From the Département de Pathologie et Biologie
Cellulaire, Université de Montréal, C.P. 6128, Succ. Centre
ville, Montréal, QC, Canada, H3C 3J7, the
Laboratoire de
Chimie des Protéines, CEA, 38054 Grenoble, France, and the
** Institute for Molecular Bioscience, Centre for Microscopy and
Microanalysis and the Department of Physiology and Pharmacology,
University of Queensland, Queensland 4072, Australia
Received for publication, February 5, 2001
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ABSTRACT |
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Flotillin-1 was recently shown to be enriched on
detergent-resistant domains of the plasma membrane called lipid rafts.
These rafts, enriched in sphingolipids and cholesterol, sequester
certain proteins while excluding others. Lipid rafts have been
implicated in numerous cellular processes including signal
transduction, membrane trafficking, and molecular sorting. In this
study, we demonstrate both morphologically and biochemically that lipid rafts are present on phagosomes. These structures are enriched in
flotillin-1 and devoid of the main phagosomes membrane protein lysosomal-associated membrane protein (LAMP1). The flotillin-1 present on phagosomes does not originate from the plasma membrane during phagocytosis but accumulates gradually on maturing phagosomes. Treatment with bafilomycin A1, a compound that inhibits the proton pump
ATPase and prevents the fusion of phagosomes with late endocytic organelles, prevents the acquisition of flotillin-1 by phagosomes, indicating that this protein might be recruited on phagosomes from
endosomal organelles. A proteomic characterization of the lipid rafts
of phagosomes indicates that actin, the Lateral assemblies of lipids, termed lipid rafts, have been
postulated to represent a general feature of the plasma membrane of
eukaryotic cells (1, 2). Rafts apparently form because of the
biophysical properties of sphingolipids and cholesterol, which pack
tightly into liquid-ordered
(lo)1 domains
that partition away from the more disorganized glycerophospholipids in
the bulk of the membrane (3). Lipid-modified proteins and some
transmembrane proteins are concentrated in the rafts while other
proteins are excluded. Lipid rafts have been implicated in many
important cellular processes, such as polarized sorting of apical
membrane proteins in epithelial cells and signal transduction (4).
Recent evidence further indicates that a raft-based mechanism might be
involved in the sorting of SNAREs to the plasma membrane and in
their function in apical membrane docking and fusion events (5). As
this is in no way an exhaustive list of the potential function of lipid
rafts, it appears that membrane subdomains represent important sites
conferring specialized properties to foci within biological membranes.
In the present study, we provide evidence showing that lipid rafts are
present on phagosomes. These specialized regions, devoid of the major
phagosomal protein LAMP1, are enriched in flotillin-1. The phagosomal
lipid rafts are unlikely to be simply transferred from the plasma
membrane to phagosomes during phagocytosis because early phagosomes
display low amounts of flotillin-1. Instead, flotillin-1 is recruited
to phagosomes during phagosome maturation, possibly through fusion with
late endocytic organelles. The identification of lipid rafts on
phagosomes suggests that specific functions occur at focal points on
the phagosome membrane. Further proteomic characterization allowed us
to identify sets of proteins indicating that phagosome lipid rafts
might be involved in signal transduction, interaction with actin, and
phagosome acidification.
Cell Culture and Phagosome Formation and Isolation--
The
murine macrophage-like cell line J774 was cultured in Dulbecco's
modified Eagle's medium high glucose (Life Technologies, Grand Island,
NY) supplemented with 10% heat-inactivated fetal bovine serum, 1%
glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin at
37 °C in a 5% CO2 atmosphere. Cells were grown to
~80% confluency in Petri dishes prior to each experiment, as described previously (6).
To form phagosomes, J774 macrophages were fed with 0.8 µm blue-dyed
latex beads (Sigma) diluted 1:50 in culture medium. Depending on the
experiment, cells were allowed to internalize beads for 30 to 90 min at
37 °C. Cells were then washed three times for 5 min with ice-cold
PBS to remove non-internalized beads and were further incubated for
increasing periods of time to obtain early and late phagosomes.
Phagosomes were then isolated on sucrose step gradients as described
previously (6). Purified phagosomes were resuspended in Laemmli or
rehydration/lysis buffers for Western blotting and two-dimensional gel
electrophoresis, respectively.
Sensitivity to Bafilomycin A1--
To determine whether
phagosome maturation is required for the acquisition of flotillin-1, we
internalized latex beads in J774 macrophages for 30 min. Cells were
then incubated for 3 h in the presence of bafilomycin A1 (0.5 µM), a drug that inhibits the vacuolar H+ ATPase and
prevents lysosome biogenesis (7), or without drug (Me2SO
vehicle only). To further determine whether the association of
flotillin-1 present on phagosomes is modulated by luminal pH, we
internalized latex beads for 60 min followed by a 3-h chase, a time
point at which flotillin-1 is present in high amounts on phagosomes
(see below). Cells were then incubated for 60 min with bafilomycin as
above. Phagosomes were then isolated and prepared for Western blotting.
Sensitivity to Pronase--
To determine whether flotillin-1 is
exposed on the cytoplasmic side of the phagosome membrane, phagosomes
(1-h pulse/3-h chase) isolated as described above were treated with
Pronase, a mixture of proteases, as described previously (8). As
demonstrated, in our previous study, Pronase treatment did not affect
proteins present in the lumen of phagosomes, such as cathepsins and
other hydrolases, indicating that phagosomes in our preparations were intact.
Triton X-114 Extraction--
The phase separation of membrane
proteins using Triton X-114 was performed according to the procedure
previously described (9) using isolated phagosomes as starting
material. Proteins from the separated phases (aqueous and detergent)
were solubilized in Laemmli buffer for Western blot analysis.
Lipid Raft Isolation--
To prepare phagosome rafts, phagosomes
were formed by internalizing latex beads for 60 min followed by
incubation in culture medium without beads for 3 h. For each
experiment, 42 × 10 cm Petri dishes were used, and phagosomes
were isolated as described above. The purified phagosome pellet was
resuspended in 1.5 ml of TNE-Triton buffer (25 mM Tris, 150 mM NaCl, 5 mM EDTA, protease inhibitor mixture
(Roche Molecular Biochemicals), pH 7.4 and 1% Triton X-100),
transferred to an Eppendorf tube and shaken for 30 min at 4 °C to
solubilize phagosomal membranes. Latex beads were then removed by
centrifugation, and the supernatant containing solubilized and
insoluble phagosome components was added to 1.5 ml of sucrose (90%) to
obtain a final concentration of 45% sucrose, which was then poured at
the bottom of an Ultraclear centrifuge tube (Beckman). Finally, 4 ml of
35% sucrose and 4 ml of 5% sucrose (with protease inhibitors) were
layered. After a 17-20-h centrifugation at 38,000 rpm (SW41 rotor) to
float the insoluble rafts, 1 ml at the 5%/35% interface containing
the rafts was collected. The proteins in this fraction were then
precipitated with methanol/chloroform according to Wessel and Flugge
(10) and resuspended in the appropriate buffers for Western blotting or
two-dimensional gel electrophoresis. In some cases, to determine the
distribution of flotillin-1 and LAMP1 in the gradient after the
flotation step, 1-ml fractions from the top of the gradient were
collected and the proteins were then precipitated by
methanol/chloroform and resuspended in Laemmli buffer for Western blot analysis.
Western Blotting--
For Western blot analysis, each sample in
a given experiment contained the same number of phagosomes determined
by evaluating the number of latex bead by FACS analysis as done
previously (11). Western blotting was performed according to standard
procedures. In the kinetic study, the membrane was cut in half between
the molecular mass markers 52 and 80. The upper part was probed with the 1D4B rat monoclonal antibody (Developmental Studies Hybrodoma Bank,
University of Iowa) directed against LAMP1. The lower part was probed
with a rabbit polyclonal antibody specific for flotillin-1. These
antibodies were raised against a synthetic peptide (Chiron Technologies, Adelaide, Australia) corresponding to the C terminus of
mouse flotillin-1 (VNHNKPLRTA) with the addition of a cysteine residue
at the N terminus for coupling to carrier protein or for preparing an
affinity purification column. Affinity purification was performed
exactly as described previously (12). Appropriate second antibodies
coupled to horseradish peroxidase were then used and the membranes were
treated for ECL (Roche Diagnostics).
The presence of flotillin-1 and LAMP1 in phagosomes and phagosome rafts
was evaluated by Western blot on the same membrane (see above). The
same amount of protein was loaded for each sample. For a
two-dimensional gel Western blot, the portion of the gel corresponding
to the area where flotillin-1 was identified was transferred to
nitrocellulose membrane and immunoblotted with the anti-flotillin-1
antibody as described above.
Immunofluorescence--
J774 macrophages were grown on
coverslips to a confluency of about 80%, at least 36 h before the
experiment. Cells were then fed or not with 3 µm latex beads (Sigma)
(3 µm beads rather than 0.8 µm beads were used to facilitate
microscopic observations) in culture medium for 30 min (1/200) or 60 min (1/400) followed by chase periods of 1, 3, and 16 h. In some
cases, cells were infected with Leishmania donovani strain
1S grown as described (13) at a concentration of 2.0 and 1.0 × 107/ml medium for 60 min, or with an lpg2 High Resolution Two-dimensional Gel Electrophoresis--
Samples
destined for two-dimensional gel electrophoresis were prepared from
cells metabolically labeled with [35S]methionine
following published protocols (6). The various samples were first
separated according to their isoelectrical point using immobilized pH
gradient strips (IPGs). Equal counts of radioactivity were loaded for
each sample in a given experiment. Loading of the samples in the first
dimension was performed by in-gel re-swelling (16). At the end of the
first dimension, the strips were equilibrated with a 10-min incubation
in equilibration solution (urea, 6 M; SDS, 2%; glycerol,
20%; Tris-HCl, 1.0 M, pH 6.8) freshly supplemented with
dithioerythritol (2% w/v) followed by a 5-min incubation in
equilibration solution freshly supplemented with iodoacetamide (2.5%
w/v), and the proteins were separated according to their molecular mass
using standard SDS-PAGE. At the end of the migration, gels were treated
for autoradiography as described previously (6). To identify some of
the lipid raft proteins according to their migration properties,
protein patterns of raft preparations were compared with a phagosome
two-dimensional gel data base in which 140 protein spots have been
identified so far (8).
In this study, we have provided evidence that lipid rafts are
present on the phagosome membrane, a key organelle involved in the
killing and degradation of intracellular pathogens (17). The existence
of lipid subdomains on phagosomes was first suggested by a proteomic
analysis indicating that proteins known to associate to lipid rafts,
including flotillin-1, are present on this organelle (8). Here, we
further demonstrate the enrichment of flotillin-1 on phagosomes by
Western blot analysis in both one- and two-dimensional gels (Fig.
1, A-C). The association of
flotillin-1 to the phagosome membrane was confirmed by Triton X-114
extraction showing that a great proportion of this protein partitioned
in the detergent phase (Fig. 1D), and Pronase proteolysis
experiments indicating that flotillin-1 (at least the C-terminal end
recognized by our antibody) is exposed on the cytoplasmic side of the
phagosome membrane (Fig. 1E). The latter experiment rules
out the possibility that flotillin-1 is simply present within the lumen
of phagosomes for degradation. Although flotillin-1 was originally
shown to accumulate in subdomains of the plasma membrane of adipocytes and neurons (18, 19), our studies using immunofluorescence analysis
failed to detect noticeable levels of flotillin-1 on the plasma
membrane of macrophages. Furthermore, biochemical (Fig. 2A) and morphological (Fig.
2B) analyses indicated that flotillin-1 is barely detectable
on early phagosomes (derived from the plasma membrane), but accumulates
on maturing phagosomes. Interestingly, our results showed that
flotillin-1 associates to phagosomes at later time points than LAMP1, a
marker normally used to define late endocytic/phagocytic structures
(Fig. 2, A and B). In cells that had not
internalized latex beads, observation at the confocal microscope
revealed that although a small part of the flotillin-1 labeling is
present on vesicles also labeled for LAMP1, most of the labeling does
not colocalize to the same vesicle populations (Fig. 2C),
suggesting that these markers are distributed on different vesicles of
a common pathway. These results also indicate that flotillin-1 is a
novel marker of late endocytic/phagocytic organelles that may
accumulate on post-LAMP structures. This is supported by results
showing that bafilomycin, a drug that inhibits the formation (or
maturation) of lysosomes (7), also inhibits the accumulation of
flotillin-1 to phagosomes (Fig. 2D).
- and
-subunits of
heterotrimeric G proteins, as well as subunits of the proton pump
V-ATPase are among the constituents of these domains. Remarkably, the
intracellular parasite Leishmania donovani can actively
inhibit the acquisition of flotillin-1-enriched lipid rafts by
phagosomes and the maturation of these organelles. These results
indicate that specialized functions required for phagolysosome
biogenesis may occur at focal points on the phagosome membrane, and
therefore represent a potential target of intracellular pathogens.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
/
mutant lacking surface LPG (14) followed by a 3-h chase. Cells were
then fixed at
20 °C in methanol/acetone (80:20) for 20 min. Fixed
cells were then washed and rehydrated in PBS two times for 5 min, two
times for 10 min, and then blocked for 10 min with PBS, 2% bovine
serum albumin (fraction V, Sigma, St-Louis, MO), 0.2% gelatin.
Coverslips were then incubated with the rabbit anti-flotillin-1
antibody and the rat anti-LAMP1 1D4B for 1 h. In the case of
Leishmania infection, cells were incubated with the CA7AE
antibody directed against the major surface glycoconjugate of
Leishmania (15) to visualize the parasites within cells and
with the anti-flotillin-1 antibody. After several washes in PBS, 1%
bovine serum albumin, coverslips were incubated with an anti-rabbit IgG
coupled to Alexa and with an anti-rat IgG coupled to Texas Red for
1 h. Coverslips were then washed in PBS, mounted on slides with
Gelvatol, and observed at the epifluorescence or confocal microscope.
Controls included the tests for interspecies cross-reaction and cells
incubated only with the secondary antibodies.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
View larger version (93K):
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Fig. 1.
Flotillin-1 is present and enriched on the
phagosome membrane. A, phagosomes were isolated from
J774 macrophages, and two-dimensional gel electrophoresis was performed
with immobilized pH gradients in the first dimension and SDS-PAGE in
the second dimension, following standard procedures. The spot
corresponding to flotillin-1 was previously identified by a proteomic
approach (see Ref. 8). B, an area of a two-dimensional gel
corresponding to the location of flotillin-1 was cut off and
transferred to nitrocellulose for immunoblotting with the
anti-flotillin-1 antibody. Several spots at the same molecular mass
with different pI were revealed. C, Western blot from
SDS-PAGE gels indicates that flotillin-1 is highly enriched on
phagosomes (Phago) compared with total cell lysate
(TCL). In each lane, equal amounts of protein
were loaded. D, Western blot analysis indicates that
flotillin-1 is partially recovered in the detergent phase of a Triton
X-114 phagosome extract, as expected for a membrane-associated protein.
The presence of flotillin-1 in the aqueous phase could imply that this
protein is loosely associated to phagosomal membrane. E,
phagosome fractions were incubated for 30 min at 37 °C in the
presence or absence of Pronase. This treatment degrades all proteins or
portion of proteins exposed on the cytoplasmic side of phagosomes. The
anti-flotillin-1 antibody, which recognizes the C-terminal portion of
the protein, failed to reveal the protein in the fraction treated with
Pronase, indicating that this part of the protein is present on the
cytoplasmic side of phagosomes.
View larger version (65K):
[in a new window]
Fig. 2.
Flotillin-1 accumulates on phagosomes during
maturation. A, Western blotting was performed on
purified phagosomes of increasing ages formed by the internalization of
latex beads for 30-min internalization/no chase (early phagosomes) to
1-h internalization/16-h chase (late phagosomes). The membrane was cut
in half to reveal LAMP1 and flotillin-1 on the same samples. Each
lane was loaded with the same number of phagosomes
determined by flow cytometry. The results indicate that flotillin-1
accumulates on maturing phagosomes and thus represents a late
phagocytic marker. B, double immunofluorescence analysis
with flotillin-1 and LAMP1 antibodies confirms that flotillin-1
accumulates on late phagosomes. It also clearly shows that flotillin-1
appears to be a later marker than LAMP1, as shown by the absence of
flotillin-1 labeling on most of the early phagosomes
(arrows). C, J774 macrophages that had not
internalized latex beads were processed as described for double
immunofluorescence. Observation by confocal microscopy indicates that
there is very little colocalization of flotillin-1 with vesicular
structures labeled for LAMP1. D, cells were fed with latex
beads for 30 min. Phagosomes were then either isolated immediately or
allowed to mature for 3 h in the presence or absence of
bafilomycin A1, an inhibitor of the vacuolar H+ ATPase, and
were processed for Western blotting. Inhibition of endovacuolar
acidification prevented the recruitment of flotillin-1 to phagosomes,
indicating that this process is pH-dependent. In contrast,
treatment of cells already containing mature flotillin-1-enriched
phagosomes (lanes 4 and 5) did not release this
protein from phagosomes, indicating that the association of flotillin-1
to phagosomes is not regulated by the luminal pH.
At high magnification, double immunofluorescence labeling clearly
indicates that flotillin-1 is present on patches of the phagosome
membrane whereas LAMP1 forms a uniform ring around the membrane of this
organelle (Fig. 3A). To
demonstrate that flotillin-1 is a general marker of phagosomes, and not
simply associated with latex-containing compartments, we showed by
immunofluorescence its presence on phagosomes housing the intracellular
parasite Leishmania (Fig. 3B). However, we
observed that only a small proportion of
Leishmania-containing phagosomes were positive for
flotillin-1. Indeed, quantitative analysis indicates that over 90% of
latex bead-containing phagosomes are positive for flotillin-1, whereas only 20% of phagosomes housing Leishmania parasites are
labeled by the antibody (Fig. 3C). We have shown previously
that the promastigote form of Leishmania parasites are able
to inhibit phagosome fusion with late endocytic organelles (14). This
inhibition is caused by the lipophosphoglycan (LPG), the major surface
glycoconjugate of Leishmania, because mutants lacking LPG
fuse extensively with late endocytic organelles (20). Accordingly, we
performed additional experiments and measured the presence of
flotillin-1 on phagosomes containing Leishmania lpg2/
mutants. The results obtained indicate that 53% of phagosomes
containing that mutant are positive for flotillin-1. This suggests that
flotillin-1 might be necessary for, or acquired through, fusion with
late endocytic organelles. Interestingly, LPG is a GPI-anchored
molecule secreted by the parasite. Its mode of action in the inhibition
of phagosome-endosome fusion was proposed to involve its insertion
through the lipidic anchor in the phagosomal membrane (14, 21). Because
GPI anchors have a strong affinity for lipid rafts (22), this process
could interfere with the formation of lipid rafts on
Leishmania-containing phagosomes or the association of
flotillin-1 to these structures. Other Leishmania
LPG-deficient mutants are currently tested in our system to further
ensure the role of that molecule in the modulation of raft
formation.
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The properties governing the association of flotillin-1 to phagosome lipid rafts are unknown. The presence in its structure of a Prohibitin Homology (PHB) Domain, also referred to as Stomatin, Prohibitin, Flotillin, HflC, and K (SPFH) Domain (23), might allow its association to lipid rafts. As the name implies, this domain is also present on prohibitin and stomatin, two membrane proteins shown to associate with Triton X-100-insoluble rafts (24, 25). Interestingly, both prohibitin and stomatin have been identified on latex bead-containing phagosomes by mass spectrometry (8). Alternatively, results by Western blot analysis on two-dimensional gels showing that flotillin-1 migrates as a series of spots of different pI suggest that flotillin-1 might be hyperphosphorylated (Fig. 1B). This feature may also provide a potential mechanism of association to lipid rafts, as shown for p56lck, whose segregation in lipid domains of the plasma membrane is linked to its phosphorylation state (26).
The presence of lipid rafts on phagosomes is surprising because these
structures have been described mainly in the Golgi apparatus and the
plasma membrane. Despite the phagosomal distribution of flotillin-1 in
raft-like structures, it was important to establish whether these
structures correspond to the biochemical definition of rafts, which is
the insolubility in Triton X-100 and low density in sucrose gradients.
To address this point, we performed raft isolation from purified
phagosomes and tested them for the presence of flotillin-1 by Western
blotting (Fig. 4A). Our
results clearly showed an enrichment of flotillin-1 in the
phagosome-rafts fraction compared with total phagosomes (Fig.
4B). In contrast, LAMP1, a major membrane protein of
phagosomes, was not detected in the phagosome-rafts fraction,
demonstrating the specificity of our extraction procedure. Our results
also showed that a significant portion of flotillin-1 present on
phagosomes is solubilized by the Triton X-100 treatment (Fig.
4A), suggesting that some of the phagosomal flotillin-1 is
not associated with lipid rafts. It is also possible that
flotillin-1-enriched rafts are partially solubilized by the detergent
because it was shown that lipid rafts displaying different
sensitivities to solubilization, depending on the detergent used, can
coexist in the same cells (27).
|
SDS-PAGE analysis indicated that several proteins are enriched in the
phagosome-rafts fraction compared with total phagosomes (Fig.
4C, arrows). To identify some of the proteins
present in lipid rafts, we used a proteomic approach. The proteins
recovered in phagosome lipid rafts isolated from
[35S]methionine-labeled cells were separated by
two-dimensional gel electrophoresis. The gels were dried and exposed
for 6 weeks and then analyzed and compared with gels of total
phagosomes. This allowed identification of phagosome proteins present
in the rafts. Using our data base of identified phagosomal proteins
(8), we were able to show that actin, the ,
1, and
2 subunits
of heterotrimeric G-proteins, as well as the A, B, and possibly the E
subunits of the vacuolar proton pump ATPase were among the major proteins of the Triton X-100 insoluble lipid rafts (Fig.
4D). At least 9 as yet unidentified proteins were also
enriched in the phagosome lipid rafts preparations (3 of which are
highlighted by question marks in Fig. 4C). Subunits of
heterotrimeric G-proteins have been identified in lipid rafts in other
studies (28). Their identification was instrumental to the proposal
that rafts are specialized sites for signal transduction (4). Our
findings suggest that signal transduction could also take place through specialized regions of the phagosome membrane. Subunits of the proton
pump ATPase have also been identified previously in Triton X-100
insoluble fractions (29), in association with proteins of the SNARE
complex, suggesting that control of fusion events (see below), through
acidification, could involve lipid rafts.
Although flotillin-1 was recently shown to be involved in insulin signaling at the plasma membrane of adipocytes (30), the functions of this protein and, more generally, of lipid rafts on phagosomes are currently unknown. An interesting feature of phagosomes is that it is an organelle unable to perform its main task, the killing and degradation of microorganisms, immediately after its formation at the plasma membrane. Indeed, the acquisition of phagosome functional properties depends on complex sets of interactions with various cellular organelles, leading to the biogenesis of phagolysosomes (31). Studies of this complex process in the last few years has put forward at least two types of interaction required for phagolysosome biogenesis. First, phagosomes must bind and move along cytoskeletal elements, both microtubules and actin filaments, to encounter and interact with other endovacuolar organelles (6, 32). Second, phagosomes must recognize and fuse with these endovacuolar organelles to allow the transfer of important microbicidal molecules to the phagosome lumen. Interestingly, data from the study of phagolysosome biogenesis, as well as analyses of lipid raft composition and function support the idea that specialized subdomains of the phagosome membrane might play key roles in both types of interactions. Biochemical analyses have shown that actin and actin-binding proteins are closely associated with phagosomes (11) and that this organelle has the ability to induce the nucleation of actin at certain foci on its membrane (32). Interestingly, the latter study demonstrated that late phagosomes are more efficient at inducing actin nucleation, in accordance with a potential role for flotillin-1 and lipid rafts in this process, which accumulate on maturing phagosomes. Proteomic analysis of phagosome lipid rafts indicated that actin is a major protein of these structures, in accordance with recent results showing that lipid rafts are the sites of actin accumulation and polymerization (33). Allen and Aderem (34) have also published results clearly showing the focal recruitment of the actin-associated molecules vinculin and paxillin to phagosomes.
The presence of molecules of the fusion machinery in membrane subdomains indicates that specialized regions of biological membranes might also favor membrane fusion (4, 35). There is increasing evidence that fusion between phagosomes and endosomes might take place preferentially at certain sites on the membrane of these organelles. Stahl and co-workers (36) have shown that phagosome-endosome fusion is initiated at "hot spots" on membranes where rab5 accumulates. Focal distribution of EEA1, a rab5 effector of endosome/phagosome fusion, was also observed at the surface of early endosomes (37). Interestingly, phagosome-endosome fusion also appears to involve transient interactions of parts of their membranes, a process referred to as kiss and run fusion (6, 31). According to the kiss and run hypothesis, fusion between these organelles is initiated by the formation of a fusion pore that allows transient exchange of luminal molecules. However, the expansion of the pore is limited and does not lead to the complete fusion of the organelles. Instead, the fusion pore closes allowing the separation of phagosomes and endosomes. Confirmation that transient fusions occur between phagosomes and endosomes was shown by the fact that molecules of different sizes present in the same endosomes are not transferred to phagosomes simultaneously (38). Instead, small molecules are transferred whereas larger molecules remain in the endosomes (39). Similar results are also observed between endosomes along the endocytic pathway (40). Remarkably, the kiss and run fusion is regulated, in part, by the small GTPase rab5, as shown by the loss of size selectivity in the transfer of solute materials from endosomes to phagosomes in Raw 264.7 macrophages expressing the active GTP-bound form of rab5 (13). Interestingly, current models of the fusion pore predict that lipidic pores could either expand irreversibly or remain open for several seconds and then close if slight changes in the membrane lipid composition were to occur (41). In this context, the presence of lipidic microdomains on the phagosome membrane could rapidly provide the lipid changes required for the fusion pore closure.
This study extends current models of lipid raft microdomain formation
to the membrane of phagosomes. Segregation of lipids and proteins
within the phagosomal membrane may provide focal points on which
complexes of signaling proteins or proteins of the fusion machinery can
assemble, and where specialized functions may occur. Phagosomes have
considerable advantages in the study of the function of lipid rafts
because these organelles can be formed and isolated at will under
various cellular conditions, and experimentally manipulated in in
vitro assays.
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ACKNOWLEDGEMENTS |
---|
We thank Christiane Rondeau for technical
assistance and Jean Léveillé for photographic work. We also
thank Robert Nabi for helpful discussion and the kind gift of some
reagents and Albert Descoteaux for the gift of lpg2
/
mutants.
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FOOTNOTES |
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* This work was supported in part by Grant MT-12951 from the Medical Research Council (MRC) of Canada and grants from FCAR Equipe (to M. D.) and from the National Health and Medical Research Council of Australia (to R. G. P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Recipient of a studentship from the Natural Sciences and Engineering Research Council of Canada.
¶ Recipient of a studentship from the MRC.
Present Address: Dept. of Biology, McGill University, Montreal, Canada.
§§ Scholar from Fonds de la recherche on Santé du Québec. To whom correspondence should be addressed. Tel.: 1 514 343-7250; E-mail: michel.desjardins@umontreal.ca.
Published, JBC Papers in Press, February 27, 2001, DOI 10.1074/jbc.M101113200
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ABBREVIATIONS |
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The abbreviations used are: lo, liquid-ordered; SNARE, soluble NSF-attachment protein receptor; PBS, phosphate-buffered saline; FACS, fluorescence-activated cell sorter; PAGE, polyacrylamide gel electrophoresis; LPG, lipophosphoglycan; GPI, glycosylphosphatidylinositol.
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REFERENCES |
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