Journal of Leukocyte Biology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published online as doi:10.1189/jlb.1104630 on February 23, 2005

Published online before print February 23, 2005
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
jlb.1104630v1
77/6/878    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Touret, N.
Right arrow Articles by Grinstein, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Touret, N.
Right arrow Articles by Grinstein, S.
(Journal of Leukocyte Biology. 2005;77:878-885.)
© 2005 by Society for Leukocyte Biology

The nature of the phagosomal membrane: endoplasmic reticulum versus plasmalemma

Nicolas Touret*, Paul Paroutis*,{dagger} and Sergio Grinstein*,{dagger},1

* Programme in Cell Biology, Hospital for Sick Children, and
{dagger} Department of Biochemistry, University of Toronto, Ontario, Canada

1Correspondence: Programme in Cell Biology, Hospital for Sick Children, 555 University Ave., Toronto, Ontario, Canada, M5G 1X8. E-mail: sga{at}sickkids.ca


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 THE CONVENTIONAL MODEL: PLASMA...
 THE NEW MODEL: PARTICLE...
 FUNCTIONAL IMPLICATIONS OF THE...
 ER OR PLASMALEMMA?
 CONCLUDING REMARKS
 REFERENCES
 
For decades, the vacuole that surrounds particles engulfed by phagocytosis was believed to originate from the plasma membrane. Conversion of the nascent phagosome into a microbicidal organelle was thought to result from the subsequent, orderly fusion of early endosomes, late endosomes, and ultimately, lysosomes with the original plasma membrane-derived vacuole. This conventional model has been challenged, if not superseded, by a revolutionary model that regards phagosome formation as resulting from the particle sliding into the endoplasmic reticulum via an opening at the base of the phagocytic cup. The merits and implications of these two hypotheses are summarized here and analyzed in light of recent results.

Key Words: phagosome • maturation • acidification • endocytosis • macrophage • neutrophil


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
THE CONVENTIONAL MODEL: PLASMA...
 THE NEW MODEL: PARTICLE...
 FUNCTIONAL IMPLICATIONS OF THE...
 ER OR PLASMALEMMA?
 CONCLUDING REMARKS
 REFERENCES
 
Phagocytosis, the engulfment of large (≥0.5 µm diameter) particles by leukocytes, plays a key role in host defense against invading microorganisms [1 , 2 ]. Particles are internalized by neutrophils or macrophages by two different processes. Some receptors trigger the extension of pseudopodia that surround the target and eventually meet and fuse at their tips. Other receptors induce tight binding of the particle to the phagocyte membrane, which is gradually displaced inwardly (dimpled), gently sinking the target into the phagocytic cell [3 ]. In either case, the ingested particle is ultimately delivered to the cell interior, contained within a membrane-bound vacuole known as phagosomes, which undergo maturation, a finely tuned sequence of fusion and fission events that progressively remodel the vacuole, rendering its membrane and contents effectively microbicidal.

For simplicity, it was assumed originally that the nascent phagocytic vacuole was composed largely of the plasmalemma, as it appeared to be derived from its extension or inward deformation. Early endosomes were reported to merge with the nascent phagosome shortly after sealing, followed by late endosomes and lysosomes that provided most of the microbicidal components to the resulting hybrid organelle, the phagolysosome. These observations accounted well for the gradual acidification that was recorded to develop inside the phagocytic vacuole during the course of maturation [4 ], as endosomes and lysosomes are endowed with proton-pumping, vacuolar-type (V)-ATPases. Conversely, this simple model could not readily explain the remarkable ability of phagocytes to ingest large and/or multiple particles without apparent loss of cellular surface area.

More recently, two alternative models have been suggested to account for the conservation of cellular surface during the course of phagocytosis, despite the size and number of ingested particles. These are illustrated diagrammatically in Figure 1 . One hypothesis states that fusion of endosomes with the plasmalemma provides the necessary membrane to maintain the surface area near constant. Early endosomes (including recycling endosomes) were first proposed to be the source of endosomal membrane [5 6 7 ], and more recent findings suggest that late endosomes may also contribute to this process [8 ]. Fusion of endosomes with the surface membrane prior to sealing can explain the rapid development of a pH gradient across newly formed phagosomes. Rapid and extensive fusion of phagosomes with endosomes would also favor presentation of antigens via class II major histocompatibility complexes (MHC II).



View larger version (50K):
[in this window]
[in a new window]
  		Figure 1. Schematic representation of the two models of phagocytosis. Upon engagement of phagocytic receptors [e.g., Fc{gamma} receptors (Fc{gamma}Rs)], the plasmalemma surrounds a target particle by extension of pseudopods. In the conventional model (left panel), the phagocytic vacuole is formed by the fusion of pseudopods at their tips and is composed largely of plasmalemmal constituents with a varying contribution of endosomes, perhaps depending on the particle size. The sealed phagosome proceeds to mature by sequential fusion of additional early and late endosomes and ultimately, lysosomes. The new model (right panel) envisages phagosome formation as resulting from the particle sliding into the endoplasmic reticulum (ER) via an opening at the base of the phagocytic cup. The nascent phagosome consists largely of ER, which remains present in the phagosome for hours.

 
A second hypothesis, which has garnered much attention [9 , 10 ], states instead that phagocytic particles slide into the ER via an opening formed at the base of the phagocytic cup. As a result, the phagocytic vacuole is composed largely of ER-derived membrane, presumably with minimal use and maximal conservation of the surface membrane. This hypothesis has proven attractive, as it not only explains the ability of phagocytes to preserve their size while ingesting multiple large particles but also provides an anatomical basis for the cross-presentation of antigens via MHC I [11 ]. Moreover, to the extent that autophagy and invasion by Legionella and other pathogens involve the formation of ER-derived vacuoles, the ER model of phagocytosis would bring some commonality to the underlying mechanisms.

In this article, we overview the evidence supporting these alternative models and try to rationalize the apparent discrepancies.


   THE CONVENTIONAL MODEL: PLASMA MEMBRANE PLUS ENDOSOMES
TOP
 ABSTRACT
 INTRODUCTION
 THE CONVENTIONAL MODEL: PLASMA...
THE NEW MODEL: PARTICLE...
 FUNCTIONAL IMPLICATIONS OF THE...
 ER OR PLASMALEMMA?
 CONCLUDING REMARKS
 REFERENCES
 
The basic, original notion that nascent phagosomes are composed primarily of invaginated plasma membrane has been tested experimentally by biochemical and microscopic means. Early studies compared the protein composition of early phagosomes with that of the plasma membrane. Purified phagosomes formed by Amoeba were found to have a composition that closely resembled that of the plasma membrane. Not only were the proteins of the two membranes similar but so were their phospholipids and sterols [12 ]. A more sophisticated approach was developed by Muller and colleagues [13 ], who iodinated phagosomal proteins selectively by covalently attaching peroxidase to latex beads. These were compared with the proteins labeled by soluble peroxidase added to intact cells, i.e., plasmalemmal components exposed to the external medium. Comparison of the two sets of proteins following sodium dodecyl sulfate-polyacrylamide gel electrophoresis separation and autoradiography led Muller’s team to conclude that the protein composition and density of the phagosomal vacuole were similar to those of the plasma membrane. In accordance with these findings, a variety of other plasma membrane proteins has been found to be represented abundantly in the phagosome, including MHC II [14 ], LRG47 [15 ], Fc{gamma} receptors (Fc{gamma}Rs), and others.

The predominant contribution of the plasma membrane to the composition of the early phagosomal vacuole is also supported by microscopic observations (e.g., see Fig. 3D ). We find that at least in the case of small to medium-sized particles (0.5–3 µm), the normalized fluorescence intensity of the limiting membrane of newly formed phagosomes is similar to that of the plasma membrane (P. Paroutis and N. Touret, unpublished observations).



View larger version (83K):
[in this window]
[in a new window]
  		Figure 3. Assessing the relationship between ER and phagosomes by fluorescence microscopy. (A) Fluorescence imaging of the localization of calnexin and LAMP1 in phagocytic cells containing ingested particles (reprinted with permission from ref. [10 ]). (B) Localization of calnexin and MHC II in phagocytic cells containing ingested particles (reprinted with permission from ref. [36 ]). (C) Localization of glucose-regulated protein 78 (GRP78) and phospholipase A2{alpha} (cPLA2{alpha}) in the vicinity of the phagosome (reprinted with permission from ref. [37 ]). (D) Localization of diacylated, plasma membrane-targeted green fluorescent protein (PM-GFP) in newly formed phagosomes (reprinted with permission from ref. [38 ]).

 
Although the concept that the plasma membrane is the sole constituent of the early phagosomal membrane may suffice to explain the uptake of single, small particles, it appears simplistic in the case of large or multiple particles. In this instance, delivery of endomembranes to the surface is required to account for the maintenance of the surface area of the cell. The near-constancy of the surface area of the cell has been validated by electrophysiological and spectroscopic experiments. In fact, both approaches demonstrated that rather than undergoing a reduction, the surface area increases, at least transiently, during phagocytosis. Holevinsky and Nelson [16 ] showed this unexpected net insertion of endomembranes into the plasmalemma as step increases in membrane capacitance, and Hackam et al. [17 ] showed it as an enhanced fluorescence of the lipophilic dye FM1-43, a marker of cell-surface area.

In view of the existing evidence that early endosomes fuse with phagosomes shortly after sealing, it was natural to postulate that the endosomal compartment might also be able to fuse with the forming phagosomal cup. The recent elucidation of the molecular machinery responsible for the fusion of various cellular compartments enabled investigators to test this hypothesis. Fusion is thought to require interaction of cognate soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE) molecules of the vesicular and target membranes. The current paradigm proposes the formation of a four-helix bundle, or SNAREpin, where an arginine on one membrane contributes one coiled-coil domain, and three Q-SNAREs (Q=glutamine) on the opposite membrane contribute three coiled-coils (synaptosome-associated protein of 25 kDa-like SNAREs contribute two coiled-coils and therefore only require one additional Q-SNARE [18 ]). SNAREpins bring the cognate bilayers in tight apposition, a critical step in membrane coalescence. After fusion, NSF, a hexameric chaperone ATPase, disassembles SNAREpins so that the individual components can be recycled for another round of fusion [19 20 21 ]. When SNARE interaction or NSF function is impaired, fusion is prevented.

Inhibition of NSF activity using dominant-negative constructs greatly decreased the phagocytic efficiency [17 , 22 ], validating the idea that membrane fusion is required for particle ingestion. However, these experiments did not directly identify the source of endomembranes that fuse with forming phagosomes, as NSF is required for dissociation of virtually all SNAREpins. More specific information has been obtained recently by monitoring the traffic of individual SNAREs and/or by inhibiting their functions with mutant alleles. Vesicle-associated membrane protein-3 (VAMP-3), which is predominantly expressed in early (recycling) endosomes, was shown to be enriched at the phagosomal cup [5 , 7 ]. Accordingly, treatment of cells with tetanus toxin, which cleaves VAMP-3, as well as other VAMP isoforms, impaired phagocytosis [17 ]. These observations suggested that delivery of early endosomes to the phagocytic cup was required for optimal particle ingestion. This notion is buttressed by studies of Rab11 function in phagocytosis. This GTPase resides primarily in recycling endosomes and is thought to promote their fusion with the plasma membrane. Cox et al. [6 ] found that impairment of normal Rab11 activity diminished the phagocytic efficiency of macrophages. Moreover, Arf6, a distinct GTPase that like Rab11, is required for recycling of early endosomes [23 ] as well as for the traffic of other compartments [24 ], is similarly necessary for optimal phagocytosis [7 ]. Together, these observations make a convincing case for the involvement of early endosomes in the closure of the phagosomal vacuole. Consistent with these observations, older studies by Russell and colleagues [25 ] had demonstrated the rapid fusion of endosomes with newly formed phagosomes. The more recent observations indicate that such fusion may start prior to phagosomal closure, seemingly contributing to pseudopod extension.

More recently, evidence was introduced that late endosomal/lysosomal membranes may also contribute to phagosome formation. Braun and colleagues [8 ] demonstrated that VAMP-7, also called TI-VAMP because of its insensitivity to tetanus toxin, is recruited to sites of phagocytosis. Moreover, they showed that expression of a dominant-negative form of this SNARE or gene silencing using small interfering RNA partially reduced the phagocytic index. These findings, which implicate late endosomes in vacuole formation, are not unprecedented. Lysosomes can be recruited to the sites where Trypanosoma cruzi invades mammalian cells, entering a host-derived vacuole that resembles the phagosomes [26 ]. Not only are lysosomal proteins delivered to the site of parasite penetration, but also, the integrity of the lysosomal compartment is required for the completion of invasion [27 ].

The observations summarized in this section can be integrated into a model where formation of the phagosomal vacuole involves invagination of the plasmalemma, which is supplemented by focal delivery of membranes from compartments of the endocytic pathway, by SNARE-mediated exocytic events. Although it has not been documented directly, we suspect that internalization of large and/or multiple particles will prove to be more tightly associated with and more dependent on endomembrane insertion, which prior to sealing of the vacuole, may be intended to maintain surface area homeostasis, although this may be construed as a teleological rationalization. Mechanistically, the ability of endosomes to fuse with the open cup formed around large particles may simply result from the additional time required for completion of particle engulfment, allowing the endomembranes to fuse with the nascent vacuole in response to receptor stimulation prior to sealing.


   THE NEW MODEL: PARTICLE DELIVERY TO THE ER
TOP
 ABSTRACT
 INTRODUCTION
 THE CONVENTIONAL MODEL: PLASMA...
 THE NEW MODEL: PARTICLE...
FUNCTIONAL IMPLICATIONS OF THE...
 ER OR PLASMALEMMA?
 CONCLUDING REMARKS
 REFERENCES
 
Although Desjardins and his colleagues (10 ) recently articulated the hypothesis that the ER participates directly in phagosome formation, a number of earlier observations were consistent with this concept. That proteins such as calnexin and calreticulin can reach the cell surface had been documented earlier [28 29 30 ], pointing to the connectivity of the ER and plasma membranes. Moreover, there was evidence that such ER proteins play a functional role in phagocytosis. Thus, antibodies directed against cell-surface calreticulin decreased particle uptake by 50–75% in macrophages [31 ], and a similar decline in phagocytosis was noted in calnexin/calreticulin double-null mutants of Dictyostelium discoideum [32 ].

Other precedents of ER involvement in particle internalization and degradation include the fate of some intracellular pathogens, such as Legionella. These bacteria elude the microbicidal activity of host cells by ensconcing within a compartment that closely resembles the ER. ER-mediated phagocytosis would provide a direct route to the lumen of this sheltered compartment, but other mechanisms are possible (see below). In addition, autophagosomes, which share several features with the phagosomes of leukocytes, were proposed early on to originate from a subcompartment of the ER [33 , 34 ]. Lastly, Rothman and colleagues [35 ] found that incorporation of Sec22 (an ER SNARE) into liposomes could support their fusion with membranes displaying the plasmalemmal SNARE Sso1/Sec9c, thereby providing a biochemical foundation for the merger of these compartments. The cumulative, pre-existing evidence, although circumstantial, made it reasonable and tempting to analyze the role of ER in phagocytosis more directly.

In Montreal, Desjardins and his colleagues made the first, specific suggestion that the ER is involved directly in the particle engulfment process. They stated explicitly that immediately after particle attachment to the cell, "The ER is recruited close to the surface, where it fuses with the plasma membrane and opens at the site of particle contact. The particle then slides into the open ER and the plasma membrane is resealed. This leads to the formation of phagosomes made largely of ER." The initial evidence in support of this revolutionary model was derived from proteomic analyses of purified phagosomal membranes. Garin et al. [9 ] identified more than 140 proteins that were associated with isolated latex bead-containing phagosomes; remarkably, these included a plethora of ER-resident proteins such as calnexin, calreticulin, binding protein (BIP), protein disulfide isomerase (PDI), and Sec61 [9 ]. Gagnon et al. [10 ], who characterized purified phagosomes by immunoblotting, validated these observations by demonstrating the enrichment of ER-resident marker proteins, such as calnexin, calreticulin, and Sec61, relative to the post-nuclear supernatant (Fig. 2A ). Kinetic profiling of the phagosomal proteins following particle ingestion revealed an association of the ER with phagosomes, not only at early time-points but also at various stages along the phagolysosome biogenesis pathway. Confirmation of these results was also obtained at the immunofluorescence level, through the visualization of calnexin on phagocytic cups and sealed phagosomes in intact cells (Fig. 3A ), as well as on purified phagosomes (Fig. 2C) .



View larger version (99K):
[in this window]
[in a new window]
  		Figure 2. Assessing the relationship between ER and phagosomes by electron microscopy. (A) Western blot analysis of calnexin, calreticulin, and Sec61p in phagosomes purified 30 min after formation (Phago), total cell lysate (TCL), post-nuclear supernatant (PNS), and total membrane fraction (Memb). Pronase digestion reveals the presence of calnexin and calreticulin in their native orientation in the phagosome. (B) Top panel: electron microscopy of immunogold (IG) labeling of calnexin in a purified phagosomal preparation. Lower panel: immunofluorescence (IF) labeling of calnexin in purified phagosomes. Asterisk indicates unidentified contaminating membranes. (C) Electron microscopy of glucose-6-phosphatase activity in the lumen of Leishmania-containing phagosomes (A–C were reprinted from [10 ] with permission). L, Leishmania; F, flagellum; NE, nuclear envelope; ER, endoplasmic reticulum. (D) Scanning electron microscopy and (E) transmission electron microscopy of purified latex bead-containing phagosomes, revealing the presence of foreign membranes attached to the phagosomal membrane. Phagosomes were prepared as described in [10 ]. Scale bars = 0.5 µm.

 
Perhaps the most suggestive evidence of ER-phagosome fusion was provided by electron microscopy analysis of purified phagosomes and intact cells. By immunogold labeling of calnexin in purified phagosomes, Gagnon et al. [10 ] were able to demonstrate the presence of ER proteins directly on vacuolar membranes in their native orientation (Fig. 2B) . It is more striking that in the presence of various inhibitors of phagocytosis and vacuolar proton pumps, apparent continuity was observed between the ER and phagocytic cups forming around latex beads, implying that the ER was recruited to the cell surface to form phagosomes. These observations were made identifying the ER cytochemically, using glucose-6-phosphatase activity as a marker, and were further extended to the uptake of red blood cells and several pathogens, including Leishmania (Fig. 2D) . Desjardins and his colleagues therefore concluded that ER-mediated phagocytosis is a general mode of particle entry into macrophages.

This novel conclusion impacts not only our understanding of the basic mechanism of phagocytosis but also may help unravel the mystery of MHC I antigen cross-presentation. Whereas exogenous antigens are normally presented by MHC II molecules, leading to the activation of CD4+ helper T cells, some exogenous antigens can also be presented by MHC I molecules, a process termed "cross-presentation." Antigens are exposed to MHC I in the lumen of the ER, leading to the activation of CD8+ cytotoxic T cells. How and where exogenous antigens become exposed to MHC I are poorly understood. Recent studies indicate that phagosomes are competent organelles for antigen cross-presentation, suggesting a possible mechanism for this process. Using fluorescence imaging, Houde et al. [11 ] provided evidence that exogenous proteins internalized by phagocytosis can be retrotranslocated to the cytoplasm side of J774 macrophages by Sec61 (Fig. 4A ). These authors further suggested that the translocated proteins are then processed by proteasomes, which are anchored to the cytoplasmic face of the phagosome (Fig. 4B) . The resulting peptides can then be delivered to the lumen of the ER (or back into the phagosome) by the transporter associated with antigen processing (TAP), where they are loaded onto and eventually presented by MHC I complexes at the cell surface, triggering a CD8+ T cell response. Guermonprez et al. [36 ] and Ackerman et al. [39 ], who studied dendritic cells, reached similar conclusions. Through this complex and sophisticated sequence, phagosomes would become a direct portal for antigen cross-presentation.



View larger version (53K):
[in this window]
[in a new window]
  		Figure 4. Assessing antigen cross-presentation via ER-phagosome fusion. (A) Retrotranslocation of luminal fluorescence to the cytosol after 30–60 min phagocytosis of latex beads coated with fluorescent ovalbumin (reprinted with permission from ref. [11 ]). (B) Immunofluorescence of proteasomes revealing their localization relative to phagosomes in whole cells and in purified phagosomes (inset). Arrows point to phagosomes (reprinted with permission from ref. [11 ]). (C) Immunofluorescence of proteasomes and of LAMP1, revealing their localization relative to phagosomes in RAW264.7 cells. A magnified version of the phagosome enclosed by the dotted line is shown in inset. (D–F) Fluorescence imaging of phagosomal fission following phagocytosis by RAW264.7 cells of latex beads coated with fluorescent ovalbumin. (D, inset) An early phagosome containing an ovalbumin-coated latex bead. The main panels show three later stages in the 30- to 60-min interval, illustrating the active fission of vesicles containing fluorescent ovalbumin. Original scale bars = 3 µm.

 

   FUNCTIONAL IMPLICATIONS OF THE ER MODEL
TOP
 ABSTRACT
 INTRODUCTION
 THE CONVENTIONAL MODEL: PLASMA...
 THE NEW MODEL: PARTICLE...
 FUNCTIONAL IMPLICATIONS OF THE...
ER OR PLASMALEMMA?
 CONCLUDING REMARKS
 REFERENCES
 
Despite the allure of the new model, several aspects of phagosomal physiology are not reconciled easily with phagosomes composed largely of ER. One of the defining characteristics of phagosomes is their ability to undergo rapid acidification, which becomes manifest shortly after sealing and is attributed to the activity of V-ATPases [4 ]. To achieve and sustain such a rapid acidification, the limiting membrane of the phagosome must not only be endowed with V-ATPases but must also be relatively impermeable to the passive efflux of protons. Studies of the mechanisms regulating pH in the secretory pathway, however, have found ER membranes to be seemingly devoid of functional V-ATPases and to be highly permeable to proton equivalents [40 ]. How phagosomes composed primarily of ER membranes manage to undergo a rapid, profound, and sustained acidification is puzzling and remains unclear.

The unambiguous and generally acknowledged occurrence of phagosomal acidification also raises questions regarding the proposed mechanism of antigen cross-presentation by phagosomes. The current model involves degradation of phagosomal contents by lysosomal enzymes, followed by retrotranslocation via Sec61. After further processing by proteasomes, peptides were proposed to be reinternalized by TAP. Although Sec61 and TAP are well-established to perform their assigned transport roles at the neutral pH that prevails in the lumen of the ER, it is not at all clear that they can function at the extremely acidic pH found in phagosomes. Indeed, the pH must be low prior to retrotranslocation if lysosomal enzymes are to degrade their substrates effectively. More research is required to validate the ability of Sec61 and TAP to function at acidic luminal pH.


   ER OR PLASMALEMMA?
TOP
 ABSTRACT
 INTRODUCTION
 THE CONVENTIONAL MODEL: PLASMA...
 THE NEW MODEL: PARTICLE...
 FUNCTIONAL IMPLICATIONS OF THE...
 ER OR PLASMALEMMA?
CONCLUDING REMARKS
 REFERENCES
 
The conventional and novel models of phagosome formation are seemingly incompatible. The former postulates that the plasmalemma is the primary constituent of the phagosomal membrane, and the model of Desjardins and colleagues specifies that early phagosomes are made largely of ER. Are these models truly incompatible? Which model is correct, and how else can some of the observations be explained?

A critical analysis of the experimental approaches used may shed some light on the discrepancy. The ER model was prompted originally by biochemical determinations of the protein composition of purified phagosomes. Although greatly aided by the characteristic buoyancy of latex beads, the method for purification of phagosomal membranes is imperfect. As with any other biochemical fractionation procedure, contaminants are an inevitable component of the final preparation. As the ER is by far the most abundant organelle, it is a likely source of contaminating membranes. The proposed close, functional interaction between phagosomes and the ER [10 ] makes it likely that physical connections may exist between these organelles, making it impossible to segregate their membranes by differential centrifugation. Indeed, scanning and transmission electron microscopy of latex bead phagosomes purified exactly as described by Desjardins’ group [10 ] reveals the presence of foreign membranes, often in close apposition with the phagosomes (Fig. 2E and 2F) . The presence of impurities in our preparations may be a reflection of our ineptitude, yet we were not able to dislodge the adherent membranes by washing, and it is not clear how they would have been removed in the experiments of the Montreal group. In fact, close scrutiny of their data reveals that adherent membranes are also associated with their phagosomes. Moreover, their proteomic analysis includes mitochondrial components, which they attribute to contamination [41 ]. Thus, by their admission, the phagosomal preparation is not entirely pure. That the ER may be a contaminant of the preparation is also suggested by the findings of Gotthardt et al. [42 ], who showed that treatment of "purified" phagosomes with adenosine 5'-triphosphate led to a five- to tenfold decrease in the amount of ER markers (PDI and calreticulin) and a concomitant enrichment for cathepsin D, a bona fide marker of phagolysosomes. In this context, the presence of ER components in the purified preparation does not necessarily imply that the phagosomal membrane is composed largely, or even partly, of ER.

Contamination of the purified phagosomal preparation, however, cannot explain the reports that ER components are found on the phagosomal membrane, when intact cells are analyzed microscopically. At the light level, calnexin and GRP78, two components of the ER, have been suggested to line the vacuole (e.g., Fig. 3A 3B 3C ). We would argue, however, that the resolution of such images is insufficient to draw conclusive results. In preliminary experiments from our laboratory using confocal microscopy, neither PDI nor Sec61 appears to colocalize with well-resolved markers of the phagosomal membrane (unpublished observations), whereas plasma membrane markers were reported earlier to be readily detectable in newly formed phagosomes (e.g., Fig. 3D ).

Limited resolution may explain the discrepancies between light micrographs, but this explanation is hardly applicable to electron micrographs. How is the presence of products of glucose-6-phosphatase in close association with phagosomal membranes explained? Clearly, the simplest explanation is that ER is indeed a component of the phagosomal membrane. However, caution must be exerted when analyzing precipitates generated by this cytochemical method. False positives can be generated by the presence of other hydrolytic enzymes, such as alkaline and acid phosphatases [43 ], which are enriched in the plasma membrane [44 ] and lysosomes [45 ], respectively. In this regard, it is worth noting that previous studies reported low glucose-6-phosphatase activity on the phagosome membrane, coupled with a tenfold enrichment of plasmalemmal reduced nicotinamide adenine dinucleotide phosphate oxidase and 5'-nucleotidase activity [46 ]. Therefore, a re-examination of the location of glucose-6-phosphatase appears justified. Ideally, such studies should avoid the use of inhibitors of phosphatidylinositol 3-kinases or of V-ATPases. These agents, which were applied to the cells in many of the experiments of Gagnon et al. [10 ], disrupt vesicular traffic and distort endomembrane compartments.

The presence of more specific markers of the ER detected by immunogold staining in electron micrographs of purified phagosomes or intact phagosomes within macrophages is difficult to dispute. Collapsed vesicles of contaminating ER, such as those seen in Figure 2E , may have conferred immunoreactivity to the isolated phagosomes, yet the presence of similar markers on the phagosomal membrane of intact cells would appear incontrovertible. Yet, this is not a universal observation. Recent experiments from our collaborators failed to detect significant amounts of ER markers in phagosomes (Marc Pypaert, N. Touret, Sergio Trombetta, and Ira Mellman, unpublished observations).

The evidence in support of antigen cross-presentation by ER-lined phagosomes should also be subject to further scrutiny. Not only do we fail to detect Sec61 on the phagosomal membrane (unpublished observations), but also, we were similarly unable to reproduce the accumulation of proteasomes or immunoproteasomes in the immediate vicinity of the phagosomal membrane (Fig. 4C) , contrary to the results of Houde et al. [11] (Fig. 4B) . These authors also reported that fluorescence derived from labeled ovalbumin, internalized with phagocytic particles, was detectable in the cytosol of phagocytes (Fig. 4A) . The fluorescence was interpreted to represent fluorescent peptides that reached the cytosol by hydrolysis and retrotranslocation across the phagosomal membrane [11 ]. However, it is also possible that the fluorescent probe was detached from the protein and diffused or was transported across the membrane by another mechanism to the cytosol. Moreover, when we attempted to repeat the experiments described by Houde et al. [11], we noted that much of the fluorescence was removed from the phagosomes by fission of vesicles containing the labeled ovalbumin or fragments thereof (Fig. 4D 4E 4F ). Vesicular fission, coupled to retrograde transport of vesicles to the Golgi complex and/or ER, may provide an alternative mechanism for antigen cross-presentation by MHC I, not requiring that the phagosome itself be composed of ER membranes. Retrograde traffic of vesicles derived from late endosomes to the trans-Golgi network is a well-established event.


   CONCLUDING REMARKS
TOP
 ABSTRACT
 INTRODUCTION
 THE CONVENTIONAL MODEL: PLASMA...
 THE NEW MODEL: PARTICLE...
 FUNCTIONAL IMPLICATIONS OF THE...
 ER OR PLASMALEMMA?
 CONCLUDING REMARKS
REFERENCES
 
In the past couple of years, the paradigm of phagocytosis that was accepted for decades was forsaken in favor of a revolutionary new model. It is our contention that disposal of the conventional model may have been premature and that additional experimental analysis will be required to ascertain the applicability of the ER model. The eagerness with which the ER model was embraced is, in part, a result of the existence of precedents such as Legionella invasion and autophagosome formation, relating ER to formation of the vacuoles. However, recent information has questioned the involvement of ER in autophagosome biogenesis [47 ]. In the case of Legionella, the merger of the parasitic vacuole with the ER-to-Golgi intermediate compartment, or ERGIC, is not disputed, but this is a late event that occurs long after internalization of the bacteria has occurred [48 ]. In this regard, sequestration of Legionella in an ERGIC-derived vacuole for the purpose of avoiding the microbicidal machinery of the host cells is just another variant of a more general strategy. Chlamydia occupies a vacuole rich in Golgi-derived lipids, Salmonella resides in a unique compartment with some late endosomal markers, and Listeria escapes into the cytosol. All of these parasites, as well as Legionella, have been traditionally thought to initially occupy a plasma membrane-derived vacuole, and no convincing evidence has been offered to dispute this notion.

Phagocytosis, phagosome maturation, and antigen presentation are keys to the innate and acquired immune responses. A clear understanding of the mode of phagosome formation is essential to rationalize these events. It is our opinion that although innovative and attractive, the ER hypothesis has not yet been proven definitively. Its validity and universality are challenged by earlier findings that seem incompatible with the ER model, as well as by the emergence of new data that validate the conventional model. A more definitive answer will only be obtained by further experimentation. These should include a dynamic and quantitative assessment of the contribution of the plasmalemma and ER membrane to the formation of early phagosomes; determination of the presence of the ER in phagosomes by quantitative immunogold electron microscopy in intact cells; assessment of the accessibility of the ER lumen to exogenous probes, which is predicted to occur upon fusion of the ER with nascent phagosomes; and biochemical determinations to validate the delivery of luminal ER proteins to the interior of the phagosomes.

Received November 2, 2004; revised January 24, 2005; accepted February 4, 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 THE CONVENTIONAL MODEL: PLASMA...
 THE NEW MODEL: PARTICLE...
 FUNCTIONAL IMPLICATIONS OF THE...
 ER OR PLASMALEMMA?
 CONCLUDING REMARKS
 REFERENCES
 

  1. Aderem, A. (2002) How to eat something bigger than your head Cell 110,5-8[CrossRef][Medline]
  2. Greenberg, S., Grinstein, S. (2002) Phagocytosis and innate immunity Curr. Opin. Immunol. 14,136-145[CrossRef][Medline]
  3. Allen, L. A., Aderem, A. (1996) Molecular definition of distinct cytoskeletal structures involved in complement- and Fc receptor-mediated phagocytosis in macrophages J. Exp. Med. 184,627-637[Abstract/Free Full Text]
  4. Lukacs, G. L., Rotstein, O. D., Grinstein, S. (1990) Phagosomal acidification is mediated by a vacuolar-type H(+)-ATPase in murine macrophages J. Biol. Chem. 265,21099-21107[Abstract/Free Full Text]
  5. Bajno, L., Peng, X. R., Schreiber, A. D., Moore, H. P., Trimble, W. S., Grinstein, S. (2000) Focal exocytosis of VAMP3-containing vesicles at sites of phagosome formation J. Cell Biol. 149,697-706[Abstract/Free Full Text]
  6. Cox, D., Lee, D. J., Dale, B. M., Calafat, J., Greenberg, S. (2000) A Rab11-containing rapidly recycling compartment in macrophages that promotes phagocytosis Proc. Natl. Acad. Sci. USA 97,680-685[Abstract/Free Full Text]
  7. Niedergang, F., Colucci-Guyon, E., Dubois, T., Raposo, G., Chavrier, P. (2003) ADP ribosylation factor 6 is activated and controls membrane delivery during phagocytosis in macrophages J. Cell Biol. 161,1143-1150[Abstract/Free Full Text]
  8. Braun, V., Fraisier, V., Raposo, G., Hurbain, I., Sibarita, J. B., Chavrier, P., Galli, T., Niedergang, F. (2004) TI-VAMP/VAMP7 is required for optimal phagocytosis of opsonized particles in macrophages EMBO J. 23,4166-4176[CrossRef][Medline]
  9. Garin, J., Diez, R., Kieffer, S., Dermine, J. F., Duclos, S., Gagnon, E., Sadoul, R., Rondeau, C., Desjardins, M. (2001) The phagosome proteome: insight into phagosome functions J. Cell Biol. 152,165-180[Abstract/Free Full Text]
  10. Gagnon, E., Duclos, S., Rondeau, C., Chevet, E., Cameron, P. H., Steele-Mortimer, O., Paiement, J., Bergeron, J. J., Desjardins, M. (2002) Endoplasmic reticulum-mediated phagocytosis is a mechanism of entry into macrophages Cell 110,119-131[CrossRef][Medline]
  11. Houde, M., Bertholet, S., Gagnon, E., Brunet, S., Goyette, G., Laplante, A., Princiotta, M. F., Thibault, P., Sacks, D., Desjardins, M. (2003) Phagosomes are competent organelles for antigen cross-presentation Nature 425,402-406[CrossRef][Medline]
  12. Ulsamer, A. G., Wright, P. L., Wetzel, M. G., Korn, E. D. (1971) Plasma and phagosome membranes of Acanthamoeba castellanii J. Cell Biol. 51,193-215[Abstract/Free Full Text]
  13. Muller, W. A., Steinman, R. M., Cohn, Z. A. (1980) The membrane proteins of the vacuolar system I. Analysis of a novel method of intralysosomal iodination J. Cell Biol. 86,292-303[Abstract/Free Full Text]
  14. Ramachandra, L., Harding, C. V. (2000) Phagosomes acquire nascent and recycling class II MHC molecules but primarily use nascent molecules in phagocytic antigen processing J. Immunol. 164,5103-5112[Abstract/Free Full Text]
  15. Martens, S., Sabel, K., Lange, R., Uthaiah, R., Wolf, E., Howard, J. C. (2004) Mechanisms regulating the positioning of mouse p47 resistance GTPases LRG-47 and IIGP1 on cellular membranes: retargeting to plasma membrane induced by phagocytosis J. Immunol. 173,2594-2606[Abstract/Free Full Text]
  16. Holevinsky, K. O., Nelson, D. J. (1998) Membrane capacitance changes associated with particle uptake during phagocytosis in macrophages Biophys. J. 75,2577-2586[Abstract/Free Full Text]
  17. Hackam, D. J., Rotstein, O. D., Sjolin, C., Schreiber, A. D., Trimble, W. S., Grinstein, S. (1998) v-SNARE-dependent secretion is required for phagocytosis Proc. Natl. Acad. Sci. USA 95,11691-11696[Abstract/Free Full Text]
  18. Ungar, D., Hughson, F. M. (2003) SNARE protein structure and function Annu. Rev. Cell Dev. Biol. 19,493-517[CrossRef][Medline]
  19. Sollner, T., Bennett, M. K., Whiteheart, S. W., Scheller, R. H., Rothman, J. E. (1993) A protein assembly-disassembly pathway in vitro that may correspond to sequential steps of synaptic vesicle docking, activation, and fusion Cell 75,409-418[CrossRef][Medline]
  20. Swanton, E., Bishop, N., Sheehan, J., High, S., Woodman, P. (2000) Disassembly of membrane-associated NSF 20S complexes is slow relative to vesicle fusion and is Ca(2+)-independent J. Cell Sci. 113,1783-1791[Abstract]
  21. Littleton, J. T., Barnard, R. J., Titus, S. A., Slind, J., Chapman, E. R., Ganetzky, B. (2001) SNARE-complex disassembly by NSF follows synaptic-vesicle fusion Proc. Natl. Acad. Sci. USA 98,12233-12238[Abstract/Free Full Text]
  22. Coppolino, M. G., Kong, C., Mohtashami, M., Schreiber, A. D., Brumell, J. H., Finlay, B. B., Grinstein, S., Trimble, W. S. (2001) Requirement for N-ethylmaleimide-sensitive factor activity at different stages of bacterial invasion and phagocytosis J. Biol. Chem. 276,4772-4780[Abstract/Free Full Text]
  23. D’Souza-Schorey, C., Li, G., Colombo, M. I., Stahl, P. D. (1995) A regulatory role for ARF6 in receptor-mediated endocytosis Science 267,1175-1178[Abstract/Free Full Text]
  24. Donaldson, J. G. (2003) Multiple roles for Arf6: sorting, structuring, and signaling at the plasma membrane J. Biol. Chem. 278,41573-41576[Free Full Text]
  25. Sturgill-Koszycki, S., Schaible, U. E., Russell, D. G. (1996) Mycobacterium-containing phagosomes are accessible to early endosomes and reflect a transitional state in normal phagosome biogenesis EMBO J. 15,6960-6968[Medline]
  26. Andrews, N. W. (1995) Lysosome recruitment during host cell invasion by Trypanosoma cruzi Trends Cell Biol. 5,133-137[CrossRef][Medline]
  27. Tardieux, I., Webster, P., Ravesloot, J., Boron, W., Lunn, J. A., Heuser, J. E., Andrews, N. W. (1992) Lysosome recruitment and fusion are early events required for trypanosome invasion of mammalian cells Cell 71,1117-1130[CrossRef][Medline]
  28. Wiest, D. L., Bhandoola, A., Punt, J., Kreibich, G., McKean, D., Singer, A. (1997) Incomplete endoplasmic reticulum (ER) retention in immature thymocytes as revealed by surface expression of "ER-resident" molecular chaperones Proc. Natl. Acad. Sci. USA 94,1884-1889[Abstract/Free Full Text]
  29. Okazaki, Y., Ohno, H., Takase, K., Ochiai, T., Saito, T. (2000) Cell surface expression of calnexin, a molecular chaperone in the endoplasmic reticulum J. Biol. Chem. 275,35751-35758[Abstract/Free Full Text]
  30. Johnson, S., Michalak, M., Opas, M., Eggleton, P. (2001) The ins and outs of calreticulin: from the ER lumen to the extracellular space Trends Cell Biol. 11,122-129[CrossRef][Medline]
  31. Ogden, C. A., deCathelineau, A., Hoffmann, P. R., Bratton, D., Ghebrehiwet, B., Fadok, V. A., Henson, P. M. (2001) C1q and mannose binding lectin engagement of cell surface calreticulin and CD91 initiates macropinocytosis and uptake of apoptotic cells J. Exp. Med. 194,781-795[Abstract/Free Full Text]
  32. Muller-Taubenberger, A., Lupas, A. N., Li, H., Ecke, M., Simmeth, E., Gerisch, G. (2001) Calreticulin and calnexin in the endoplasmic reticulum are important for phagocytosis EMBO J. 20,6772-6782[CrossRef][Medline]
  33. Dunn, W. A., Jr (1990) Studies on the mechanisms of autophagy: formation of the autophagic vacuole J. Cell Biol. 110,1923-1933[Abstract/Free Full Text]
  34. Klionsky, D. J. (2005) The molecular machinery of autophagy: unanswered questions J. Cell Sci. 118,7-18[Abstract/Free Full Text]
  35. McNew, J. A., Parlati, F., Fukuda, R., Johnston, R. J., Paz, K., Paumet, F., Sollner, T. H., Rothman, J. E. (2000) Compartmental specificity of cellular membrane fusion encoded in SNARE proteins Nature 407,153-159[CrossRef][Medline]
  36. Guermonprez, P., Saveanu, L., Kleijmeer, M., Davoust, J., Van Endert, P., Amigorena, S. (2003) ER-phagosome fusion defines an MHC class I cross-presentation compartment in dendritic cells Nature 425,397-402[CrossRef][Medline]
  37. Ackerman, A. L., Kyritsis, C., Tampe, R., Cresswell, P. (2003) Early phagosomes in dendritic cells form a cellular compartment sufficient for cross presentation of exogenous antigens Proc. Natl. Acad. Sci. USA 100,12889-12894[Abstract/Free Full Text]
  38. Wu, M. M., Grabe, M., Adams, S., Tsien, R. Y., Moore, H. P., Machen, T. E. (2001) Mechanisms of pH regulation in the regulated secretory pathway J. Biol. Chem. 276,33027-33035[Abstract/Free Full Text]
  39. Desjardins, M., Celis, J. E., van Meer, G., Dieplinger, H., Jahraus, A., Griffiths, G., Huber, L. A. (1994) Molecular characterization of phagosomes J. Biol. Chem. 269,32194-32200[Abstract/Free Full Text]
  40. Gotthardt, D., Warnatz, H. J., Henschel, O., Bruckert, F., Schleicher, M., Soldati, T. (2002) High-resolution dissection of phagosome maturation reveals distinct membrane trafficking phases Mol. Biol. Cell 13,3508-3520[Abstract/Free Full Text]
  41. Nichols, B. A., Setzer, P. Y., Bainton, D. F. (1984) Glucose-6-phosphatase as a cytochemical marker of endoplasmic reticulum in human leukocytes and platelets J. Histochem. Cytochem. 32,165-171[Abstract]
  42. Matsubara, S., Yamada, T., Minakami, H., Watanabe, T., Takizawa, T., Sato, I. (1999) Polymorphonuclear leukocytes in the fetal membranes are activated in patients with preterm delivery: ultrastructural and enzyme-histochemical evidence Placenta 20,185-188[CrossRef][Medline]
  43. Obermuller, S., Kiecke, C., von Figura, K., Honing, S. (2002) The tyrosine motifs of LAMP 1 and LAP determine their direct and indirect targeting to lysosomes J. Cell Sci. 115,185-194[Abstract/Free Full Text]
  44. Berton, G., Bellavite, P., de Nicola, G., Dri, P., Rossi, F. (1982) Plasma membrane and phagosome localization of the activated NADPH oxidase in elicited peritoneal macrophages of the guinea-pig J. Pathol. 136,241-252[CrossRef][Medline]
  45. Mizushima, N., Yamamoto, A., Hatano, M., Kobayashi, Y., Kabeya, Y., Suzuki, K., Tokuhisa, T., Ohsumi, Y., Yoshimori, T. (2001) Dissection of autophagosome formation using Apg5-deficient mouse embryonic stem cells J. Cell Biol. 152,657-668[Abstract/Free Full Text]
  46. Roy, C. R., Tilney, L. G. (2002) The road less traveled: transport of Legionella to the endoplasmic reticulum J. Cell Biol. 158,415-419[Abstract/Free Full Text]
  47. Girotti, M., Evans, J. H., Burke, D., Leslie, C. C. (2004) Cytosolic phospholipase A2 translocates to forming phagosomes during phagocytosis of zymosan in macrophages J. Biol. Chem. 279,19113-19121[Abstract/Free Full Text]
  48. Botelho, R. J., Teruel, M., Dierckman, R., Anderson, R., Wells, A., York, J. D., Meyer, T., Grinstein, S. (2000) Localized biphasic changes in phosphatidylinositol-4,5-bisphosphate at sites of phagocytosis J. Cell Biol. 151,1353-1368[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Int ImmunolHome page
Y. Sawatani, T. Miyamoto, S. Nagai, M. Maruya, J. Imai, K. Miyamoto, N. Fujita, K. Ninomiya, T. Suzuki, R. Iwasaki, et al.
The role of DC-STAMP in maintenance of immune tolerance through regulation of dendritic cell function
Int. Immunol., July 24, 2008; (2008) dxn082v1.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Biol. CellHome page
V. Braun, C. Deschamps, G. Raposo, P. Benaroch, A. Benmerah, P. Chavrier, and F. Niedergang
AP-1 and ARF1 Control Endosomal Dynamics at Sites of FcR mediated Phagocytosis
Mol. Biol. Cell, December 1, 2007; 18(12): 4921 - 4931.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
L. D. Rogers and L. J. Foster
The dynamic phagosomal proteome and the contribution of the endoplasmic reticulum
PNAS, November 20, 2007; 104(47): 18520 - 18525.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
D. J. Kusner, C. R. Thompson, N. A. Melrose, S. M. Pitson, L. M. Obeid, and S. S. Iyer
The Localization and Activity of Sphingosine Kinase 1 Are Coordinately Regulated with Actin Cytoskeletal Dynamics in Macrophages
J. Biol. Chem., August 10, 2007; 282(32): 23147 - 23162.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. ProteomicsHome page
C. Burlak, A. R. Whitney, D. J. Mead, T. Hackstadt, and F. R. DeLeo
Maturation of Human Neutrophil Phagosomes Includes Incorporation of Molecular Chaperones and Endoplasmic Reticulum Quality Control Machinery
Mol. Cell. Proteomics, April 1, 2006; 5(4): 620 - 634.
[Abstract] [Full Text] [PDF]

Home page
 J. Exp. Med.Home page
T. A.M. Groothuis and J. Neefjes
The many roads to cross-presentation
J. Exp. Med., November 21, 2005; 202(10): 1313 - 1318.
[Abstract] [Full Text] [PDF]

Home page
 J. Leukoc. Biol.Home page
E. Gagnon, J. J. Bergeron, and M. Desjardins
ER-mediated phagocytosis: myth or reality?
J. Leukoc. Biol., June 1, 2005; 77(6): 843 - 845.
[Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
jlb.1104630v1
77/6/878    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Touret, N.
Right arrow Articles by Grinstein, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Touret, N.
Right arrow Articles by Grinstein, S.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS