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Contribution of phosphatidylserine to membrane surface charge and protein targeting during phagosome maturation
Correspondence to Sergio Grinstein: sga{at}sickkids.ca
During phagocytosis, the phosphoinositide content of the activated membrane decreases sharply, as does the associated surface charge, which attracts polycationic proteins. The cytosolic leaflet of the plasma membrane is enriched in phosphatidylserine (PS); however, a lack of suitable probes has precluded investigation of the fate of this phospholipid during phagocytosis. We used a recently developed fluorescent biosensor to monitor the distribution and dynamics of PS during phagosome formation and maturation. Unlike the polyphosphoinositides, PS persists on phagosomes after sealing even when other plasmalemmal components have been depleted. High PS levels are maintained through fusion with endosomes and lysosomes and suffice to attract cationic proteins like c-Src to maturing phagosomes. Phagocytic vacuoles containing the pathogens Legionella pneumophila and Chlamydia trachomatis, which divert maturation away from the endolysosomal pathway, are devoid of PS, have little surface charge, and fail to recruit c-Src. These findings highlight a function for PS in phagosome maturation and microbial killing.
© 2009 Yeung et al.
This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.jcb.org/misc/terms.shtml). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).
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After sealing, phagosomes undergo rapid and extensive remodeling of their membrane and contents. This process, called maturation, converts phagosomes into effective microbicidal and degradative organelles. Maturation entails further changes in the lipid composition of the phagosomal membrane, raising the possibility of additional fluctuations in surface charge and, consequently, in protein targeting. However, the major polyphosphoinositides are unlikely to contribute significantly to the charge of maturing phagosomes. PI[4,5]P2 is undetectable when phagosomes seal and has not been reported to reappear during maturation, and PI[3,4,5]P3 disappears from nascent phagosomes within 1 min of sealing (Marshall et al., 2001). In contrast, little is known about phosphatidylserine (PS), the most abundant anionic phospholipid (Vance and Steenbergen, 2005). Because it is asymmetrically distributed, PS is thought to be a major contributor to the surface charge of the inner aspect of the plasmalemma (Leventis and Silvius, 1998). Nevertheless, the fate of this lipid during phagosome formation and maturation has not been studied in any detail due in all likelihood to the lack of suitable means of detection.
We recently designed a genetically encoded biosensor for PS that can be used to monitor the distribution and dynamics of the phospholipid in intact, live cells (Yeung et al., 2008). The probe consists of a discoidin-type C2 domain, which binds selectively to PS, attached to a green or red fluorescent protein. In this study, we used such chimeras to examine the fate of PS during the formation and maturation of phagosomes. In addition, we monitored the surface charge of maturing phagosomes and compared the properties of vacuoles containing inert particles with those containing pathogens that survive intracellularly by eluding the microbicidal machinery of phagocytes.
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The extent to which the phagosomal surface potential was reduced was assessed in more detail using a probe with a net charge of 5+. This probe was shown earlier to associate with the plasma membrane but also with a subpopulation of endomembranes that have an intermediary degree of negative surface charge (Fig. 1 B and Fig. S1 B; Yeung et al., 2008). Interestingly, both the nascent and mature phagosome were labeled by this probe (Fig. 1, B and E), suggesting that the phagosomal membrane was not totally devoid of charge. Of note, the loss of the acylated (Palm) membrane marker was greater than that of the 5+ probe after 1 h (Fig. 1 E), suggesting that the surface charge is conferred by constituents acquired during maturation and not solely by those retained from the original surface membrane. Indeed, the transmembrane protein GT46 that demarcated the forming and early phagosome (Fig. S2 D) was virtually absent from phagosomes that underwent maturation for 1 h (Fig. S2 E), implying that extensive remodeling of the phagosomal membrane had occurred by this time. This observation suggests that the charge associated with the phagosome after 1 h is dictated primarily by components acquired in the course of maturation.
PI 3-phosphate (PI[3]P) is not required for charge maintenance on the phagosome
We next examined the mechanisms contributing to the charge of the maturing phagosome. Because phosphoinositides were found to be important for the plasmalemmal targeting of proteins with polybasic domains (Heo et al., 2006), we tested whether they contribute also to charge maintenance during phagosome maturation. Although neither PI[4,5]P2 nor PI[3,4,5]P3 have been detected in maturing phagosomes, PI[3]P is evident during the first 10–15 min of the maturation process, disappearing thereafter (Fig. 2, A [middle] and D; Vieira et al., 2001). To assess whether PI[3]P is necessary for the maintenance of charge on maturing phagosomes, macrophages were treated with the PI 3-kinase inhibitor wortmannin after multiple particles had been ingested. As expected, the inhibitor rapidly terminated the synthesis of PI[3]P, as judged by the dissociation of the 2FYVE (Fab1/YOTB/Vac1/EEA1) construct that binds specifically to this inositide (Fig. 2, B [middle] and D; Gillooly et al., 2001). However, the association of the 5+ probe persisted (Fig. 2, B and C), implying that PI[3]P is not the major determinant of the negative surface charge of maturing phagosomes.
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We therefore reanalyzed the distribution of PS during maturation using as a probe the C2 domain of lactadherin, which was shown earlier to be highly specific (Andersen et al., 2000; Yeung et al., 2008). Indeed, discoidin family C2 domains are stereospecific, capable of differentiating between the L- and D-stereoisomers of phosphoserine (Gilbert and Drinkwater, 1993; Shi et al., 2004). A GFP-tagged form of the C2 domain of lactadherin (GFP-Lact-C2) was expressed in macrophages, and its distribution was analyzed during the course of phagosome formation and maturation using spinning-disc confocal microscopy. As shown in Fig. 3 and Fig. S4, GFP-Lact-C2 is found at the plasma membrane as well as in intracellular organelles that were identified earlier as components of the endocytic pathway (Yeung et al., 2008). During the course of phagocytosis, GFP-Lact-C2 was associated with the phagosomal membrane for at least 1 h (Fig. 3 C). The levels of PS, as estimated by the density of GFP-Lact-C2 per unit area, were similar to those found in the plasma membrane (Fig. 3 D and Video 2).
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To more directly confirm the occurrence of fusion of internal organelles bearing PS with the maturing phagosomes, we labeled endocytic membranes by pulsing the cells for 15 min with the membrane-associated impermeant dye FM4-64 (Fig. S4, A–C). The labeled membranes overlapped extensively with the PS endomembrane compartment (the Manders colocalization coefficient for FM4-64/Lact-C2 was 0.933). During the course of maturation, phagosomes acquired FM4-64 (Fig. S4 B, arrows), which is indicative of fusion with endocytic membranes. Because the overwhelming majority of the FM4-64–labeled structures were also labeled by Lact-C2, such fusion must have delivered PS to the phagosomal membrane. Indeed, while acquiring videos, we were able to capture fusion events of phagosomes with structures labeled with both FM4-64 and Lact-C2 (Fig. S4 C).
Despite the ongoing delivery of PS-containing vesicles to the phagosome, neither its surface area nor its PS content increases visibly during maturation. This is probably attributable to the concomitant removal of membranous material, which in all likelihood includes PS. This notion was verified by labeling the membrane at the time of phagocytosis with cholera toxin subunit B. As shown in Fig. S4 (D–F), tubules (arrows) colabeled by Lact-C2 and cholera toxin were observed to extend from the phagosome and eventually undergo fission. Together, these observations indicate that, although its overall concentration is nearly constant during maturation, PS is continuously delivered to and removed from phagosomes during the process.
We also studied the fate of PS in phagosomes formed by ingestion of latex beads. In this case, PS similarly persisted in the phagosomes, although at a somewhat reduced level (Fig. S1, F–I). Of note, the association of the 5+ probe was also reduced in these phagosomes (Fig. S1 B). The mechanisms underlying the difference between phagosomes that contain red blood cells versus latex beads and other synthetic particles is unclear (Oh and Swanson, 1996), but the parallel behavior of the GFP-Lact-C2 and 5+ probes in both cases supports the notion that PS is an important determinant of the charge of maturing phagosomes.
Intracellular pathogens modify the PS content of phagosomes
We inferred from the previous experiments that PS is delivered to the phagosome during maturation through fusion with compartments of the endocytic pathway. To test this hypothesis, we exploited as phagocytic targets pathogenic microorganisms that, while entering plasma membrane–derived vacuoles as do inert particles, subsequently coopt the cellular machinery to divert traffic away from the endolysosomal pathway. Legionella pneumophila is one such intracellular pathogen. After entry into the host cell, L. pneumophila propagates in a unique compartment derived largely from the endoplasmic reticulum (Swanson and Isberg, 1995). This phenotype was readily replicated in the RAW macrophages used in this study. As illustrated in Fig. 4 A, 4–6 h after infection, L. pneumophila occupies a vacuole enriched in sec61
, an ER marker. Importantly, the L. pneumophila–containing vacuole was devoid of PS, as indicated by the absence of mRFP-Lact-C2 (Fig. 4, B and D). The paucity of Lact-C2 was likely the result of bacterial effectors that are delivered to the host cell where they actively divert membrane traffic (Shin and Roy, 2008). This could be demonstrated by killing the bacteria before phagocytosis. When macrophages were allowed to phagocytose PFA-fixed L. pneumophila, the resulting phagosomes matured normally, acquiring PS (Fig. 4, C and D).
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The contribution of PS to the endomembrane distribution of Src is further illustrated by experiments using C. trachomatis as the phagocytic target. As shown in Fig. 5, C. trachomatis inclusion vacuoles contain PS at early but not late stages. The same pattern was noted for Src, which was present on vacuoles 4–6 h after infection (Fig. 6, C and F) but not after 18 h (Fig. 6, D and G). The absence of Src from the late C. trachomatis vacuoles is in all likelihood the result of their reduced surface charge, as the vacuoles also fail to bind the 5+ probe (Fig. 6, E and G).
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Discussion |
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9% of the total lipid content of isolated phagosomes (Desjardins et al., 1994). A fraction of the PS originally present in nascent phagosomes most likely derives from the plasma membrane, which invaginates to provide the bulk of the membrane of early phagosomes. This is confirmed by the presence in early phagosomes of a variety of plasmalemmal markers regardless of whether they are diacylated, transmembrane, or GPI-linked proteins (Fig. 1 and Fig. S2; Yeung et al., 2006b). However, PS persists in the membrane of maturing phagosomes even after the plasmalemmal markers have been depleted by fusion and selective fission events (Fig. S4). Previously, we reported that early and late endosomes as well as lysosomes contain PS in their cytosolic-facing monolayer (Yeung et al., 2008). In this study, we observed that PS-positive endosomes were continuously being delivered to the phagosomes as they mature (Fig. S4 and Video 2). Based on these results, we concluded that the PS content of the phagosome is dictated by the maturation sequence and is liable to be affected when the process is subverted by pathogens.
Although PI[4,5]P2 is eliminated from maturing phagosomes, the persistence of PS raised the intriguing possibility that the phagosome may not be totally devoid of charge. Unlike the highly cationic 8+ probes, which are almost quantitatively bound by the highly negative plasmalemma, probes of intermediate charge (e.g., 5+) are not fully partitioned to the plasma membrane and permit the visualization of organelles with lower yet significant charge. Using GFP-5+, we found that the cytosolic leaflet of phagosomes is indeed negatively charged throughout the maturation process. Phosphoinositides do not contribute significantly to this charge; PI[4,5]P2 is not detectable in formed phagosomes, and PI[3,4,5]P3 persists for 
1 min after sealing. PI[3]P, which is formed subsequently, and its derivative, PI[3,5]P2, are not essential to confer charge to maturing phagosomes, which bind comparable amounts of the 5+ probe both before and after treatment with inhibitors of the class III PI 3-kinase (Fig. 2). These findings are most likely explained by the postulate that PS is the primary determinant of the phagosomal charge. However, the possible contribution of other anionic lipids such as PA and lysobis-PA should not be neglected, pending the development of suitable probes to detect these species.
Although the correlation between phagosomal PS and surface charge is strong, it is difficult to establish unambiguously the causal relationship between these parameters. Mammalian cells cannot be fully depleted of PS by either genetic deletion of the PS synthase isoforms or by pharmacological means. However, in the case of phagosomes, the subversion of the maturation process by certain intracellular pathogens provides an opportunity to test the role of PS. To avert killing, virulent microorganisms like L. pneumophila and C. trachomatis coopt the machinery of host cells to divert maturation away from the endolysosomal pathway. As shown in Figs. 4–6
, the mature pathogen-containing vacuoles are devoid of detectable PS and, importantly, are also devoid of either the 5+ probe or the similarly charged Src. These findings further support the notion that PS is a key contributor to the charge of phagosomes and invasion vacuoles.
Regardless of its contribution to the surface charge, the presence of PS in phagosomes is expected to have important functional consequences. In this regard, we have preliminary evidence that yeast that are deficient in the PS synthase gene (cho1), and therefore lack PS, have defective vacuolar acidification (Fig. S5 A), and their endocytic pathway has abnormal appearance and traffic as revealed by FM4-64 pulse-chase analyses (Fig. S5, B and C). Although the mechanisms accounting for the aberrant phenotype remain to be explored, they likely include the failed targeting of PS-binding proteins.
A sizable number of proteins contain C2 domains that bind PS with varying degrees of selectivity. Several of these, such as protein kinase C and phospholipase C isoforms, are engaged in signal transduction, whereas others, like the synaptotagmins, control membrane fusion events. Both phenomena are central to the maturation of endosomes and phagosomes. Similarly, regardless of whether PS is its main determinant, the negative charge of phagosomes will serve to target proteins with polycationic clusters or polybasic domains, particularly those that also contain hydrophobic moieties. Indeed, our work reveals that the phagosomal distribution of one such molecule, the tyrosine kinase c-Src, shows strong correlation with the presence of PS in the phagosomal membrane. Similarly, PS may be a critical determinant in the distribution of small GTPases of the Rab and Rho superfamilies, which have been shown to be guided electrostatically to cellular membranes (Heo et al., 2006) and are important participants in phagosome formation and maturation.
In summary, we found that PS enters phagosomes as the plasma membrane invaginates and that its concentration is maintained at a comparatively high level by ongoing fusion with endosomes and lysosomes. Being exposed to the cytosolic aspect of the membrane, PS will serve to recruit to the phagosome proteins containing PS-selective C2 domains. Moreover, by conferring onto the phagosomal surface a considerable negative charge, PS will contribute to the recruitment of cationic proteins. The congregation of these proteins on the phagosomal surface likely remodels this organelle and directs its maturation.
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Materials and methods |
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Cell culture, transfection, and treatment
RAW264.7 macrophages from American Type Culture Collection were grown as previously described (Yeung et al., 2008). Transient transfection of cDNA plasmids was performed using FuGene HD (Roche) as described previously (Yeung et al., 2008). Where indicated, the cells were treated with 200 nM wortmannin (EMD) for 15 min to inhibit PI 3-kinase.
Synchronous phagocytosis assay
RAW macrophages were grown on circular glass coverslips placed inside 6-well plates. Sheep RBCs (sRBCs) obtained from MP Biomedicals were opsonized with rabbit anti-RBC IgG (MP Biomedicals) as described previously (Botelho et al., 2000). To synchronously initiate phagocytosis, opsonized RBCs were sedimented onto the RAW macrophages by centrifugation for 1 min at 1,000 rpm using a tabletop centrifuge (Beckman Coulter). After rapidly washing nonadherent RBCs, the cells were bathed in Hepes-buffered medium RPMI 1640 (Wisent Inc.), and phagocytosis was allowed to proceed for 1, 5, or 60 min at 37°C. Coverslips were then transferred from the 6-well plate to an Attofluor live cell imaging chamber (Invitrogen) for microscopy. To arrest further phagosome maturation, the cells were bathed in ice-cold Hepes-buffered medium RPMI 1640 during the course of image acquisition. Synchronous phagocytosis of 3-µm latex beads was performed similarly and has been previously described (Yeung et al., 2006b).
L. pneumophila infection protocol
L. pneumophila (strain LpO2) and L. pneumophila expressing GFP-flaA (MB355; Hammer and Swanson, 1999) were cultured to the postexponential phase in ACES yeast extract broth as described previously (provided by M. Swanson, University of Michigan Medical School, Ann Arbor, MI; Byrne and Swanson, 1998). RAW macrophages were infected with L. pneumophila for 2 h at a multiplicity of infection of 10 followed by washing to remove excess bacteria and a further 2–4-h incubation at 37°C. For immunostaining, the infected cells were fixed and permeabilized with cold methanol, blocked with 5% goat serum, and incubated sequentially with rabbit anti–L. pneumophila antibody (1:2,000 dilution; provided by R. Isberg, Tufts University, Boston, MA) followed by Cy3-conjugated donkey anti–rabbit antibody (1:1,000; Jackson ImmunoResearch Laboratories).
C. trachomatis infection protocol
Frozen stocks of C. trachomatis serotype L2 elementary bodies were prepared as previously described (Tse et al., 2005). For infection, frozen vials containing C. trachomatis were thawed, and elementary bodies were added to RAW macrophages grown on coverslips at the bottom of 6-well plates. The plates were spun for 20 min at 2,000 rpm before incubation for 18 h at 37°C. Cells were fixed with 8% PFA (Electron Microscopy Sciences) for 2 h C. trachomatis within RAW macrophages were visualized using the fluorescent DNA stain Draq5 as suggested by the manufacturer (Biostatus).
Image acquisition and analysis
All fluorescence images were acquired using a microscope (Axiovert 200M; Carl Zeiss, Inc.) equipped with a 63x/1.40 NA oil immersion lens (Carl Zeiss, Inc.), charge-coupled device camera (C9100-13; Hamamatsu Photonics), and a spinning-disc confocal system (Quorum) as described previously (Yeung et al., 2008). Images were captured and analyzed using Volocity software (PerkinElmer). The ratio of the fluorescence intensity of the phagosome to that of the bulk, unengaged plasma membrane was calculated as described previously (Yeung et al., 2006b). In brief, regions of interest were defined in the phagosomal membrane, unengaged plasma membrane, and cytosol, and their mean fluorescence intensities were measured. After subtracting the cytosolic contribution, the excess fluorescence associated to the phagosome and bulk membranes was estimated, and their ratio was then calculated. A value of 1 indicates similar probe density at the phagosomal and bulk plasma membrane. A value of 0 indicates complete loss of the probe from the phagosomal membrane. For quantification of Lact-C2 staining of C. trachomatis– or L. pneumophila–containing vacuoles, the fluorescence associated with the vacuolar membrane was either scored as positive or negative and was reported as the percentage of the total number of vacuoles counted.
Analysis of the lipid selectivity of the anti-PS antibody
Nucleosil 120–3 C18 beads (3 µm; Richard Scientific) coated with PC or PC plus 20% PE, 20% PS, 20% PA, 2% PI, or 2% PI[4,5]P2 were prepared as described previously (Yeung et al., 2008). Lipid-coated beads were incubated with an Alexa Fluor 488–conjugated mouse anti-PS antibody (Millipore) for 1 h in 20 mM Tris-HCl, pH 7. An FACS scan flow cytometer (BD) was used to analyze fluorescence associated with the beads.
Lipid-binding analysis of annexin-V
Large unilamellar vesicles (LUVs) of dansyl-PE:PC (2:98), dansyl-PE:PC:PE (2:78:20), dansyl-PE:PC:PS (2:78:20), dansyl-PE:PC:PA (2:78:20), dansyl-PE:PC:PG (2:78:20), dansyl-PE:PC:PI(4)P (2:96:2), or dansyl-PE:PC:PI(4,5)P2 (2:96:2) were prepared using a liposome extruder (Avestin) as described previously (Yeung et al., 2006b). The binding of annexin-V to the LUVs was measured using a fluorescence resonance energy transfer (FRET) assay. In brief, purified human placental annexin-V (Sigma-Aldrich) was added to LUVs suspended in a buffer containing 10 mM Hepes and 100 µM Ca2+, pH 7.4. FRET between the tryptophan residues of annexin-V and the dansylated lipids was recorded using a spectrophotometer (F-2500; Hitachi) with excitation at 280 nm and emission at 505 nm. FRET resulting from binding of annexin to liposomes was measured in the presence and absence of calcium, which is required for annexin binding. The FRET signal observed in the absence of calcium, indicative of nonspecific binding, was subtracted from the signal recorded in the presence of calcium. To facilitate comparison between experiments, the data are normalized to the binding to PC liposomes recorded in the absence of calcium.
Phenotypic assessment of the endocytic pathway in yeast
To assess vacuole acidification in vivo, yeast cells were stained with quinacrine as described previously (Weisman et al., 1987) with minor modifications. In brief, wild-type or PS synthase–deficient (cho1) yeast cells grown to the early logarithmic phase were harvested by centrifugation at 2,000 g and incubated in YPD (1% yeast extract, 2% peptone, and 2% glucose), pH 7.6, for 30 min. Cells were harvested and resuspended in 50 mM phosphate-buffered saline, pH 7.6, containing 2% glucose, and quinacrine was added to a final concentration of 200 µM to cells. After 5 min, the cells were harvested, resuspended in PBS-2% glucose, and examined immediately by epifluorescence and differential interference contrast microscopy.
To assess the traffic and appearance of the endocytic pathway, a pulse-chase analysis using the lipophilic styryl dye FM4-64 was used as described previously (Vida and Emr, 1995). In brief, cells were grown to early logarithmic phase in YPD medium at 30°C, harvested, and resuspended at 20 OD600 U/ml in YPD medium. To pulse the cells with FM4-64, the dye was added to cells at a final concentration of 32 µM from a 16-mM stock in DMSO and incubated at 4°C for 30 min while rotating. The cells were next sedimented, resuspended in YPD medium, and incubated at 30°C for the indicated times. After this chase period, the cells were harvested, resuspended in PBS, placed on ice, and examined microscopically as described in Image acquisition and analysis.
Online supplemental material
Fig. S1 shows an assessment of the surface charge of maturing phagosomes during latex bead phagocytosis. Fig. S2 shows PS and phagosomal membrane charge during L. pneumophila and C. trachomatis vacuole maturation. Fig. S3 shows lipid-binding specificity of the anti-PS antibody and annexin-V. Fig. S4 shows membrane fusion and fission during phagosomal maturation. Fig. S5 shows phenotypic assessment of the endocytic pathway in wild-type and PS-deficient yeast. Video 1 shows distribution of charge and membrane markers during phagosome formation and maturation. Video 2 shows distribution of PS during phagosome formation and maturation. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200903020/DC1.
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Acknowledgments |
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Submitted: 5 March 2009
Accepted: 4 May 2009
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References |
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Andersen, M.H., H. Graversen, S.N. Fedosov, T.E. Petersen, and J.T. Rasmussen. 2000. Functional analyses of two cellular binding domains of bovine lactadherin. Biochemistry. 39:6200–6206.[CrossRef][Medline]
Botelho, R.J., M. Teruel, R. Dierckman, R. Anderson, A. Wells, J.D. York, T. Meyer, and S. Grinstein. 2000. Localized biphasic changes in phosphatidylinositol-4,5-bisphosphate at sites of phagocytosis. J. Cell Biol. 151:1353–1368.
Brumell, J.H., and M.A. Scidmore. 2007. Manipulation of rab GTPase function by intracellular bacterial pathogens. Microbiol. Mol. Biol. Rev. 71:636–652.
Byrne, B., and M.S. Swanson. 1998. Expression of Legionella pneumophila virulence traits in response to growth conditions. Infect. Immun. 66:3029–3034.
Desjardins, M., J.E. Celis, G. van Meer, H. Dieplinger, A. Jahraus, G. Griffiths, and L.A. Huber. 1994. Molecular characterization of phagosomes. J. Biol. Chem. 269:32194–32200.
Donepudi, M., and M.D. Resh. 2008. c-Src trafficking and co-localization with the EGF receptor promotes EGF ligand-independent EGF receptor activation and signaling. Cell. Signal. 20:1359–1367.[CrossRef][Medline]
Gilbert, G.E., and D. Drinkwater. 1993. Specific membrane binding of factor VIII is mediated by O-phospho-L-serine, a moiety of phosphatidylserine. Biochemistry. 32:9577–9585.[CrossRef][Medline]
Gillooly, D.J., A. Simonsen, and H. Stenmark. 2001. Cellular functions of phosphatidylinositol 3-phosphate and FYVE domain proteins. Biochem. J. 355:249–258.[CrossRef][Medline]
Greenfield, J.J., and S. High. 1999. The Sec61 complex is located in both the ER and the ER-Golgi intermediate compartment. J. Cell Sci. 112:1477–1486.[Abstract]
Hammer, B.K., and M.S. Swanson. 1999. Co-ordination of legionella pneumophila virulence with entry into stationary phase by ppGpp. Mol. Microbiol. 33:721–731.[CrossRef][Medline]
Heo, W.D., T. Inoue, W.S. Park, M.L. Kim, B.O. Park, T.J. Wandless, and T. Meyer. 2006. PI(3,4,5)P3 and PI(4,5)P2 lipids target proteins with polybasic clusters to the plasma membrane. Science. 314:1458–1461.
Kanai, F., H. Liu, S.J. Field, H. Akbary, T. Matsuo, G.E. Brown, L.C. Cantley, and M.B. Yaffe. 2001. The PX domains of p47phox and p40phox bind to lipid products of PI(3)K. Nat. Cell Biol. 3:675–678.[CrossRef][Medline]
Leventis, R., and J.R. Silvius. 1998. Lipid-binding characteristics of the polybasic carboxy-terminal sequence of K-ras4B. Biochemistry. 37:7640–7648.[CrossRef][Medline]
Majeed, M., E. Caveggion, C.A. Lowell, and G. Berton. 2001. Role of Src kinases and Syk in Fcgamma receptor-mediated phagocytosis and phagosome-lysosome fusion. J. Leukoc. Biol. 70:801–811.
Marshall, J.G., J.W. Booth, V. Stambolic, T. Mak, T. Balla, A.D. Schreiber, T. Meyer, and S. Grinstein. 2001. Restricted accumulation of phosphatidylinositol 3-kinase products in a plasmalemmal subdomain during Fc
receptor-mediated phagocytosis. J. Cell Biol. 153:1369–1380.
McLaughlin, S., and D. Murray. 2005. Plasma membrane phosphoinositide organization by protein electrostatics. Nature. 438:605–611.[CrossRef][Medline]
Niedergang, F., and P. Chavrier. 2004. Signaling and membrane dynamics during phagocytosis: many roads lead to the phagos(R)ome. Curr. Opin. Cell Biol. 16:422–428.[CrossRef][Medline]
Oh, Y.K., and J.A. Swanson. 1996. Different fates of phagocytosed particles after delivery into macrophage lysosomes. J. Cell Biol. 132:585–593.
Peyron, P., I. Maridonneau-Parini, and T. Stegmann. 2001. Fusion of human neutrophil phagosomes with lysosomes in vitro: involvement of tyrosine kinases of the Src family and inhibition by mycobacteria. J. Biol. Chem. 276:35512–35517.
Pralle, A., P. Keller, E.L. Florin, K. Simons, and J.K. Horber. 2000. Sphingolipid–cholesterol rafts diffuse as small entities in the plasma membrane of mammalian cells. J. Cell Biol. 148:997–1008.
Raynal, P., and H.B. Pollard. 1994. Annexins: the problem of assessing the biological role for a gene family of multifunctional calcium- and phospholipid-binding proteins. Biochim. Biophys. Acta. 1197:63–93.[Medline]
Roy, M.O., R. Leventis, and J.R. Silvius. 2000. Mutational and biochemical analysis of plasma membrane targeting mediated by the farnesylated, polybasic carboxy terminus of K-ras4B. Biochemistry. 39:8298–8307.[CrossRef][Medline]
Salcedo, S.P., and D.W. Holden. 2005. Bacterial interactions with the eukaryotic secretory pathway. Curr. Opin. Microbiol. 8:92–98.[CrossRef][Medline]
Shi, J., C.W. Heegaard, J.T. Rasmussen, and G.E. Gilbert. 2004. Lactadherin binds selectively to membranes containing phosphatidyl-L-serine and increased curvature. Biochim. Biophys. Acta. 1667:82–90.[Medline]
Shin, S., and C.R. Roy. 2008. Host cell processes that influence the intracellular survival of Legionella pneumophila. Cell. Microbiol. 10:1209–1220.[CrossRef][Medline]
Swanson, J.A., and A.D. Hoppe. 2004. The coordination of signaling during Fc receptor-mediated phagocytosis. J. Leukoc. Biol. 76:1093–1103.
Swanson, M.S., and R.R. Isberg. 1995. Association of Legionella pneumophila with the macrophage endoplasmic reticulum. Infect. Immun. 63:3609–3620.
Tse, S.M., D. Mason, R.J. Botelho, B. Chiu, M. Reyland, K. Hanada, R.D. Inman, and S. Grinstein. 2005. Accumulation of diacylglycerol in the Chlamydia inclusion vacuole: possible role in the inhibition of host cell apoptosis. J. Biol. Chem. 280:25210–25215.
Vance, J.E., and R. Steenbergen. 2005. Metabolism and functions of phosphatidylserine. Prog. Lipid Res. 44:207–234.[CrossRef][Medline]
Vida, T.A., and S.D. Emr. 1995. A new vital stain for visualizing vacuolar membrane dynamics and endocytosis in yeast. J. Cell Biol. 128:779–792.
Vieira, O.V., R.J. Botelho, L. Rameh, S.M. Brachmann, T. Matsuo, H.W. Davidson, A. Schreiber, J.M. Backer, L.C. Cantley, and S. Grinstein. 2001. Distinct roles of class I and class III phosphatidylinositol 3-kinases in phagosome formation and maturation. J. Cell Biol. 155:19–25.
Weisman, L.S., R. Bacallao, and W. Wickner. 1987. Multiple methods of visualizing the yeast vacuole permit evaluation of its morphology and inheritance during the cell cycle. J. Cell Biol. 105:1539–1547.
Yeung, T., G.E. Gilbert, J. Shi, J. Silvius, A. Kapus, and S. Grinstein. 2008. Membrane phosphatidylserine regulates surface charge and protein localization. Science. 319:210–213.
Yeung, T., B. Ozdamar, P. Paroutis, and S. Grinstein. 2006a. Lipid metabolism and dynamics during phagocytosis. Curr. Opin. Cell Biol. 18:429–437.[CrossRef][Medline]
Yeung, T., M. Terebiznik, L. Yu, J. Silvius, W.M. Abidi, M. Philips, T. Levine, A. Kapus, and S. Grinstein. 2006b. Receptor activation alters inner surface potential during phagocytosis. Science. 313:347–351.
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