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J. Biol. Chem., Vol. 276, Issue 38, 35512-35517, September 21, 2001
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From the Institut de Pharmacologie et de Biologie Structurale, CNRS
UMR 5089, 205 Route de Narbonne, 31077 Toulouse, France
Received for publication, May 15, 2001
The intracellular killing of microorganisms in
phagocytes involves the fusion of lysosomes containing bactericidal
factors with phagosomes, and several intracellular pathogens are able to inhibit this fusion event. In this study, we report the
reconstitution of phagosome-lysosome fusion in vitro, using
an assay based on resonance energy transfer between fluorescent
phospholipid analogues that were inserted into whole human
NB4-neutrophil membranes from liposomes containing positively charged
lipids. Cytosol was required for fusion, and fusion was stimulated
3-fold if this cytosol had been prepared from neutrophils activated by
using opsonized zymosan or a combination of the calcium ionophore
(A23187) and phorbol myristate acetate (PMA). Fusion was inhibited by
the addition of PP1, an inhibitor of Src family protein kinases, or
GTP Neutrophils, monocytes, and macrophages take up bacteria by
phagocytosis. Phagosomes fuse with endosomal and lysosomal
compartments, delivering enzymes, bactericidal proteins, and proton
pumps to the phagosomes (1). These factors are thought to play a major role in the intracellular killing of microorganisms (2). Contributing to the intracellular survival of mycobacteria, phagosomes containing ingested live mycobacteria do not reach a pH lower than 6.3, as they do
not acquire the lysosomal proton pump (3) and do not fuse with
lysosomes while continuing to fuse with endosomes (4-7). Little is
known about the mechanism of phagosome-lysosome fusion and its
inhibition by mycobacteria, although some of the proteins involved in
these processes have been identified (8-10).
We have previously shown that in human neutrophils, fusion between
phagosomes and azurophil granules (specialized lysosomes) does not
occur during infection with mycobacteria (11). Upon phagocytosis of
zymosan by human neutrophils or macrophages, followed by
phagosome-lysosome fusion, a tyrosine kinase of the Src family, Hck,
was found to be activated on lysosomes and translocated to phagosomes
(12-14). Translocation and activation did not take place during
phagocytosis of mycobacteria, suggesting a role for this protein in
regulating the biogenesis of phagolysosomes (11).
The major experimental problem in studying the mechanism of
phagosome-lysosome fusion is the lack of an efficient in
vitro assay for fusion (15). Moreover, it is difficult to purify
lysosomes from macrophages, lacking a specific marker for this
compartment. In this study, we took advantage of the fact that in
neutrophils, lysosomes have specific markers such as myeloperoxidase
and a high density and are thus easy to isolate by fractionation of the
postnuclear supernatant on a discontinuous Percoll gradient (16).
However, we have previously shown that centrifugation through Percoll
removes peripheral proteins from the surface of vesicles (12, 17).
Differential centrifugation avoids this problem, but neutrophils
contain other types of granules that could potentially contaminate the
lysosome preparation. Therefore, we have used NB4 cells that contain
only one type of granule, the azurophil granule/lysosome (18). These
promyeolytic human cells were differentiated to neutrophil-like
cells by means of all-trans retinoic acid (14,
19). We developed a method to label NB4 cells with the fluorescent
probes N-(lissamine rhodamine B sulfonyl)
phosphatidylethanolamine
(N-Rhodamine-PE)1
and N-(7-nitro-2,1,3-benzoxadiazol-4-yl)
phosphatidylethanolamine (N-NBD-PE). We then measured fusion
between labeled lysosomes that were isolated from these cells and
unlabeled phagosomes.
Cell Labeling and Organelle Purification--
Dry lipid films
were produced from a mixture of N-NBD-PE,
N-Rhodamine-PE, and 1,2-dioleoyl-3-N,
N,N,-trimethylammoniumpropane (DOTAP) (2:2:1 molar ratio; Avanti
Polar Lipids, Birmingham, AL) and taken up into double-distilled water
to produce liposomes. The promyelocytic human cells NB4 (obtained from
the Institut National de Santé et de la Recherche Médicale,
Ref. 19) were grown and differentiated into neutrophils by
all-trans retinoic acid as previously described (14). 156 nmol of the lipids in 500 µl of water were added to 2.5 × 107 cells in a volume of 25 ml of tissue culture medium
(MEM), buffered with 10 mM HEPES pH 7.4 (MEM/HEPES) for 15 min at 37 °C. The cells were washed by centrifugation and incubated
in culture medium for 50 min at 37 °C, after which A23187 and PMA
were added to final concentrations of 5 µM and 100 ng/ml,
respectively. After an additional 10 min at 37 °C, the cells were
collected by centrifugation, suspended in 1 ml of buffer and subjected
to nitrogen at 375 psi for 10 min (14). After cavitation, a crude
lysosomal pellet was produced from a postnuclear supernatant by
centrifugation at 12,000 × g for 15 min (14).
Phagosomes containing 3-µm latex beads (Polysciences, Eppelheim,
Germany), which had been incubated overnight at 20 °C with human
serum from healthy volunteers to increase the extent of phagocytosis,
were produced by the method of DesJardins et al. (1).
Briefly, cells (5 × 106/ml in MEM/HEPES) were
incubated at a ratio of 15 beads per cell for 90 min at 37 °C,
resuspended in 2 ml of sucrose 250 mM, 10 mM
HEPES pH 7.4, and cavitated at 350 psi for 10 min. After cavitation, sucrose was added to the postnuclear supernatant to 40% w/w and loaded
between the 62 and 35% layers of a 62:40:35:25:10% (w/w) sucrose
gradient. After centrifugation in a SW41 rotor (Beckman) at
100,000 × g for 1 h, latex bead-containing
phagosomes were recovered from the 10 to 25% interface (1). Sucrose
was then removed from the phagosomes by pelleting the phagosomes at
50,000 × g for 15 min at 4 °C, in 10 ml of
phosphate-buffered saline and resuspended in fusion buffer as
described in the legend to Fig. 3. For thin layer chromatography,
lipids were extracted according to Folch et al. (20)
and run on silica gel plates using chloroform/methanol/water (60:25:4)
as a solvent. Spots were visualized by excitation with broad spectrum
UV light.
Fluorescence Measurements--
Fusion was measured using a
Photon Technologies International (PTI, South Brunswick, NJ) or SAFAS
flx (Monte Carlo, Monaco) fluorometer, with continuous stirring in a
thermostated cuvette at excitation and emission wavelengths of 465 and
530 nm, respectively. For calibration of the fluorescence scale, the
initial residual fluorescence intensity was set to zero and the
intensity at infinite probe solution 100%. The latter value was
obtained after lysis of the liposomes with Triton X-100 (0.5% v/v)
with correction for the quenching of NBD by this detergent (21), as
described before (22). To prepare cytosol, a postnuclear supernatant of cells fractionated as described for lysosome preparation was
centrifuged at 100,000 × g, and the supernatant
was used. Spectra were obtained by excitation at 465 nm, using
2-nm slits throughout, on the PTI fluorimeter.
The phospholipid analogues N-NBD-PE and
N-Rhodamine-PE are widely used to quantify membrane fusion
with liposomes by a resonance energy transfer assay (21), because they
do not spontaneously exchange between membranes by lipid transfer, in
contrast to other probes that may give false positive results (23).
However, no efficient method existed for introducing these probes into
biological membranes. In order to incorporate them into the membranes
of live cells, we produced dry lipid films consisting of
N-NBD-PE, N-Rhodamine-PE, and the positively
charged lipid DOTAP (2:2:1 molar ratio) and added pure water, producing
liposomes as described under "Experimental Procedures." DOTAP is
widely used to transfect cells because it mixes with the lipids of
cellular membranes, delivering the DNA into the cell (24). After 15 min
of incubation of NB4 cells with these liposomes at 4 °C, we found
that cellular plasma membranes were labeled by the fluorescent probes
(Fig. 1a). After 15 min of
incubation at 37 °C, followed by a chase in the absence of liposomes
for 1 h at 37 °C, we found that intracellular vesicles were
labeled, most likely by endocytosis of plasma membrane material (Fig.
1, b and c). Fluorescent NB4-neutrophils retained their capacity to mount bactericidal responses, as demonstrated by
measuring superoxide generated by NADPH oxidase from cells stimulated
by the calcium ionophore A23187 and PMA (results not shown). To isolate
fluorescent lysosomes, cells labeled using a pulse-chase protocol were
activated by incubation for 10 min at 37 °C in the presence of
A23187 and PMA according to Welch and Maridonneau-Parini (14), a
treatment that activates NADPH oxidase and stimulates lysosome
exocytosis. Subsequently, the cells were lysed by nitrogen cavitation
as described under "Experimental Procedures," and the fluorescent
lysosomes were isolated by differential centrifugation (14).
Fluorescence spectra of the lysosomes (Fig. 2a) showed that both probes
were present in the granule fraction, and resonance energy transfer
from NBD to Rhodamine (21) was observed. By comparison with a
calibration curve generated from phosphatidylcholine liposomes
containing the fluorescent probes in different concentrations, at a 1:1
ratio, (not shown) it was estimated that the concentration of the
probes was about 1.6 mol % each with respect to total membrane lipids,
implying that the liposomal material was diluted 25 times by mixing
with the lysosomal material. This also means that the concentration of
DOTAP in the labeled lysosomal membranes was about 0.8 mol %. Notice
that the DOTAP-, N-NBD-PE-, and
N-Rhodamine-PE-containing liposomes that were used to label
the cells are not at all fluorescent at the excitation wavelength of
N-NBD-PE because of quenching and self-quenching of the
probes at these high concentrations (Fig. 2a). The
fluorescent probes were not degraded or otherwise chemically altered by
their incorporation into lysosomes (Fig. 2b). Unlabeled
phagosomes then were produced by nitrogen cavitation of NB4 cells that
had taken up latex beads, separated from other cellular material by
flotation of the latex bead-containing phagosomes on sucrose density
gradients (1), and pelleted by centrifugation to remove the
sucrose.
Upon mixing of labeled lysosomes with unlabeled phagosomes at 37 °C,
little or no increase in fluorescence, measured as described under
"Experimental Procedures," was seen, indicating that there was no
fusion, exchange of fluorescent material, or DOTAP-mediated lipid
mixing between the membranes under these circumstances (Fig. 3a). However, when cytosol
from differentiated, but not activated NB4 cells was added, an increase
in fluorescence was observed after a lag of 2-3 min. Dilution of the
lysosomal membrane lipids into the much larger phagosomal membrane by
fusion would result in the almost complete dequenching of the NBD, and
therefore a 1% increase in fluorescence would mean that 1% of the
lysosomes fused with the phagosomes. In the presence of cytosol, the
initial rate of fluorescence increase was 4.2%/h (Fig. 3c).
Importantly, addition of cytosol from NB4 cells activated by using a
calcium ionophore and PMA tripled the activity to 12.6%/h (Fig.
3a), reaching a final level of 25% after several hours.
Similar results were obtained using cytosol prepared from cells
activated by phagocytosis of zymosan opsonized with human serum (Fig.
3b). After 1 h in the presence of non-activated
cytosol, the fluorescence increase was 2.5%, compared with 8.2% for
activated cytosol. We found that the removal of sucrose from the
phagosome preparation was essential for activity. Lowering the
concentration of phagosomes decreased the rate and extent of fusion,
whereas increasing the concentration of lysosomes had no effect,
indicating that a limited number of lysosomes fuse with phagosomes. The
increase in fluorescence was not due to chemical changes in the probes
caused by their incubation with cytosol (Fig. 2b).
Fusion of Human Neutrophil Phagosomes with Lysosomes in
Vitro
INVOLVEMENT OF TYROSINE KINASES OF THE Src FAMILY AND INHIBITION
BY MYCOBACTERIA*
, and
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
S. We have previously reported that the biogenesis of
phagolysosomes in human neutrophils is inhibited by
mycobacteria. Here we show that cytosol from cells having
internalized live (not heat-killed) Mycobacterium smegmatis
or cytosol simply incubated with mycobacteria inhibited fusion,
indicating that soluble factors are involved in mycobacterial
inhibition of phagosome-lysosome fusion.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (40K):
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Fig. 1.
Labeling of the plasma membrane by liposomes
and subsequent endocytosis. Incubation of NB4 cells with liposomes
for 15 min at 4 (a) or 37 °C, followed by a 1-h chase in
the absence of liposomes at 37 °C (b, c).
Cells were rapidly placed between glass slides and coverslips, and
microsocopic observations of cells were immediately made at the
wavelength of rhodamine (515-560 excitation, 590 nm long pass filter
emission) using PL-Fluotar × 40 (a, b) or a
PL Apo × 100 (c) oil immersion objectives on a Leica
DM IRB/E fluorescence microscope.
View larger version (24K):
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Fig. 2.
Dilution of the liposomal fluorescent probes
in the membrane of lysosomes and their chemical stability during pulse
chase and fusion. Panel a, emission spectra of liposomes
(a, b) and lysosomes (c, d)
before (b, c) and after (a,
d) addition of the detergent Triton X-100, with excitation
of N-NBD-PE at 465 nm, normalized to the NBD peak after
addition of Triton. Notice that the N-NBD-PE present in
liposomes is completely quenched by the N-Rhodamine-PE and
self-quenched at these high concentrations, and therefore the liposomes
are not fluorescent when excited at the wavelength of
N-NBD-PE. The somewhat lower Rhodamine peak after liberation
of the probes from the lysosomes is probably due to absorption by
proteins present in the sample. Panel b, thin layer
chromatogram, showing the fluorescence of the probes visualized by
illumination with ultraviolet light. Lane 1, N-NBD-PE standard; lane 2, lipid extract from
lysosomes, labeled by the pulse-chase protocol; lane 3, from
lysosomes incubated with activated cytosol for 30 min at 37 °C,
lane 4 from lysosomes incubated with non-activated cytosol
for 30 min at 37 °C, lane 5, N-Rhodamine-PE standard; lanes 2-4,
upper spot: N-Rhodamine-PE, lower spot:
N-NBD-PE. Standards contain 0.5 nmol and the other lanes, 20 nmol of phospholipids.
View larger version (20K):
[in a new window]
Fig. 3.
Fusion between labeled lysosomes and
unlabeled phagosomes from human neutrophils. Panel a, 3.2 µg (protein) of a phagosome preparation were mixed with 5 µg of a
lysosome preparation in buffer containing 145 mM KCl, 2.5 mM Mg Cl2, 10 mM HEPES/KOH, 70 µM GTP, 200 µM ATP, and an ATP-regenerating
system as described in Ref. 37 at pH 7.4, 37 °C in a total volume of
1.6 ml (curve 3). Additionally, the mixture contained 300 µg of cytosol (curve 2) or cytosol from neutrophils
activated by A23187 and PMA (curve 1). Panel b,
conditions as in panel a, but in the presence of cytosol
from neutrophils activated by phagocytosis of opsonized zymosan
(curve 1) or non-activated cytosol (curve 2). The
addition of GTP and ATP or an ATP-regenerating system was not essential
for fusion. Panel c, average initial rates of fluorescence
increase ± 1 S.D.; the number of phagosome/lysosome preparations
is indicated.
The above experiments were performed with a rather crude preparation of lysosomes obtained by differential centrifugation. Considering that phagosome-lysosome fusion has been much harder to reconstitute in vitro than phagosome-endosome fusion (15), the observed activity could have been due to contamination of this preparation by endosomes. Endosomes were separated from the crude lysosome preparation and identified by the presence of endosomal markers as previously published (25). Endosomes were found to induce an increase in fluorescence when mixed with phagosomes; a 3.2-µg preparation of endosomes produced a 9.6%/h increase in fluorescence, compared with 12%/h for 3.2 µg of purified lysosomes. The endosomal fraction was found to represent 2.1% of the protein present in the purified lysosomal fraction. Therefore, the crude and purified lysosome preparations had similar fusion activities, and a maximum of 1.6% of the total increase in fluorescence measured with the crude lysosome preparation was contributed by contaminating endosomes.
The addition of GTP and ATP or an ATP-regenerating system to the cytosol was not essential for the fluorescence increase, in contrast to what was reported for phagosome-lysosome fusion in permeabilized J774 cells (26). However, in our system, activated cytosol was added at concentrations that produced maximal extents of fluorescence increase. In the presence of less cytosol, the initial rate of fluorescence increase was similar, but the final extent of fusion was lower, indicating that factors contained in the cytosol are present in sufficient quantities to support the activity initially, but soon become exhausted.
Intercellular membrane traffic is regulated by GTPases. These are
thought to act as switches, actuated by a conformational change in the
protein upon GTP hydrolysis. To test the involvement of such proteins,
we used the non-hydrolysable GTP analogue GTPS. It was found
that GTPS reduced the initial rate of fluorescence increase induced
by activated cytosol to the level of non-activated cytosol (Fig.
4). Inhibition at a comparable GTPS
concentration was seen for phagosome-lysosome fusion in permeabilized
J774 cells (26) while fusion of endosomes with phagosomes was shown to be activated by GTPS (27), confirming that fusion with endosomes does not contribute significantly to the measured increase in fluorescence.
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During phagocytosis-linked lysosome fusion in neutrophils and macrophages, the Src family kinase Hck translocates to phagosomes and is activated. Src family kinases are specifically inhibited by PP1 (28). To test the involvement of these proteins, phagosomes and lysosomes were pretreated with PP1, and fusion was measured in the presence of activated cytosol and PP1. It was found that PP1 inhibited the fluorescence increase with a half-maximal concentration of 1 µM (Fig. 4). Half-maximal concentrations for Src kinase inhibition in PMA stimulated intact T cells were around 26 µM (28); in human neutrophils, Hck activity was reported to be completely blocked at 10 µM (29).
Together, these data indicate that specific, efficient
phagosome-lysosome fusion was reconstituted in our assay, allowing the
quantitative measurement of fusion in real time. M. smegmatis inhibits the biogenesis of phagolysosomes in human
neutrophils as much as pathogenic mycobacteria (11). We then sought to
reconstitute the specific inhibitory effect of mycobacteria on
phagosome-lysosome fusion in vitro. To this end, NB4 cells
were infected with live M. smegmatis (50 bacteria per cell)
by incubation for 30 min at 37 °C, followed by three washes. This
incubation inhibited lysosome exocytosis as witnessed by the lack of
-glucuronidase release during the incubation, compared with
-glucuronidase release from NB4-neutrophils incubated with opsonized
zymosan instead of mycobacteria (data not shown). We then prepared
cytosol from these M. smegmatis-infected cells and measured
fusion in the presence of mixtures of cytosol from these cells and
cytosol activated as described above, compared with mixtures of
activated cytosol with non-activated cytosol from uninfected cells. The
lysosomes and latex bead-containing phagosomes used in these fusion
experiments were prepared from non-infected cells, as in the
experiments described above. Cytosol from infected cells was found to
have a dose-dependent inhibitory effect on fusion (Fig.
5a), thus indicating that it
contains factors inhibitory of phagosome-lysosome fusion after a brief
incubation of cells with mycobacteria. Inhibition could be due to
factors associated with or secreted by mycobacteria, passing into the cytosol through the membrane of the phagosomes, or the neutrophils could produce these factors after interaction with mycobacteria. To
test these possibilities, cells were first incubated with heat-killed M. smegmatis, and then cytosol was prepared from these cells
(Fig. 5b). No inhibition of fusion was seen. We then
incubated either activated cytosol or a preparation of the plasma
membranes plus the cytosol of activated cells with live M. smegmatis and used the cytosol to assay fusion. In both cases,
fusion was reduced to the level of non-activated cytosol. These data
suggest that factors directly involved in inhibiting fusion are present
in the cytosol.
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DISCUSSION |
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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The method that we have developed for the labeling of cells with a pair of phospholipid analogues allows quantitative and kinetic measurement of phagosome-lysosome fusion in vitro, without the false positive results that are often produced by assays based on other fluorescent probes that are more easily incorporated into membranes (23). The fusion that we measured had the hallmarks of specific phagosome-lysosome fusion; it was not significantly due to contaminating endosomes and it was stimulated by cytosol prepared from cells activated by A23187 and PMA or opsonized zymosan, agents known to mobilize and induce fusion of lysosomes with the plasma membrane and phagosomes, respectively (11, 14). Phagocytosis signaling pathways are not well understood; receptor clustering, tyrosine or serine/threonine phosphorylation of substrates such as MARCKS, Ca2+ ions, and arachidonic acid are thought to be involved (30, 31). Inhibition of phagolysosome biogenesis by PP12 and other less selective tyrosine kinase inhibitors (33) suggest the involvement of tyrosine phosphorylation in intact human neutrophils. In addition we found that activation of Hck, a tyrosine kinase mostly expressed in phagocytes, correlates with phagosome-lysosome fusion in neutrophils (11, 14). In the present study, we report that PP1 reduces phagosome-lysosome fusion induced by cytosol from activated cells to the level of non-activated cytosol, providing clear evidence for the involvement of Src family tyrosine kinases in the activation of fusion.
Similar to PP1, M. smegmatis also inhibited fusion induced by activated cytosol to the level of non-activated cytosol. It seems as though there are two different types of lysosome-phagosome fusion: one that can be inhibited by these treatments and is only found in activated cells and a non-inhibitable component. However, we think that the non-inhibitable component is due to cytosol that is activated in a fraction of the cells by the act of preparing the cytosol itself, involving various manipulations of the cells, like centrifugation. We have made similar observations using neutrophils isolated from blood; a fraction of the lysosomal enzymes is secreted by non-activated cells (11).
In this paper, we provide evidence that factors associated with or released by live mycobacteria are present in the cytosol and inhibit phagosome-lysosome fusion. These factors acted when mycobacteria were simply incubated with cytosol, indicating that their phagocytosis is not required for inhibition. Once enclosed in phagosomes, these factors would have to pass the phagosomal membrane to inhibit fusion. Phagosomes containing M. bovis BCG were shown to be permeable to dextrans as large as 70 kDa from the cytoplasm in murine macrophages (34), and a number of mycobacterial surface proteins are released from phagosomes into subcellular compartments (35). Therefore, mycobacterial factors that could be inhibitors of fusion or affect fusion-regulating proteins may be released into the cytosol from phagosomes.
Additionally, the entry pathway of mycobacteria into its host cell determines the fate of the phagosome (13, 36, 32), for example by triggering specific signaling involving host cell proteins and a peripheral murine membrane protein called TACO involved in inhibition of phagolysosome biogenesis in macrophages recruited to the sites of mycobacterial entry and retained on phagosomes containing mycobacteria (9, 10). In this study, we provide evidence that inhibition of phagosome-lysosome fusion in human neutrophils also involves mycobacterial factors acting in the cytosol.
In conclusion, the in vitro reconstitution of fusion and a
quantitative and kinetic assay have allowed us to identify the role of
Src family tyrosine kinases in phagosome-lysosome fusion stimulated by
cytosol from activated cells. Moreover, the in vitro assay
could facilitate the search for the mycobacterial factors involved in
inhibition of fusion and help to unravel the mechanism of fusion.
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ACKNOWLEDGEMENTS |
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We thank Joke Orsel and Ingrid Bartoldus for preliminary experiments, Donato Zipeto for help with the figures and critical reading of the manuscript, and Sébastien Carreno for help with the photographs.
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FOOTNOTES |
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* This research was supported by the Région Midi-Pyrénées, the Fondation pour la Recherche Médicale (FRM), the Association pour la Recherche sur le Cancer, the comité scientifique SIDACTION of the FRM, the project program "du normal au pathologique" of the CNRS and the microbiology program of the French ministry of education, research and technology.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.
To whom correspondence may be addressed. Tel.: 33 5 61 17 54 63;
E-mail: stegmann@ipbs.fr (to T. S.) or Tel.: 33 5 61 17 54 58;
E-mail: maridono@ipbs.fr (to I. M-P.); Fax 33 5 61 17 59 94.
Published, JBC Papers in Press, July 19, 2001, DOI 10.1074/jbc.M104399200
2 C. Cougoule and I. Maridonneau-Parini, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are:
N-Rhodamine-PE, N-(lissamine Rhodamine B
sulfonyl) phosphatidylethanolamine;
DOTAP, 1,2
dioleoyl-3-N,N,N,-trimethylammoniumpropane;
N-NBD-PE, N-(7-nitro-2,1,3-benzoxadiazol-4-yl)
phosphatidylethanolamine;
PMA, phorbol myristate acetate;
MEM, minimum
essential medium;
GTPS, guanosine
5'-3-O-(thio)triphosphate.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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