Gagnon et al., 2002). To reconcile these seemingly disparate hypotheses, we used a combination of biochemical, fluorescence imaging, and electron microscopy techniques to quantitatively and dynamically assess the contribution of the plasmalemma and of the ER to phagosome formation and maturation. We could not verify even a transient physical continuity between the ER and the plasma membrane, nor were we able to detect a significant contribution of the ER to forming or maturing phagosomes in either macrophages or dendritic cells. Instead, our data indicate that the plasma membrane is the main constituent of nascent and newly formed phagosomes, which are progressively remodeled by fusion with endosomal and eventually lysosomal compartments as phagosomes mature into acidic, degradative organelles.     

Fusion between phagosomes, early and late endosomes: a role for actin in fusion between late, but not early endocytic organelles

Actin is implicated in membrane fusion, but the precise mechanisms remain unclear. We showed earlier that membrane organelles catalyze the de novo assembly of F-actin that then facilitates the fusion between latex bead phagosomes and a mixture of early and late endocytic organelles. Here, we correlated the polymerization and organization of F-actin with phagosome and endocytic organelle fusion processes in vitro by using biochemistry and light and electron microscopy. When membrane organelles and cytosol were incubated at 37 degrees C with ATP, cytosolic actin polymerized rapidly and became organized into bundles and networks adjacent to membrane organelles. By 30-min incubation, a gel-like state was formed with little further polymerization of actin thereafter. Also during this time, the bulk of in vitro fusion events occurred between phagosomes/endocytic organelles. The fusion between latex bead phagosomes and late endocytic organelles, or between late endocytic organelles themselves was facilitated by actin, but we failed to detect any effect of perturbing F-actin polymerization on early endosome fusion. Consistent with this, late endosomes, like phagosomes, could nucleate F-actin, whereas early endosomes could not. We propose that actin assembled by phagosomes or late endocytic organelles can provide tracks for fusion-partner organelles to move vectorially toward them, via membrane-bound myosins, to facilitate fusion.     

Role of phosphatidylinositol 3-kinase and Rab5 effectors in phagosomal biogenesis and mycobacterial phagosome maturation arrest

Phagosomal biogenesis is a fundamental biological process of particular significance for the function of phagocytic and antigen-presenting cells. The precise mechanisms governing maturation of phagosomes into phagolysosomes are not completely understood. Here, we applied the property of pathogenic mycobacteria to cause phagosome maturation arrest in infected macrophages as a tool to dissect critical steps in phagosomal biogenesis. We report the requirement for 3-phosphoinositides and acquisition of Rab5 effector early endosome autoantigen (EEA1) as essential molecular events necessary for phagosomal maturation. Unlike the model phagosomes containing latex beads, which transiently recruited EEA1, mycobacterial phagosomes excluded this regulator of vesicular trafficking that controls membrane tethering and fusion processes within the endosomal pathway and is recruited to endosomal membranes via binding to phosphatidylinositol 3-phosphate (PtdIns[3]P). Inhibitors of phosphatidylinositol 3'(OH)-kinase (PI-3K) activity diminished EEA1 recruitment to newly formed latex bead phagosomes and blocked phagosomal acquisition of late endocytic properties, indicating that generation of PtdIns(3)P plays a role in phagosomal maturation. Microinjection into macrophages of antibodies against EEA1 and the PI-3K hVPS34 reduced acquisition of late endocytic markers by latex bead phagosomes, demonstrating an essential role of these Rab5 effectors in phagosomal biogenesis. The mechanism of EEA1 exclusion from mycobacterial phagosomes was investigated using mycobacterial products. Coating of latex beads with the major mycobacterial cell envelope glycosylated phosphatidylinositol lipoarabinomannan isolated from the virulent Mycobacterium tuberculosis H37Rv, inhibited recruitment of EEA1 to latex bead phagosomes, and diminished their maturation. These findings define the generation of phosphatidylinositol 3-phosphate and EEA1 recruitment as: (a) important regulatory events in phagosomal maturation and (b) critical molecular targets affected by M. tuberculosis. This study also identifies mycobacterial phosphoinositides as products with specialized toxic properties, interfering with discrete trafficking stages in phagosomal maturation.     

Morphometric and cytochemical studies of Dictyostelium discoideum in vegetative phase. Digestive system and membrane turnover

The morphometric analysis of growing cells shows that the membranes of the digestive apparatus have a surface area equal to the cell surface area. After yeast phagocytosis, the surface area of the membrane surrounding the ingested yeast is equal to 40% of the surface area of the cell membrane. In spite of this internalization, the cell surface remains constant. Its renewal is insured by the translocation of the membrane of the digestive system, the surface area that concomitantly decreases by 40%. This means that the influx of plasma membrane is continually compensated for by the same outflow of internal membranes. During this turnover, the characteristic polysaccharide stainability (two different stains were used) of the plasma membrane is maintained after internalization, at the level of the digestive system, despite the presence of hydrolases in the digestive vacuoles. The cytochemical demonstration of acid phosphatase shows that this enzyme penetrates into phagosomes by fusion between phagosomes and vacloles of various sizes. The debris of digested yeast are released into the culture medium after 2 h. This process of defecation is accompanied by the appearance of new pinocytotic vacuoles, which indicates that the uptake of axenic medium has resumed. A model of membrane turnover is proposed to explain these observations.     

The phagosome proteome: insight into phagosome functions

Phagosomes are key organelles for the innate ability of macrophages to participate in tissue remodeling, clear apoptotic cells, and restrict the spread of intracellular pathogens. To understand the functions of phagosomes, we initiated the systematic identification of their proteins. Using a proteomic approach, we identified >140 proteins associated with latex bead-containing phagosomes. Among these were hydrolases, proton pump ATPase subunits, and proteins of the fusion machinery, validating our approach. A series of unexpected proteins not previously described along the endocytic/phagocytic pathways were also identified, including the apoptotic proteins galectin3, Alix, and TRAIL, the anti-apoptotic protein 14-3-3, the lipid raft-enriched flotillin-1, the anti-microbial molecule lactadherin, and the small GTPase rab14. In addition, 24 spots from which the peptide masses could not be matched to entries in any database potentially represent new phagosomal proteins. The elaboration of a two-dimensional gel database of >160 identified spots allowed us to analyze how phagosome composition is modulated during phagolysosome biogenesis. Remarkably, during this process, hydrolases are not delivered in bulk to phagosomes, but are instead acquired sequentially. The systematic characterization of phagosome proteins provided new insights into phagosome functions and the protein or groups of proteins involved in and regulating these functions.     

Temporal changes of lysosome and phagosome pH during phagolysosome formation in macrophages: studies by fluorescence spectroscopy

Intravascular pH was measured within the lysosomes and newly formed phagosomes in cultured mouse peritoneal macrophages. The kinetics of pH change in both vacuolar systems was quantitatively determined within a large cell population by fluorescence spectroscopy. Additionally, pH changes within individual phagosomes were followed semiquantitatively using indicator dyes. Two novel findings were made. Firstly, the pH in new phagosomes was transiently driven alkaline (higher than physiological) even when the external medium was buffered at pH 6.5. Secondly, perturbations of phagosome-lysosome fusion had little effect upon phagosomal pH changes, even though the compounds used markedly altered the pH of the lysosomes in resting and phagocytosing cells.     

CYTOCHEMICAL OBSERVATIONS ON THE RELATIONSHIP BETWEEN LYSOSOMES AND PHAGOSOMES IN KIDNEY AND LIVER BY COMBINED STAINING FOR ACID PHOSPHATASE AND INTRAVENOUSLY INJECTED HORSERADISH PEROXIDASE

After incubation of formalin-fixed, frozen sections of kidney and liver from peroxidase-treated rats in an azo dye medium for acid phosphatase, and after subsequent incubation of the same sections with benzidine, phagosomes were stained blue and lysosomes were stained red in the same cells. It was observed that newly formed phagosomes were separate from preexisting lysosomes in the tubule cells of the kidney and in the Kupffer cells of the liver at early periods after treatment with peroxidase. At later periods, the color reactions for acid phosphatase and peroxidase occurred in the same granules. The reaction of peroxidase decreased gradually and disappeared from the phago-lysosomes after 2 to 3 days, whereas the reaction for acid phosphatase persisted. In the liver, most of the injected protein was concentrated in large phagosomes located at the periphery of the cells lining the sinusoids. The peribiliary lysosomes showed a relatively weak reaction for peroxidase in the proximity of the portal veins. After pathological changes of permeability, phagosomes and lysosomes lost their normal location and fused, in the interior of many liver cells, to form large vacuoles or spheres. The effects of a reduced load of peroxidase and the effects of the pretreatment with another protein (egg white) on the phago-lysosomes of the kidney were tested. The relationship of the fusion of phagosomes with lysosomes to the size of normal and pathological phago-lysosomes was discussed.     

Acid sphingomyelinase is required for efficient phago-lysosomal fusion

The acid sphingomyelinase (ASMase) localizes to the lumen of endosomes, phagosomes and lysosomes as well as to the outer leaflet of the plasma membrane and hydrolyses sphingomyelin to ceramide and phosphorylcholine. Using the facultative intracellular bacterium Listeria monocytogenes , we show that maturation of phagosomes into phagolysosomes is severely impaired in macrophages genetically deficient for ASMase. Unlike in wild-type macrophages, phagosomes containing L. monocytogenes in ASMase^−/− macrophages remained positive for the late phagosomal markers mannose-6-phosphate receptor (M6PR) and Rab7 for at least 2 h and, correspondingly, showed delayed acquisition of lysosomal markers like lysosome associated membrane protein 1 (Lamp1). The transfer of lysosomal fluid phase markers into phagosomes containing L. monocytogenes was severely impaired in ASMase^−/− macrophages and decreased with increasing size of the cargo. Moreover, phagosomes containing L. monocytogenes from ASMase^−/− cells acquired significantly less listeriocidal proteases cathepsin D, B and L. The results of this study suggest that ASMase is required for the proper fusion of late phagosomes with lysosomes, which is crucial for efficient transfer of lysosomal antibacterial hydrolases into phagosomes.