Cytosolic Access of Mycobacterium tuberculosis: Critical Impact of Phagosomal Acidification Control and Demonstration of Occurrence In Vivo

Mycobacterium tuberculosis (Mtb) uses efficient strategies to evade the eradication by professional phagocytes, involving—as recently confirmed—escape from phagosomal confinement. While Mtb determinants, such as the ESX-1 type VII secretion system, that contribute to this phenomenon are known, the host cell factors governing this important biological process are yet unexplored. Using a newly developed flow-cytometric approach for Mtb, we show that macrophages expressing the phagosomal bivalent cation transporter Nramp-1, are much less susceptible to phagosomal rupture. Together with results from the use of the phagosome acidification inhibitor bafilomycin, we demonstrate that restriction of phagosomal acidification is a prerequisite for mycobacterial phagosomal rupture and cytosolic contact. Using different in vivo approaches including an enrichment and screen for tracking rare infected phagocytes carrying the CD45.1 hematopoietic allelic marker, we here provide first and unique evidence of M. tuberculosis-mediated phagosomal rupture in mouse spleen and lungs and in numerous phagocyte types. Our results, linking the ability of restriction of phagosome acidification to cytosolic access, provide an important conceptual advance for our knowledge on host processes targeted by Mtb evasion strategies.     

Dynamic ca(2+)changes in neutrophil phagosomes A source for intracellular ca(2+)during phagolysosome formation?

An increase in cytosolic Ca(2+)concentration periphagosomally is critical for phagolysosomal formation and neutrophil elimination of microbes. The Ca(2+)increase could be achieved through release of Ca(2+)from mobilized intracellular stores. Alternatively, Ca(2+)that passively enter the phagosome during phagocytosis could be provided by the phagosome. Intraphagosomal Ca(2+)changes in single human neutrophils was measured during phagocytosis of serum opsonized Fura-2-conjugated zymosan particles, using a digital image processing system for microspectrofluorometry. A decrease in phagosomal Ca(2+)down to nanomolar concentrations was seen within minutes following phagosomal closure. Blockage of plasma membrane Ca(2+)channels by econazole abolished this decrease. The fluorescence properties of Fura-2 zymosan were retained after phagocytosis and stable to pH changes, reactive oxygen species, and proteolytic enzymes. We suggest that Ca(2+)ions present in the phagosome enter the cell cytosol through Ca(2+)channels in the phagosomal membrane, achieving a localized Ca(2+)rise that is important for phagosome processing.     

Microfilaments and microtubules regulate recycling from phagosomes

It is clear that the uptake of large particles is driven by a finely controlled rearrangement of the actin cytoskeleton. Here, we present evidence that myosin motors and microtubules also participate in the Fcgamma-mediated internalization process in macrophages. During phagocytosis, a substantial amount of plasma membrane is internalized without a net reduction in cell surface area, implying an active mechanism for membrane recycling. Despite the importance of this recycling pathway in phagosome maturation and in the retrieval of immunogenic peptides from phagosomes, the cytoskeletal requirements are largely unknown. To study this vesicle-mediated recycling transport, we used a biochemical assay and we developed a method to follow this process by confocal fluorescence microscopy. Interestingly, recycling from the phagosomal compartment was increased when the actin cortex was thinned by inhibitors of F-actin. In contrast, depolymerization of microtubules diminished both phagocytosis and recycling from phagosomes. Our results suggest that actin and microtubules are needed not only for phagosome biogenesis but also at other steps along the phagocytic pathway.     

Magnetic phagosome motion in J774A.1 macrophages: influence of cytoskeletal drugs

The role of the different cytoskeletal structures like microfilaments (MF), microtubuli (MT), and intermediate filaments (IF) in phagosome motion is unclear. These cytoskeletal units play an important role in macrophage function (migration, phagocytosis, phagosome transport). We investigated ferromagnetic phagosome motions by cell magnetometry. J774A.1 macrophages were incubated with 1.3-microm spherical magnetite particles for 24 h, after which more than 90% of the particles had been phagocytized. Phagosome motions can be caused either by the cell itself (relaxation) or by applying magnetic twisting forces, yielding cell stiffness and viscoelastic properties of the cytoskeleton. Apparent viscosity of the cytoplasm was non-Newtonian and showed a shear-rate-dependent power law behavior. Elastically stored energy does not force the magnetic phagosomes back to their initial orientation: 57% of the twisting shear was not recoverable. Cytoskeletal drugs, like Cytochalasin D (CyD, 2 - 4 microM), Colchicine (CoL, 10 microM), or Acrylamide (AcL, 40 mM) were added in order to disturb the different cytoskeletal structures. AcL disintegrates IF, but affected neither stochastic (relaxation) nor directed phagosome motions. CyD disrupts MF, resulting in a retarded stochastic phagosome motion (relative decay 0.53 +/- 0.01 after 5 min versus 0.34 +/- 0.01 in control), whereas phagosome twisting shows only a small response with a 9% increase of stiffness and a small reduction of recoverable strain. CoL depolymerizes the MT, inducing a moderately accelerated relaxation (relative decay 0.28 +/- 0.01 after 5 min) and a 10% increase of cell stiffness, where the pure viscous shear is increased and the viscoelastic recoil is inhibited by 40%. Combining the two drugs conserves both effects. After disintegrating either MF or MT, phagosome motion and cytoskeletal stiffness reflect the behavior of either MT or MF, respectively. The results verify that the dominant phagosome transport mechanism is MF-associated. MT depolymerization by CoL induces an activation of the F-actin synthesis, which may induce an accelerated relaxation and an increase of stiffness. Cell mechanical properties are not modulated by MF depolymerization, whereas MT depolymerization causes a loss of viscous resistance and a loss of cell elasticity. The mean energy for stochastic phagosome transport is 5*10(-18) Joules and corresponds to a force of 7 pN on a single 1.3-microm phagosome.     

Ethanol suppresses phagosomal adhesion maturation, Rac activation, and subsequent actin polymerization during FcγR-mediated phagocytosis

Clinical and laboratory investigations have provided evidence that ethanol suppresses normal lung immunity. Our initial studies revealed that acute ethanol exposure results in transient suppression of phagocytosis of Pseudomonas aeruginosa by macrophages as early as 3 h after initial exposure. Focusing on mechanisms by which ethanol decreases macrophage Fcγ-receptor (FcγR) phagocytosis we targeted the study on the focal adhesion and cytoskeletal elements that are necessary for phagosome progression. Ethanol inhibited macrophage phagocytosis of IgG-coated bead recruitment of actin to the site of the phagosome, dampened the phosphorylation of vinculin, but had no effect on paxillin phosphorylation suggesting a loss in "phagosomal adhesion" maturation. Moreover, our observations revealed that FcγR-phagocytosis induced Rac activation, which was increased by only 50% in ethanol exposed cells, compared to 175% in the absence of ethanol. This work is the first to show evidence of the cellular mechanisms involved in the ethanol-induced suppression of FcγR-mediated phagocytosis.     

Interleukin-4 alters early phagosome phenotype by modulating class I PI3K dependent lipid remodeling and protein recruitment

Phagocytosis is a complex process that involves membranelipid remodeling and the attraction and retention of key effector proteins. Phagosome phenotype depends on the type of receptor engaged and can be influenced by extracellular signals. Interleukin 4 (IL-4) is a cytokine that induces the alternative activation of macrophages (MΦs) upon prolonged exposure, triggering a different cell phenotype that has an altered phagocytic capacity. In contrast, the direct effects of IL-4 during phagocytosis remain unknown. Here, we investigate the impact of short-term IL-4 exposure (1 hour) during phagocytosis of IgG-opsonized yeast particles by MΦs. By time-lapse confocal microscopy of GFP-tagged lipid-sensing probes, we show that IL-4 increases the negative charge of the phagosomal membrane by prolonging the presence of the negatively charged second messenger PI(3,4,5)P3. Biochemical assays reveal an enhanced PI3K/Akt activity upon phagocytosis in the presence of IL-4. Blocking the specific class I PI3K after the onset of phagocytosis completely abrogates the IL-4-induced changes in lipid remodeling and concomitant membrane charge. Finally, we show that IL-4 direct signaling leads to a significantly prolonged retention profile of the signaling molecules Rac1 and Rab5 to the phagosomal membrane in a PI3K-dependent manner. This protracted early phagosome phenotype suggests an altered maturation, which is supported by the delayed phagosome acidification measured in the presence of IL-4. Our findings reveal that molecular differences in IL-4 levels, in the extracellular microenvironment, influence the coordination of lipid remodeling and protein recruitment, which determine phagosome phenotype and, eventually, fate. Endosomal and phagosomal membranes provide topological constraints to signaling molecules. Therefore, changes in the phagosome phenotype modulated by extracellular factors may represent an additional mechanism that regulates the outcome of phagocytosis and could have significant impact on the net biochemical output of a cell.     

Phagocytosis of IgG-coated polystyrene beads by macrophages induces and requires high membrane order

The biochemical composition and biophysical properties of cell membranes are hypothesized to affect cellular processes such as phagocytosis. Here, we examined the plasma membranes of murine macrophage cell lines during the early stages of uptake of immunoglobulin G (IgG)-coated polystyrene particles. We found that the plasma membrane undergoes rapid actin-independent condensation to form highly ordered phagosomal membranes, the biophysical hallmark of lipid rafts. Surprisingly, these membranes are depleted of cholesterol and enriched in sphingomyelin and ceramide. Inhibition of sphingomyelinase activity impairs membrane condensation, F-actin accumulation at phagocytic cups and particle uptake. Switching phagosomal membranes to a cholesterol-rich environment had no effect on membrane condensation and the rate of phagocytosis. In contrast, preventing membrane condensation with the oxysterol 7-ketocholesterol, even in the presence of ceramide, blocked F-actin dissociation from nascent phagosomes and particle uptake. In conclusion, our results suggest that ordered membranes function to co-ordinate F-actin remodelling and that the biophysical properties of phagosomal membranes are essential for phagocytosis.     

Motion and twisting of magnetic particles ingested by alveolar macrophages in the human lung: effect of smoking and disease

Background

Magnetic microparticles being ingested by alveolar macrophages can be used as a monitor for intracellular phagosome motions and cytoskeletal mechanical properties. These studies can be performed in the human lung after voluntary inhalation. The influence of cigarette smoking and lung diseases on cytoskeleton dependent functions was studied.

Methods

Spherical 1.3 μm diameter ferrimagnetic iron oxide particles were inhaled by 17 healthy volunteers (40 – 65 years), 15 patients with sarcoidosis (SAR), 12 patients with idiopathic pulmonary fibrosis (IPF), and 18 patients with chronic obstructive bronchitis (COB). The retained particles were magnetized and aligned in an external 100 mT magnetic field. All magnetized particles induce a weak magnetic field of the lung, which was detected by a sensitive SQUID (superconducting quantum interference device) sensor. Cytoskeletal reorganizations within macrophages and intracellular transport cause stochastic magnetic dipole rotations, which are reflected in a decay of the magnetic lung field, called relaxation. Directed phagosome motion was induced in a weak magnetic twisting field. The resistance of the cytoplasm to particle twisting was characterized by the viscosity and the stiffness (ratio between stress to strain) of the cytoskeleton.

Results

One week after particle inhalation and later macrophage motility (relaxation) and cytoskeletal stiffness was not influenced by cigarette smoking, neither in healthy subjects, nor in the patients. Patients with IPF showed in tendency a faster relaxation (p = 0.06). Particle twisting revealed a non-Newtonian viscosity with a pure viscous and a viscoelastic compartment. The viscous shear was dominant, and only 27% of the shear recoiled and reflected viscoelastic properties. In patients with IPF, the stiffness was reduced by 60% (p < 0.02). An analysis of the shear rate and stress dependence of particle twisting allows correlating the rheological compartments to cytoskeletal subunits, in which microtubules mediate the pure viscous (non-recoverable) shear and microfilaments mediate the viscoelastic (recoverable) behavior. The missing correlation between relaxation and particle twisting shows that both stochastic and directed phagosome motion reflect different cytoskeletal mechanisms.

Conclusion

Faster relaxation and a soft cytoskeleton in patients with IPF indicate alterations in cytoskeleton dependent functions of alveolar macrophages, which may cause dysfunction's in the alveolar defense, like a slower migration, a retarded phagocytosis, a disturbed phagosome lysosome fusion and an impaired clearance.

     

Localized biphasic changes in phosphatidylinositol-4,5-bisphosphate at sites of phagocytosis

Phagocytosis requires localized and transient remodeling of actin filaments. Phosphoinositide signaling is believed to play an important role in cytoskeletal organization, but it is unclear whether lipids, which can diffuse along the membrane, can mediate the focal actin assembly required for phagocytosis. We used imaging of fluorescent chimeras of pleckstrin homology and C1 domains in live macrophages to monitor the distribution of phosphatidylinositol-4,5-bisphosphate (4,5-PIP(2)) and diacylglycerol, respectively, during phagocytosis. Our results reveal a sequence of exquisitely localized, coordinated steps in phospholipid metabolism: a focal, rapid accumulation of 4,5-PIP(2) accompanied by recruitment of type Ialpha phosphatidylinositol phosphate kinase to the phagosomal cup, followed by disappearance of the phosphoinositide as the phagosome seals. Loss of 4,5-PIP(2) correlated with mobilization of phospholipase Cgamma (PLCgamma) and with the localized formation of diacylglycerol. The presence of 4, 5-PIP(2) and active PLCgamma at the phagosome was shown to be essential for effective particle ingestion. The temporal sequence of phosphoinositide metabolism suggests that accumulation of 4,5-PIP(2) is involved in the initial recruitment of actin to the phagocytic cup, while its degradation contributes to the subsequent cytoskeletal remodeling.     

The human macrophage sodium channel NaV1.5 regulates mycobacteria processing through organelle polarization and localized calcium oscillations

Phagocytosis and intracellular processing of mycobacteria by macrophages are complex cellular processes that require spatial and temporal coordination of particle uptake, organelle movement, activation of signaling pathways, and channel-mediated ionic flux. Recent work demonstrated that human macrophage NaV1.5, an intracellular voltage-gated sodium channel expressed on late endosomes, enhances endosomal acidification and phagocytosis. Here, using bacillus Camille-Guerin (BCG) as a model of mycobacterial infection, we examined how this channel regulates phagocytosis and phagosome maturation in human macrophages. Knockdown of NaV1.5 reduced high capacity uptake of labeled BCG. BCG-containing, NaV1.5-expressing cells demonstrated localization of NaV1.5 and Rab-7 positive endosomes and mitochondria to periphagosome regions that was not observed in NaV1.5-deficient cells. Knockdown of the channel reduced the initial calcium response following bacterial challenge and prevented the generation of prolonged and localized calcium oscillations during phagosome maturation. Inhibition of the mitochondrial Na(+) /Ca(2+) exchanger also prevented prolonged calcium oscillations during phagosome maturation. These results suggest that NaV1.5 and mitochondrial-dependent calcium signaling regulate mycobacteria phagocytosis and phagosome maturation in human macrophages through spatial-temporal coordination of calcium signaling within a unique subcellular region.     

Rab GTPases regulating phagosome maturation are differentially recruited to mycobacterial phagosomes

Mycobacterium tuberculosis (M. tb) is an intracellular pathogen that can replicate within infected macrophages. The ability of M. tb to arrest phagosome maturation is believed to facilitate its intracellular multiplication. Rab GTPases regulate membrane trafficking, but details of how Rab GTPases regulate phagosome maturation and how M. tb modulates their localization during inhibiting phagolysosome biogenesis remain elusive. We compared the localization of 42 distinct Rab GTPases to phagosomes containing either Staphylococcus aureus or M. tb. The phagosomes containing S. aureus were associated with 22 Rab GTPases, but only 5 of these showed similar localization kinetics as the phagosomes containing M. tb. The Rab GTPases responsible for phagosome maturation, phagosomal acidification and recruitment of cathepsin D were examined in macrophages expressing the dominant-negative form of each Rab GTPase. LysoTracker staining and immunofluorescence microscopy revealed that Rab7, Rab20 and Rab39 regulated phagosomal acidification and Rab7, Rab20, Rab22b, Rab32, Rab34, Rab38 and Rab43 controlled the recruitment of cathepsin D to the phagosome. These results suggest that phagosome maturation is achieved by a series of interactions between Rab GTPases and phagosomes and that differential recruitment of these Rab GTPases, except for Rab22b and Rab43, to M. tb-containing phagosomes is involved in arresting phagosome maturation and inhibiting phagolysosome biogenesis.     

The Protein Tyrosine Phosphatase SHP-1 Regulates Phagolysosome Biogenesis

The process of phagocytosis and phagosome maturation involves the recruitment of effector proteins that participate in phagosome formation and in the acidification and/or fusion with various endocytic vesicles. In the current study, we investigated the role of the Src homology region 2 domain-containing phosphatase 1 (SHP-1) in phagolysosome biogenesis. To this end, we used immortalized bone marrow macrophages derived from SHP-1-deficient motheaten mice and their wild-type littermates. We found that SHP-1 is recruited early and remains present on phagosomes for up to 4 h postphagocytosis. Using confocal immunofluorescence microscopy and Western blot analyses on purified phagosome extracts, we observed an impaired recruitment of lysosomal-associated membrane protein 1 in SHP-1-deficient macrophages. Moreover, Western blot analyses revealed that whereas the 51-kDa procathepsin D is recruited to phagosomes, it is not processed into the 46-kDa cathepsin D in the absence of SHP-1, suggesting a defect in acidification. Using the lysosomotropic agent LysoTracker as an indicator of phagosomal pH, we obtained evidence that in the absence of SHP-1, phagosome acidification was impaired. Taken together, these results are consistent with a role for SHP-1 in the regulation of signaling or membrane fusion events involved in phagolysosome biogenesis.     

Dual functions of Bruton's tyrosine kinase and Tec kinase during Fcgamma receptor-induced signaling and phagocytosis

Tec family nonreceptor tyrosine kinases are expressed by hematopoietic cells, activate phospholipase C (PLC)gamma, and regulate cytoskeletal rearrangement, yet their role in FcgammaR-induced signaling and phagocytosis remains unknown. We demonstrate in this study that Bruton's tyrosine kinase (Btk) and Tec, the only Tec kinases expressed by RAW 264.7 cells, are activated throughout phagocytosis. Activated Btk and Tec kinase accumulate at an early stage at the base of phagocytic cups and inhibition of their activity by the specific inhibitor LFM-A13 or expression by small interfering RNA significantly inhibited FcgammaR-induced phagocytosis. Similarly, a significant role for these kinases in phagocytosis was found in primary macrophages. FcgammaR-induced activation of Mac-1, which is required for optimal phagocytosis, was markedly inhibited and our findings suggest that the roles of kinases Btk and Tec in Mac-1 activation account for their functions in the early stages of phagocytosis. Initial activation of PLCgamma2, the predominant PLC isoform in RAW 264.7 cells, is dependent on Syk. In contrast, a late and prolonged activation of PLCgamma2 was dependent on Btk and Tec. We found accumulation of diacylglycerol (DAG), a PLCgamma product, in phagosome membranes, and activated Btk, but not Tec, colocalized with phagosomal DAG. Inhibition of Tec family kinase activity increased the level of DAG in phagosomes, suggesting a negative regulatory role for Btk. Tec, in contrast, clustered at sites near phagosome formation. In summary, we elucidated that Tec family kinases participate in at least two stages of FcgammaR-mediated phagocytosis: activation of Mac-1 during ingestion, and after phagosome formation, during which Btk and Tec potentially have distinct roles.     

Phthiocerol Dimycocerosates of M. tuberculosis Participate in Macrophage Invasion by Inducing Changes in the Organization of Plasma Membrane Lipids

Phthiocerol dimycocerosates (DIM) are major virulence factors of Mycobacterium tuberculosis (Mtb), in particular during the early step of infection when bacilli encounter their host macrophages. However, their cellular and molecular mechanisms of action remain unknown. Using Mtb mutants deleted for genes involved in DIM biosynthesis, we demonstrated that DIM participate both in the receptor-dependent phagocytosis of Mtb and the prevention of phagosomal acidification. The effects of DIM required a state of the membrane fluidity as demonstrated by experiments conducted with cholesterol-depleting drugs that abolished the differences in phagocytosis efficiency and phagosome acidification observed between wild-type and mutant strains. The insertion of a new cholesterol-pyrene probe in living cells demonstrated that the polarity of the membrane hydrophobic core changed upon contact with Mtb whereas the lateral diffusion of cholesterol was unaffected. This effect was dependent on DIM and was consistent with the effect observed following DIM insertion in model membrane. Therefore, we propose that DIM control the invasion of macrophages by Mtb by targeting lipid organisation in the host membrane, thereby modifying its biophysical properties. The DIM-induced changes in lipid ordering favour the efficiency of receptor-mediated phagocytosis of Mtb and contribute to the control of phagosomal pH driving bacilli in a protective niche.     

Infection of macrophages with Mycobacterium tuberculosis induces global modifications to phagosomal function

The phagosome is a central mediator of both the homeostatic and microbicidal functions of a macrophage. Following phagocytosis, Mycobacterium tuberculosis (Mtb) is able to establish infection through arresting phagosome maturation and avoiding the consequences of delivery to the lysosome. The infection of a macrophage by Mtb leads to marked changes in the behaviour of both the macrophage and the surrounding tissue as the bacterium modulates its environment to promote its survival. In this study, we use functional physiological assays to probe the biology of the phagosomal network in Mtb‐infected macrophages. The resulting data demonstrate that Mtb modifies phagosomal function in a TLR2/TLR4‐dependent manner, and that most of these modifications are consistent with an increase in the activation status of the cell. Specifically, superoxide burst is enhanced and lipolytic activity is decreased upon infection. There are some species‐ or cell type‐specific differences between human and murine macrophages in the rates of acidification and the degree of proteolysis. However, the most significant modification is the marked reduction in intra‐phagosomal lipolysis because this correlates with the marked increase in the retention of host lipids in the infected macrophage, which provides a potential source of nutrients that can be accessed by Mtb.     

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.     

Involvement of heterotrimeric G proteins in phagocytosis and recycling from the phagosomal compartment.

Phagocytosis is a receptor-mediated process by which specialized cell types engulf large extracellular particles. Phagosome maturation involves a series of intracellular membrane fusion and budding events resulting in the delivery of particles to compartments enriched in lysosomal hydrolases where they are digested. Substantial amounts of plasma membrane and many phagosomal proteins, such as receptors, rapidly recycle to the plasma membrane following phagosome formation. Despite the importance of this recycling pathway in phagosome maturation and in the retrieval of immunogenic peptides from phagosomes, the molecular machinery involved is largely unknown. To assess the participation of GTPases in phagocytosis and recycling from phagosomes we used aluminum fluoride (AIF(-)(4)), which activates the GDP-bound form of stimulatory and inhibitory trimeric G proteins. AlF(-)(4) inhibited both the uptake to and the recycling from the phagosomal compartment. Cholera toxin, which activates Galphas, and pertussis toxin, which uncouples Gi and Go from receptors, were effective inhibitors of phagocytosis. However, both toxins stimulated recycling from phagosomes. These results suggest that more than one GTP-binding protein participates either directly or indirectly not only in phagocytosis, but also in maturation and recycling from phagosomes, and thereby assign a role for heterotrimeric G proteins in controlling traffic through the phagocytic pathway.     

Dynamics and function of phospholipase D and phosphatidic acid during phagocytosis

Phospholipase D (PLD) produces phosphatidic acid (PA), an established intracellular signalling lipid that has been also implicated in vesicular trafficking, and as such, PLD could play multiple roles during phagocytosis. Using an RNA interference strategy, we show that endogenous PLD1 and PLD2 are necessary for efficient phagocytosis in murine macrophages, in line with results obtained with wild-type constructs and catalytically inactive PLD mutants which, respectively, enhance and inhibit phagocytosis. Furthermore, we found that PA is transiently produced at sites of phagosome formation. Macrophage PLD1 and PLD2 differ in their subcellular distributions. PLD1 is associated with cytoplasmic vesicles, identified as a late endosomal/lysosomal compartment, whereas PLD2 localizes at the plasma membrane. In living cells undergoing phagocytosis, PLD1 vesicles are recruited to nascent and internalized phagosomes, whereas PLD2 is only observed on nascent phagosomes. These results provide evidence that both PLD isoforms are required for phagosome formation, but only PLD1 seems to be implicated in later stages of phagocytosis occurring after phagosomal internalization.     

Superoxide production at phagosomal cup/phagosome through beta I protein kinase C during Fc gamma R-mediated phagocytosis in microglia

Protein kinase C (PKC) plays a prominent role in immune signaling. To elucidate the signal transduction in a respiratory burst and isoform-specific function of PKC during FcgammaR-mediated phagocytosis, we used live, digital fluorescence imaging of mouse microglial cells expressing GFP-tagged molecules. betaI PKC, epsilonPKC, and diacylglycerol kinase (DGK) beta dynamically and transiently accumulated around IgG-opsonized beads (BIgG). Moreover, the accumulation of p47(phox), an essential cytosolic component of NADPH oxidase and a substrate for betaI PKC, at the phagosomal cup/phagosome was apparent during BIgG ingestion. Superoxide (O(2)(-)) production was profoundly inhibited by G?6976, a cPKC inhibitor, and dramatically increased by the DGK inhibitor, R59949. Ultrastructural analysis revealed that BIgG induced O(2)(-) production at the phagosome but not at the intracellular granules. We conclude that activation/accumulation of betaI PKC is involved in O(2)(-) production, and that O(2)(-) production is primarily initiated at the phagosomal cup/phagosome. This study also suggests that DGKbeta plays a prominent role in regulation of O(2)(-) production during FcgammaR-mediated phagocytosis.     

Reconstitution of recycling from the phagosomal compartment in streptolysin O-permeabilized macrophages: role of Rab11

By phagocytosis, macrophages engulf large particles, microorganisms and senescent cells in vesicles called phagosomes. Many internalized proteins rapidly shuttle back to the plasma membrane following phagosome biogenesis. Here, we report a new approach to the study of recycling from the phagosomal compartment: streptolysin O- (SLO) permeabilized macrophages. In this semi-intact cell system, energy and cytosol are required to efficiently reconstitute recycling transport. Addition of GDPbetaS strongly inhibits this transport step, suggesting that a GTP-binding protein modulates the dynamics of cargo exit from the phagosomal compartment. GTPases of the Rab family control vesicular trafficking, and Rab11 is involved in transferrin receptor recycling. To unravel the role of Rab11 in the phagocytic pathway, we added recombinant proteins to SLO-permeabilized macrophages. Rab11:S25N, a negative mutant, strongly diminishes the release of recycled proteins from phagosomes. In contrast, wild type Rab11 and its positive mutant (Rab11:Q70L) favor this vesicular transport event. Using biochemical and morphological assays, we confirm that overexpression of Rab11:S25N substantially decreases recycling from phagosomes in intact cells. These findings show the requirement of a functional Rab11 for the retrieval to the plasma membrane of phagosomal content. SLO-permeabilized macrophages likely constitute a useful tool to identify new molecules involved in regulating transport along the phagocytic pathway.