From the Cell Biology Programme, Research Institute,
The Hospital for Sick Children, Toronto, Ontario M5G 1X8, the
Department of Biochemistry, University of Toronto,
Toronto, Ontario, Canada M5S1A8, the ¶ Department of
Medicine, University of Pennsylvania School of Medicine and
Biotechnology Laboratory, Philadelphia,
Pennsylvania 19104-4283, and the
Biotechnology Laboratory,
University of British Columbia, Vancouver, British Columbia,
V6T1Z3 Canada
Received for publication, August 25, 2000, and in revised form, November 16, 2000
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ABSTRACT |
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Bacterial invasion, like the process of
phagocytosis, involves extensive and localized protrusion of the host
cell plasma membrane. To examine the molecular mechanisms of the
membrane remodeling that accompanies bacterial invasion,
soluble NSF attachment protein
receptor (SNARE)-mediated membrane traffic was studied in
cultured cells during infection by Salmonella typhimurium. A green fluorescent protein-tagged chimera of VAMP3, a SNARE
characteristic of recycling endosomes, was found to accumulate at sites
of Salmonella invasion. To analyze the possible role of
SNARE-mediated membrane traffic in bacterial infection, invasion was
measured in cells expressing a dominant-negative form of
N-ethylmaleimide-sensitive factor (NSF), an essential regulator of membrane fusion.
Inhibition of NSF activity did not affect cellular invasion by S. typhimurium nor the associated membrane remodeling. By contrast,
Fc Invasive bacterial species gain access to intracellular
compartments, where they survive and replicate by subverting host cell
function. Although it is not yet clear how a variety of bacterial species induce the formation of the vacuoles that engulf them, it has
been established that the process of invasion has much in common with
that of phagocytosis (1-3). Both processes typically involve membrane
remodeling events, namely pseudopodial extension during phagocytosis
and ruffling during bacterial invasion, and both phenomena are
associated with changes in the actin cytoskeleton of the host cell (1).
As well, like phagosomes, bacteria-containing vacuoles can undergo a
maturation process (2, 4). However, whereas phagocytosis and invasion
are superficially similar, it is not clear whether the molecular
mechanisms of internalization are shared.
Current models of phagocytosis suggest that phagosomes are generated by
receptor-mediated "zippering" of the host cell plasma membrane
against the surface of opsonized particles, accompanied by the active
extension of pseudopodia (5-8). A source of intracellular membrane is
required for the membrane remodeling that allows for pseudopod
formation, and recent evidence suggests that specific endosomal
compartments are involved in particle engulfment (9-11). It is now
apparent that the soluble NSF
attachment protein receptor (SNARE)1 protein VAMP3, an
integral component of the recycling endosome, plays an important role
in phagocytosis, possibly participating in localized fusion events at
the site of phagosome formation and thus driving pseudopodial extension
(12).
It is not known if endosomal compartments have a central role in the
process of bacterial invasion, but the extensive membrane remodeling
that accompanies cellular infection by Salmonella
typhimurium suggests that invasion by this bacterial pathogen may
mechanistically parallel phagocytosis. In the current study, we
examined the role of SNARE-mediated membrane fusion in S. typhimurium invasion. Because of the established similarities with
phagocytosis, the role of VAMP3 in invasion was analyzed first.
However, because the exact intracellular compartments and the
repertoire of SNARE proteins that may be involved in bacterial invasion
are not known, the role of membrane fusion was also investigated
through the general inhibition of SNARE-mediated membrane traffic,
specifically by blocking the activity of
N-ethylmaleimide-sensitive factor (NSF). NSF is an ATPase/chaperone that is
responsible for the disassembly of SNARE complexes and is therefore an
essential regulator of intracellular membrane fusion events (13). We
generated a mutant form of NSF (E329Q-NSF) that is deficient in ATP
hydrolysis and therefore inhibits the function of endogenous NSF,
resulting in a dominant-negative effect (14, 15). We found that acute expression of E329Q-NSF inhibits phagocytosis in a macrophage cell
line. By contrast, invasion of S. typhimurium into
epithelial cells was not impaired by the dominant-negative NSF;
however, maturation of Salmonella-containing vacuoles (SCVs)
was inhibited.
Reagents and Antibodies--
Sheep red blood cells (SRBC), goat
anti-SRBC IgG, and N-ethylmaleimide (NEM) were purchased
from ICN. Human IgG was purchased from Baxter Healthcare Corp.
Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum
(FBS) were obtained from Wisent (Montreal, Quebec, Canada). Brefeldin A
and G418 were obtained from Calbiochem and Fugene 6 from Roche
Molecular Biochemicals. Tetramethylrhodamine isothiocyanate-transferrin
(Tfn) and tetramethylrhodamine-phalloidin were from Molecular Probes.
Restriction enzymes were obtained from New England Biolabs. Latex beads
(3 µm diameter), ATP, and p-nitrophenyl phosphate (pNPP)
and all other reagents were purchased from Sigma.
Polyclonal antibody to
To produce an antiserum to NSF, His6-tagged recombinant NSF
protein was expressed in bacteria and purified according to Whiteheart et al. (15). The protein was then mixed with Freund's
adjuvant and used to immunize rabbits by following standard procedures. The NSF antibody was affinity-purified from rabbit serum by binding to
and eluting from an NSF-coupled Affi-Gel 10 column (Bio-Rad).
cDNA Constructs and Mutagenesis--
pEGFP-N1 was purchased
from CLONTECH Laboratories Inc. (Palo Alto, CA).
The acylation motif of Lyn kinase fused to GFP (PM-GFP) was a kind gift
of Dr. Tobias Meyer (Stanford University, Palo Alto, CA). The VAMP3-GFP
chimera is described elsewhere (12). Wild-type (WT) NSF in pQE-9
(pQE-9-NSF; Whiteheart et al. (15)) was a generous gift of
Dr. S. Whiteheart (University of Kentucky). WT NSF was subcloned into
pcDNA3.1 (Invitrogen, Carlsbad, CA) using a multistep strategy.
First, the 3'-coding region was excised from pQE-9-NSF using
HindIII and ligated into pcDNA3.1 (pcDNA3.1-3'NSF). Second, a fragment encompassing most of the coding region of NSF, including the ATG start codon, was excised from pQE-9-NSF using BamHI and NheI and ligated independently into
pcDNA3.1 (pcDNA3.1-5'NSF). Third, pcDNA3.1-5'NSF was
digested with XbaI and NheI, and this fragment
was then ligated into a pcDNA3.1-3'NSF backbone that had been
digested with NheI, yielding a complete and contiguous coding sequence of NSF within pcDNA3.1 (pcDNA3.1-NSF). The
fidelity of this, and all NSF constructs employed in this study, was
confirmed by DNA sequence analysis.
Generation of an NSF gene bearing a point mutation in the ATPase domain
of the D1 region of NSF was accomplished by polymerase chain
reaction-mediated, plasmid-based, site-specific mutagenesis. To replace
the glutamate at position 329 of NSF with glutamine (Gln substituted at
Glu329), pQE-9-NSF was used as the target vector. Two
complementary polymerase chain reaction primers containing the desired
point mutation in NSF were made and used along with the QuikChange
Site-directed Mutagenesis Kit from Stratagene Cloning Systems.
Transformed bacterial clones were screened by DNA sequencing to
identify clones containing the mutated form of the NSF gene in pQE9.
This clone of mutant NSF cDNA was designated E329Q NSF. To
introduce the E329Q mutation into NSF in pcDNA3.1,
BspEI/BstEII digestion of E329Q NSF in pQE-9 was
used to generate a cDNA cassette, containing the mutated region of
NSF, that was subcloned into like digested WT NSF in pcDNA3.1.
ATPase Assay--
NSF constructs in pQE9 were expressed in
Escherichia coli, and the His6-tagged proteins
were purified as described above. Purified wild-type and E329Q NSF
proteins were used for in vitro ATPase assays using pNPP as
a chromogenic ATP analogue, based on the method of Mignaco et
al. (16). Briefly, 5 mM ATP and 6 mM pNPP
(final concentrations) were freshly added to a buffer of 25 mM Tris-HCl, pH 7.5, 100 mM KCl, 0.5 mM dithiothreitol, 0.65 mM Cell Culture and Transfections--
RAW 264.7 and COS-1 cells
were obtained from the American Tissue Culture Collection, and
Fc Transferrin Uptake and Indirect Immunofluorescence--
To study
transferrin endocytosis, cells were serum-starved for 1 h in DMEM
and subsequently incubated with 50 µg/ml labeled Tfn for 30-45 min,
after which excess Tfn was washed away. For indirect
immunofluorescence, cells were fixed with 4% paraformaldehyde/PBS at
room temperature for 20-30 min. If permeabilization was necessary for
staining of intracellular markers, fixed cells were bathed in 0.1%
Triton X-100/PBS for 10 min. Cells were then blocked with 5% skim milk
powder/PBS for at least 1 h. For staining, cells were incubated in
the presence of primary antibody, at 37 °C, for 11/2 h,
washed, incubated with secondary antibody for 1 h, washed, and
mounted using DAKO mounting medium (DAKO Corp.).
Western Blotting--
For analysis of VAMP3-GFP expression and
cleavage by TeTx, transfected cells were lysed in PBS, pH 7.4, containing 1% Triton X-100, 0.1% SDS, 0.5% deoxycholate, and
protease inhibitors. Lysates were cleared by centrifugation at
10,000 × g for 10 min, and the protein concentrations
of the extracts were determined using the Bio-Rad protein assay.
Samples were boiled in sample buffer, and the proteins were separated
by SDS-polyacrylamide (10%) gel electrophoresis under nonreducing
conditions. Proteins were electrophoretically transferred to
polyvinylidene difluoride membranes (Immobilon P, Millipore), and the
membranes were then blocked with 5% milk powder in TBS-T (20 mM Tris-HCl, 137 mM NaCl, 0.1% Tween 20, pH 7.6). The membranes were probed with a polyclonal antibody to GFP,
washed, and incubated with a horseradish peroxidase-conjugated goat
anti-rabbit secondary antibody. After washing, the labeled proteins
were detected using an enhanced chemiluminescence (Amersham Pharmacia Biotech).
Flow Cytometry and Quantitative Image Analysis--
To analyze
cell surface Fc receptor expression, transfected RAW cells were
incubated with PE-labeled anti-Fc receptor antibody for 1 h on
ice. The cells were then fixed in 4% paraformaldehyde and analyzed
using a Becton Dickinson FACStar Plus flow cytometer and CELLQUEST
software (Becton Dickinson). The filter settings were 530/30 and 575/26
nm for GFP and PE, respectively.
Quantitation of NSF expression and transferrin labeling in RAW cells
and of surface Fc receptor expression in COS-2A cells was done by
examining labeled cells with a Leica DM IRB inverted microscope. Cell
Images were captured using WinView 32 software (Princeton Instruments)
and subsequently analyzed using NIH Image software. Individual cell
boundaries were defined manually, and fluorescence intensity was
integrated over calculated cell areas.
Phagocytosis and Particle-binding Assays--
For phagocytosis
in RAW 264.7 cells, 3-µm latex beads were opsonized with human IgG,
1 h room temperature, and washed with PBS to remove unbound IgG.
Opsonized beads were then added to RAW cells plated in DMEM containing
10% FBS and incubated for 30 min at 37 °C. Unbound beads were then
washed away, and internalized beads were quantified under Nomarski
optics. For phagocytosis in COS-2A cells, SRBCs were opsonized with
goat anti-SRBC IgG for 1 h at 37 °C. After washing with PBS,
opsonized SRBCs were added to COS-2A cells plated in complete medium
and incubated for 30 min or 1 h at 37 °C. Extracellular SRBCs
were removed by hypotonic lysis (water, 30 s), and internalized
SRBCs were quantified as above. When assessing phagocytosis in cells
transfected with EGFP, it was possible to quantify internalized
particles using fluorescence microscopy due to the exclusion of GFP
from the interior of the phagosomes. In this way, cells could be fixed
after phagocytosis and counter-stained, for example with NSF antibody
in experiments where cells had been cotransfected with NSF cDNA.
To assess particle binding efficiency, transfected RAW 264.7 cells were
incubated with opsonized SRBCs for 30 min at 4 °C. Nonadherent SRBCs
were washed away with PBS, and the number of bound SRBCs/cell was
quantified under light microscopy.
Bacterial Infection--
The S. typhimurium strain
1344 was used and has been described previously (20). Overnight
bacterial cultures were diluted 1:30 into fresh Luria-Burtani broth and
incubated at 37 °C, shaking for 3 h. Bacteria were pelleted by
centrifugation at 10,000 rpm for 2 min and then resuspended in PBS. The
bacteria were diluted in Earle's buffered salt solution (EBSS), pH
7.4, and added to COS-1 cells that had been plated on coverslips at a
density of ~50 bacteria per cell. Infection was carried out at
37 °C for 15 min. Excess bacteria were washed away with PBS, and the
cell monolayers were then incubated in growth medium containing 100 µg/ml gentamicin for 15 min at 37 °C. The cells were processed for
immunofluorescence by fixing in 2.5% paraformaldehyde/PBS for 10 min,
washing, and blocking with 1% BSA/2% donkey serum/PBS. Samples were
stained for external bacteria by incubating with rabbit anti-S.
typhimurium LPS antibody followed by Alexa 594-conjugated donkey
anti-rabbit antibody. Subsequent to this, the samples were again
incubated in blocking buffer, this time containing 0.1% saponin. The
samples were then stained for total bacteria (external and
internalized) with the same primary antibody followed by Alexa 350-conjugated donkey anti-rabbit antibody. For examination of vacuole
maturation, fixed and permeabilized samples were stained for total
bacteria with the anti-LPS antibody and Alexa 594-conjugated donkey
anti-rabbit antibody and counterstained for LAMP-1 with the monoclonal
anti-human LAMP-1 antibody and FITC-conjugated goat anti-mouse IgG.
VAMP3-GFP Localizes to Sites of Salmonella Invasion--
Previous
studies from our laboratory demonstrated the focal exocytosis of
VAMP3-containing vesicles at sites of particle internalization during
phagocytosis (12). Here, we examined the possibility that
VAMP3-containing vesicles may play a role in the membrane remodeling
that accompanies cellular invasion by S. typhimurium. Cells
expressing a chimeric VAMP3-GFP construct (12) were subjected to
invasion by S. typhimurium as described under
"Experimental Procedures." Shortly after infection (6-8 min), the
distribution of VAMP3-GFP was monitored by fluorescence microscopy,
whereas the localization of bacteria was detected by indirect
immunofluorescence using an antibody to S. typhimurium
lipopolysaccharide (anti-LPS). In resting COS-1 cells VAMP3-GFP was
present in vesicles that were observable throughout the cytoplasm but
accumulated particularly in a juxta-nuclear cluster, typical of
recycling endosomes (Fig. 1a),
consistent with previously published observations (21). Comparatively
little VAMP3 was present on the cell surface in otherwise untreated
cells. In cells undergoing invasion by S. typhimurium, in
contrast, VAMP3-GFP was seen to accumulate at or near the plasmalemma
at sites of bacterial attachment (Fig. 1, b and
c).
Accumulation of VAMP3-GFP occurred both at sites where
Salmonella bacteria were external and at sites where the
bacteria had been internalized. However, although VAMP3-GFP accumulated
at these sites, it was not clear if this represented only intracellular movement of the fluorescent protein or mobilization and exocytosis of
this SNARE at sites of invasion. To examine this possibility, we tested
for the appearance of the COOH terminus of VAMP3-GFP (bearing the GFP
moiety) on the cell surface by staining the cells with an antibody
against GFP. Following invasion, COS-1 cells transfected with VAMP3-GFP
were gently fixed (using 2.5% paraformaldehyde) but not permeabilized.
Staining of the cells then revealed external GFP (Fig. 1d)
at sites of VAMP3-GFP accumulation (Fig. 1e) that corresponded with sites of attachment of external bacteria (Fig. 1f). These observations indicate that accumulation of
VAMP3-GFP can occur prior to internalization of S. typhimurium and that the accumulation of this chimera is
accompanied by its insertion into the plasma membrane.
Tetanus Toxin Blocks Recruitment of VAMP3-GFP to Sites of
Salmonella Internalization but Does Not Inhibit Invasion--
The
localization and plasmalemmal insertion of VAMP3-GFP at sites of
bacterial invasion suggest that membrane fusion events mediated by this
SNARE may contribute to the membrane remodeling that is involved in
bacterial invasion. To test this possibility, a cDNA encoding the
light chain of the tetanus toxin (TeTx) was cotransfected into COS-1
cells along with VAMP3-GFP. To confirm that cotransfection of these two
vectors resulted in significant cleavage of VAMP3-GFP, transfected cell
populations were harvested and examined by Western blotting with an
anti-GFP antibody. Fig. 2 shows an
anti-GFP Western blot of extracts from mock-transfected COS-1 cells
(lane 1) and COS-1 cells transfected with EGFP alone (lane 2), VAMP3-GFP alone (lane 3), or VAMP3-GFP
plus TeTx (lane 4). Comparison of lanes 3 and
4 reveals that coexpression of TeTx and VAMP3-GFP results in
essentially the complete cleavage of the VAMP3-GFP chimera. The two
lower bands in lanes 3 and 4 are protein species,
possibly products of degradation, that are not influenced by
coexpression of tetanus toxin.
To study the effect of TeTx on the mobilization of VAMP3-GFP that
accompanies Salmonella invasion, COS-1 cells were
transfected with both VAMP3-GFP and TeTx prior to invasion assays with
S. typhimurium. As depicted in Fig.
3b, the expression of TeTx
altered the distribution of VAMP3-GFP within COS-1 cells. Tetanus toxin is known to cleave the cytoplasmic portion of VAMP3, and the observed redistribution of this SNARE is indicative of the accumulation of the
transmembrane/lumenal domain-GFP moiety of the chimera in the Golgi
apparatus (22). Most important, in COS cells coexpressing VAMP3-GFP and
TeTx, accumulation of VAMP3-GFP at sites of S. typhimurium invasion was markedly reduced (Fig. 3, b and
c).
The observation that TeTx blocked the accumulation of VAMP3 near sites
of Salmonella invasion prompted us to test whether TeTx
would also block Salmonella invasion, in a manner analogous to its known effect on phagocytosis (10). To quantitate the effect of
TeTx on invasion, transfected cells were exposed to S. typhimurium, and the fraction of cells harboring intracellular bacteria was estimated as described under "Experimental Procedures" (see also Fig. 3, d-f). Surprisingly, whereas expression of
TeTx altered the basal distribution of VAMP3 and prevented its
accumulation at sites of bacterial internalization, it had no
discernible effect on the number of cells invaded by
Salmonella (Fig. 4). As well, the number of bacteria that invaded individual COS-1 cells was not
affected by the expression of tetanus toxin (data not shown). Furthermore, expressing EGFP, alone, or EGFP and TeTx in COS-1 cells
had no effect on Salmonella invasion (not shown). These results suggest that, although VAMP3 accumulates at sites of invasion, accumulation and function of this protein are not required for S. typhimurium entry into the cells.
Characterization and Expression of Dominant-negative NSF--
The
finding that TeTx had no effect on Salmonella invasion
contrasts with previous studies that demonstrated a role of VAMP proteins in phagocytosis (10). These results are not necessarily incompatible because, although VAMP3 may be required for phagocytosis, other SNARE proteins, possibly including TeTx-insensitive isoforms of
VAMP, may be essential for S. typhimurium invasion. It is
impractical and in some cases impossible to use specific toxins to test
the function of all the SNARE proteins expressed in COS-1 cells that may be involved in Salmonella invasion. Instead, a general
inhibitor of SNARE-mediated membrane traffic was sought. A logical
target for this purpose was NSF, an ATPase/chaperone protein
responsible for the disassembly of SNARE complexes and an essential
regulator of membrane fusion events. To inhibit NSF activity, we
introduced a point mutation in the Chinese hamster NSF gene which
resulted in the substitution of glutamine for glutamic acid at residue 329 (E329Q) within the catalytic site of the D1 domain of NSF. E329Q-NSF has been described elsewhere and shown to form hexamers that
bind ATP but do not hydrolyze it (14, 15). When mixed with wild-type
protein, E329Q-NSF forms mixed hexamers that also lack activity,
leading to a dominant-negative effect. To verify the effectiveness of
the mutation, purified, recombinant wild-type, and mutant NSFs were
assayed for nucleotidase activity in vitro, as described
under "Experimental Procedures." Wild-type NSF had an activity of
0.382 ± 0.019 µmol pNPP/mg/h (means ± S.E.). Wild-type NSF, incubated with 2.5 mM NEM, had an activity of
0.05 ± 0.023 µmol of pNPP/mg/h, and the activity of E329Q-NSF
was 0.142 ± 0.011 µmol of pNPP/mg/h. Consistent with the
findings of others (15), the intrinsic activity of E329Q NSF was
~30% that of wild-type NSF. The remaining nucleotidase activity of
E329Q-NSF is attributable to the second catalytic site in the D2 domain
of the protein (15).
To characterize the function of E329Q-NSF in intact cells, we chose to
express the mutant protein in a macrophage cell line (RAW 264.7). This
provided us with the opportunity to compare NSF function in
phagocytosis, a receptor-mediated event initiated by the macrophage,
and in invasion, an opsonin receptor-independent process triggered by
Salmonella. Wild-type and mutant NSF genes in pcDNA 3.1 were expressed in mouse macrophages by transient transfection along
with EGFP. As illustrated in Fig. 5,
b and d, the endogenous levels of NSF (see
EGFP-negative cells) were barely detectable by immunofluorescence, but
the ectopic expression of either wild-type (WT) or mutant NSF was
clearly observable. Quantitation of the expression of E329Q-NSF (Fig.
6) revealed that 5 h after
transfection the amount of the dominant-negative NSF was slightly
increased over that of the endogenous protein. By 8 h
post-transfection, the level of ectopic expression of NSF was ~3
times the endogenous NSF (Fig. 6).
We next assessed the biological effects of the dominant-negative NSF
in vivo by monitoring transferrin uptake. Transferrin is
internalized by a receptor-mediated, clathrin-dependent
process and cycles through endosomal compartments before returning to the cell surface. By incubating cells in the presence of
rhodamine-labeled transferrin, we were able to determine that RAW cells
overexpressing WT NSF internalized transferrin in a manner that was
indistinguishable from untransfected cells (Fig. 5, e and
f). In contrast, transferrin uptake was strikingly decreased
in cells expressing E329Q-NSF (Fig. 5, g and h).
A detailed quantitation of this effect is presented in Fig. 6, as a
function of time after transfection. The level of transferrin uptake
(as a percentage of control cells) was reduced by ~60% 5 h
after transfection and by ~95% after 8 h. Expression of
E329Q-NSF also inhibited the lysosomal accumulation of material normally internalized by fluid phase and altered the morphology of the
Golgi complex in a manner resembling the effects of brefeldin A (not
illustrated). Comparable levels of expression of WT-NSF were without
effect on the uptake of transferrin (Fig. 6) or fluid phase markers or
on Golgi morphology. These experiments indicate that ectopic expression
of NSF to levels that can cause altered SNARE function can be attained
shortly after transfection (within 8 h). Importantly, such brief
periods of expression preserved cellular viability and had no
discernible effect on several aspects of cell function. Specifically,
the acidity of lysosomes, assessed by partition of fluorescent weak
bases, was preserved, as was the electrical potential across the
mitochondrial inner membrane, estimated using rhodamine 143 (not
shown). Unless indicated otherwise, an 8-h transfection protocol was
used thereafter.
Dominant-negative NSF Inhibits Phagocytosis in
Macrophages--
The phagocytic ability of RAW cells expressing either
WT or E329Q-NSF, along with EGFP, was examined using IgG-opsonized
latex beads. As seen in Fig. 7,
a and b, RAW cells transfected with WT-NSF were
capable of phagocytosis as efficiently as untransfected neighboring
cells. Phagocytosis by cells transfected with WT-NSF was also similar
to that in cells transfected with EGFP and pcDNA 3.1 vector alone
(see quantitation in Fig. 7b). In contrast, RAW cells
transfected with E329Q-NSF ingested significantly fewer beads (Fig. 7,
a and b). The phagocytic indices for WT-NSF and E329Q-NSF transfectants were 205 ± 26 and 66 ± 11, respectively (Fig. 7b), equivalent to 68% inhibition.
Similar results were obtained using IgG-opsonized red blood cells.
It was conceivable that inhibition of phagocytosis by E329Q-NSF
resulted from impairment of constitutive secretion, causing depletion
of proteins that turn over rapidly. This possibility was tested by
exposing the cells to brefeldin A, a potent inhibitor of ARF GTPase
activity and COP I function (23), for 3 h prior to phagocytosis
assays. This time is comparable to the period of expression of
inhibitory concentrations of E329Q-NSF (see Fig. 6). Incubation with
brefeldin A for 3 h had no detectable effects on phagocytosis
(Fig. 7b), despite inducing very rapid dispersal of the
Golgi complex. Thus, the impairment of phagocytosis in RAW cells caused
by dominant-negative NSF is not due to the inhibition of constitutive secretion.
Dominant-negative NSF Does Not Block Fc
Actin cup formation also proceeded normally in cells transfected with
E329Q-NSF. Actin remodeling is a well characterized post-receptor event
that occurs early and transiently in the process of phagocytosis
(24-26). Staining of F-actin in RAW cells exposed to opsonized
particles for 5 min showed that the reorganization of actin fibers in
nascent phagosomal cups was indistinguishable in WT or mutant NSF
transfectants (Fig. 8c). Collectively, these results
indicate that impairment of phagocytosis by the dominant-negative NSF
occurred at a step distal to Fc receptor activation and is independent
of actin reorganization.
Dominant-negative NSF Inhibits VAMP3-GFP Recruitment to Sites of
Salmonella Internalization but Does Not Block Salmonella-induced
Membrane Ruffling--
Based on the inhibitory effect of
dominant-negative NSF on phagocytosis reported above, we anticipated
that inhibition of NSF activity would also have adverse effects on
Salmonella invasion. To ensure that the dominant-negative
NSF construct was having inhibitory effects in the cells used for
invasion assays, COS-1 cells were cotransfected with EGFP and either
WT-NSF or E329Q-NSF for 8 h, and transferrin uptake was
determined. As in RAW cells, E329Q-NSF impaired the intracellular
accumulation of transferrin in COS-1 cells (data not shown). Also,
immunofluorescent staining of
We next cotransfected COS-1 cells with VAMP3-GFP and either WT-NSF or
E329Q-NSF and performed Salmonella invasion assays as described above. As seen in Fig.
9b, coexpression of E329Q-NSF influenced the intracellular distribution of VAMP3-GFP (compare Fig.
9b with Fig. 1a) and prevented the localization
of VAMP3-GFP to sites of Salmonella attachment (Fig. 9,
b and c). Coexpression of WT-NSF did not affect
the expression pattern of VAMP3-GFP nor did it influence its
redistribution to Salmonella-associated ruffles (not
shown).
To determine whether membrane ruffling had been blocked by expression
of E329Q-NSF, we next expressed either the WT or mutant NSF together
with a cDNA encoding a chimera of GFP and the acylation motif of
the membrane-associated kinase Lyn (PM-GFP). This construct has been
described elsewhere and has been demonstrated to label preferentially
the plasma membrane, making it a useful marker of plasmalemmal
redistribution (27). The basal pattern of PM-GFP is shown in Fig.
9d. This relatively homogeneous plasmalemmal distribution
was not altered by coexpression of WT-NSF. PM-GFP was found to
accumulate at sites of Salmonella invasion (not
illustrated), in accordance with the reported formation of membrane
ruffles. Coexpression with E329Q-NSF had subtle effects on the resting distribution of PM-GFP in COS-1 cells but, importantly, did not alter
the redistribution of PM-GFP to sites of Salmonella invasion (Fig. 9, e and f). Together, the results
presented in Fig. 9 indicate that dominant-negative NSF blocked the
participation of SNARE-mediated membrane traffic in the membrane
redistribution that accompanies Salmonella invasion but did
not eliminate membrane ruffling.
NSF Activity Is Not Required for Invasion of COS-1 cells by S. typhimurium but Is Necessary for Particle Ingestion by Phagocytically
Competent COS-1 Cells--
The combined observations that
dominant-negative NSF inhibited the phagocytosis of opsonized particles
but did not block Salmonella-induced membrane ruffling
prompted us to quantify Salmonella invasion in COS-1 cells
expressing E329Q-NSF. COS-1 cells were cotransfected with EGFP and
either WT or E329Q-NSF prior to assaying Salmonella invasion
as described in Fig. 1. As seen in Fig.
10a, expression of either WT
or dominant-negative NSF had no effect on the invasion of S. typhimurium into COS-1 cells. The percentage of cells invaded was
not affected by expression of mutant NSF (Fig. 10a) nor was the number of Salmonella bacteria per invaded cell altered
(not shown). Similar results were obtained when HeLa cells were used, when NSF constructs were coexpressed with VAMP3-GFP or PM-GFP, and when
transient expression of E329Q-NSF was extended to 16-h periods.
The differential effects of E329Q-NSF on phagocytosis and bacterial
invasion may reflect differences in the cell types used, rather than in
the mechanisms underlying the two processes. To address this
possibility, we conferred phagocytic capability to COS-1 cells by
stable transfection of Fc Dominant-negative NSF Impairs SCV Maturation--
SCVs are known
to undergo a maturation process resulting in the insertion of specific
endomembrane components into the SCV membrane along with changes in
vacuolar contents (2, 28). The appearance of lysosomal-associated
membrane protein 1 (LAMP-1) in the vacuolar membrane is a marker of
such maturation. To examine the possible role of NSF in vacuolar
maturation, we examined the distribution of LAMP-1 in infected cells
using indirect immunofluorescence microscopy. Fig.
11 shows COS-1 cells cotransfected with
EGFP and WT or E329Q-NSF that have been infected with S. typhimurium. The percentage of S. typhimurium-containing vacuoles that were positively stained for
LAMP-1 was significantly reduced in cells expressing dominant-negative
NSF (Fig. 11e). The expression of E329Q NSF had no effect on
the level of expression of LAMP-1, as determined by Western blot, or on
the basal distribution of this lysosomal marker (data not shown). It is
important to note that the inhibition of LAMP-1 acquisition is likely
to be underestimated in these experiments because scoring was done on
an all-or-none basis, not taking relative degrees of staining into
account. Thus, while internalization of S. typhimurium into
COS-1 cells was not impeded by inhibition of NSF function, expression
of dominant-negative NSF in these cells did perturb the maturation of
the resultant SCVs.
Our findings indicate that NSF-regulated membrane fusion is
required to support Fc receptor-mediated phagocytosis but not bacterial
invasion. Specifically, whereas expression of dominant-negative NSF
inhibited particle engulfment both in macrophages and phagocytically competent fibroblastic cells, invasive bacterial internalization was
not significantly altered. This does not rule out the possibility that
other AAA proteins related to NSF, such as p97 (29), are active in
response to Salmonella invasion, and this alternative is
currently being investigated. Importantly, however, inactive NSF did
impair the maturation of Salmonella-containing vacuoles at a
stage after bacterial internalization was complete. These results
suggest that membrane traffic has different roles during these two processes.
The finding that NSF function is required for optimal phagocytosis is
consistent with an emerging body of evidence that suggests that
regulated fusion of endomembranes with the plasmalemma is essential for
phagocytosis (10, 11, 30-32). Recent work from our laboratory
indicates that VAMP3-containing vesicles participate in the focal
exocytosis that occurs at sites of particle engulfment during
phagocytosis (12), and together with the results presented here, these
data are compatible with a model wherein NSF-regulated, SNARE-mediated
membrane fusion drives exocytic events that are essential for the
completion of particle engulfment. This requirement is independent of
particle size, as phagocytosis of IgG-opsinized, invasion-null
Salmonella bacteria (~1 µm long) was also blocked by
expression of dominant-negative
NSF.2 Fusion of intracellular
vesicles with the plasma membrane may be required primarily to provide
additional bilayer area required for pseudopod extension.
Alternatively, it is possible that fusion of endomembranes is needed to
deliver components of the molecular machinery or signaling complexes
necessary for completion of the particle engulfment process. The
examination of phagocytosis-associated downstream signaling events in
the presence of dominant-negative NSF will be informative in this regard.
Whereas mechanisms of bacterial invasion are currently the subject of
intensive study, it is not yet clear how a variety of bacterial species
induce the formation of the vacuoles that engulf them. In the case of
Salmonella species, it is known to involve the type III
secretion system encoded for by the genes of pathogenicity island-1.
However, the functionality of many of the gene products in this system
is still poorly understood (2, 33, 34). It has been established that
Salmonella invasion can be accompanied by localized membrane
ruffling and membrane protrusions that resemble the pseudopods observed
during phagocytosis. The current study suggests that, although VAMP3
does localize to Salmonella-induced ruffles, this
localization is not required for the formation of ruffles (see results
with PM-GFP, Fig. 8). Furthermore, internalization of bacteria during
the invasion of cells by S. typhimurium was not impaired by
TeTx or by inhibition of NSF, revealing an important contrast between
phagocytosis and Salmonella invasion.
Collectively, these findings suggest that the ruffling noted during
invasion may be largely driven by rearrangement of the underlying
cytoskeleton and does not require membrane fusion. This model is in
agreement with previous observations that indicate that invasion by
S. typhimurium occurs independently of the
phosphatidylinositol 3-kinase activity (PI3K) of the host (1, 35). This
enzyme is an acknowledged mediator of membrane traffic in animal cells. Remarkably, inhibition of PI3K effectively precludes phagocytosis (31,
36), highlighting the differences between the latter process and
bacterial invasion. It is also possible that membrane fusion events are
involved in vacuole formation but that these are triggered by bacterial
proteins that are independent of host NSF and PI3K and therefore
insensitive to E329Q-NSF and to wortmannin, respectively. Although it
has been shown that bacterial proteins are required for the fusion of
Chlamydia trachomatis-containing inclusions (37), no
fusogenic bacterial products have been described in
Salmonella species.
After internalization of some invasive bacterial species, the vacuoles
formed can undergo a series of fission and fusion events involving
endomembrane compartments such as endosomes and lysosomes (2). S. typhimurium is known to reside initially in vacuoles that
transiently bear the markers EEA1 and transferrin receptor and
subsequently become enriched in markers of later compartments such as
the vacuolar H+-ATPase and LAMP-1. We found that the
percentage of S. typhimurium-containing vacuoles that were
positive for LAMP-1 was significantly reduced in cells expressing
dominant-negative NSF. These findings are consistent with recent
studies that indicate roles for both NSF and Rab proteins in the
fusion of SCVs and early endosomes in vitro (38). We are
currently working to elucidate the roles of specific SNAREs involved in
SCV maturation; however, the present study reveals a clear mechanistic
difference between the NSF-dependent maturation of vacuoles
and the NSF-independent internalization of the bacteria. Therefore, our
results are consistent with a model of bacterial invasion whereby
S. typhimurium autonomously invades target cells and
subsequently commandeers intracellular machinery to manipulate its
own microenvironment.
receptor-mediated phagocytosis was greatly reduced in the
presence of the mutant NSF. Most important, dominant-negative NSF
significantly impaired the fusion of Salmonella-containing
vacuoles with endomembranes. These observations indicate that the
membrane protrusions elicited by Salmonella invasion,
unlike those involved in phagocytosis, occur via an NSF-independent
mechanism, whereas maturation of Salmonella-containing
vacuoles is NSF-dependent.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-mannosidase II was a generous gift of Dr.
Marilyn Farquhar (University of California, San Diego). The
phycoerythrin (PE)-labeled antibody to mouse Fc
II/III receptors (CD16/CD32) was purchased from PharMingen. Rabbit polyclonal antiserum to S. typhimurium lipopolysaccharide was purchased from
Difco. Monoclonal antibody to human LAMP-1 was obtained from the
Developmental Studies Hybridoma Bank. Fluorescein isothiocyanate
(FITC)- and tetramethylrhodamine isothiocyanate (TRITC)-conjugated
donkey anti-mouse IgG and donkey anti-rabbit IgG were purchased from Jackson ImmunoResearch Laboratories. Polyclonal antibody to GFP, Alexa
350-, and Alexa 594-conjugated donkey anti-rabbit antibodies were
purchased from Molecular Probes. Horseradish peroxidase-conjugated goat
anti-rabbit antibody was obtained from Pierce.
-mercaptoethanol,
1 mM MgCl2, and 10% glycerol. With this was
mixed 20-25 µg/ml purified NSF protein, and the samples were
incubated at 37 °C for 1-2 h. Reactions were quenched with NaOH, to
a final concentration of 60 mM, and absorbance at 425 nm
was read on an Hitachi U-2000 spectrophotometer. Activities were
calculated by determining quantities of pNPP produced based on a molar
extinction coefficient of 1.1 × 104
M
1 cm
1
(16). For controls with NEM, protein samples were incubated in the
presence of 2.5 mM NEM for 15 min on ice prior to addition to the assay.
RIIA-transfected COS-1 cells (COS-2A) have been described
previously (8, 17-19). All cells were cultured in DMEM supplemented
with 10% FBS and incubated at 37 °C and 5% CO2. COS-2A
were periodically maintained in the presence of 200 µg/ml G418. For
cell-based assays, cells were grown on 25-mm glass coverslips to
50-75% confluence. Transfections of exponentially growing populations
of cells were done with the Fugene 6 reagent according to the
manufacturer's instructions. Cells were transfected with either WT NSF
or E329Q NSF alone or cotransfected with EGFP and then incubated,
depending upon the assay, for periods of 5, 8, or 16 h prior to
analysis. For cotransfections, a mass ratio of EGFP:NSF cDNA of
10:1 was used, and immunofluorescent staining of the cells was
performed to confirm that greater than 95% of EGFP-positive cells were
ectopically expressing NSF.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (37K):
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Fig. 1.
VAMP3-GFP accumulates at sites of
Salmonella invasion. VAMP3-GFP fluorescence and
indirect immunofluorescent labeling of GFP and S. typhimurium in COS-1 cells that were transfected with VAMP3-GFP
are shown. a, VAMP3-GFP distribution in a COS-1 cell that
was not exposed to S. typhimurium; b, a cell that
was infected. c, S. typhimurium, labeled with
antibody to LPS, in same field of view as b. d, VAMP3-GFP
fluorescence in a nonpermeabilized cell incubated with S. typhimurium and corresponding image of external GFP (e)
detected with an anti-GFP antibody. f, external
Salmonella in same field of view as d and
e. Arrowheads point to Salmonella in
c and f and to corresponding locations in cells
in b and e, respectively. Scale bars
represent 10 µm.
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Fig. 2.
Tetanus toxin cleaves cotransfected
VAMP3-GFP. Proteins from COS-1 cell extracts were separated by
SDS-polyacrylamide gel electrophoresis (10% gel) and analyzed by
Western blotting with antibody to GFP. Equal amounts of total protein
were loaded from mock-transfected cells (lane 1), cells
transfected with EGFP (lane 2), cells transfected with
VAMP3-GFP alone (lane 3), and cells transfected with
VAMP3-GFP and tetanus toxin (lane 4). Upper
arrowhead points to VAMP3-GFP (~43 kDa) in lane 3 and
lower arrowhead points to novel band (major cleavage
product) in lane 4. Differences in the amounts of
GFP-containing proteins in the different lanes are due to differences
in the transfection efficiencies of the different samples (~60% in
lane 2, ~35% in lane 3, and ~10% in
lane 4).
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Fig. 3.
Tetanus toxin prevents accumulation of
VAMP3-GFP at sites of S. typhimurium invasion.
VAMP3-GFP fluorescence and indirect immunofluorescent labeling of
S. typhimurium in COS-1 cells transfected with VAMP3-GFP, a
DIC image of cell expressing VAMP3-GFP and TeTx after
Salmonella infection. VAMP3-GFP distribution in same cell as
a is shown in b and Salmonella is
shown in c. d, VAMP3-GFP distribution and corresponding
image of external (e) and total (f)
Salmonella in TeTx-transfected cell. Arrowheads
point to bacteria in c and to corresponding location in
b (note lack of VAMP3-GFP accumulation).
Arrowheads in f point to internal bacteria.
Scale bar in f represents 10 µm.
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Fig. 4.
Tetanus toxin does not impair
Salmonella invasion. Percentages of COS-1 cells
containing internalized bacteria were determined for cells expressing
GFP, VAMP3-GFP alone, or VAMP3-GFP and TeTx. Means and standard errors
of three determinations are shown, representing >100 cells per
condition.
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Fig. 5.
Expression of WT and mutant NSF in RAW
macrophages. RAW 264.7 cells were transfected with a combination
of EGFP and either WT-NSF (a and b) or E329Q-NSF
(c and d). Alternatively, cells were transfected
with WT-NSF (e and f) or E329Q-NSF (g
and h) constructs alone. a-d, cells were fixed
after 8-h transfections and stained with anti-NSF antiserum and a
TRITC-conjugated secondary antibody. EGFP (a and
c) and NSF (b and d) images from the
same fields of view are shown. e-h, at 8 h
post-transfection, serum-starved cells were labeled with
TRITC-transferrin and then fixed and stained for NSF using a
FITC-conjugated secondary antibody. NSF (e and g)
and transferrin (f and h) distributions in
corresponding fields of view are shown. Arrows indicate
transfected cells. Scale bar represents 10 µm.
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Fig. 6.
Transient, acute expression of E329Q-NSF in
RAW cells impairs transport of transferrin. Untransfected RAW
cells or cells at 5 and 8 h after transfection with E329Q-NSF were
labeled with TRITC-transferrin and stained for NSF as above. The levels
of NSF expression (open squares) and the transferrin content
(closed circles) in the cells were then determined, relative
to control cells. The 8-h time point for transferrin uptake in cells
transfected with WT-NSF is also shown (open circle). Data
points are means ± S.E. from three independent experiments
representing more than 40 randomly chosen cells measured for each point
plotted.
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Fig. 7.
Expression of dominant-negative NSF inhibits
phagocytosis. a, RAW 264.7 cells were transiently
transfected with a combination of EGFP and either WT or E329Q-NSF.
After 8 h, cells were subjected to phagocytosis assays using
opsonized latex beads. Corresponding fields of view, using fluorescent
and DIC microscopy, are shown. Arrows indicate internalized
beads, and arrowheads indicate external beads. Scale
bar, 10 µm. b, quantitation of phagocytosis in
transfected RAW cells. Phagocytic index represents average number of
beads ingested per 100 cells. Control samples were transfected with
pcDNA 3.1 vector alone along with EGFP and treated with or without
5 µg/ml brefeldin A for 3 h. Means ± S.E. are taken from
at least three experiments for each treatment, and the number of cells
examined is indicated in parentheses.
Receptor
Function--
To define further the nature of the phagocytic defect in
RAW cells expressing mutant NSF, we examined the level of Fc receptor expression on the surface of transfected RAW cells. Flow cytometry of
RAW cells expressing WT and E329Q-NSF revealed that transfected cells
had levels of Fc
II/III receptors on their surface that were similar
to those of untransfected cells (Fig.
8a). The presence of receptors
and their ability to engage opsonized particles was also assessed by
particle binding assays using both latex beads and red blood cells. As
seen in Fig. 7a and quantified in Fig. 8b, RAW
cells expressing WT or E329Q-NSF bound opsonized particles with equal
efficiency.
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Fig. 8.
NSF inhibition does not alter
Fc receptor function. a,
quantitation of cell surface Fc
receptor expression using flow
cytometry. Levels of receptor expression in cells transfected with EGFP
alone or in combination with WT or E329Q-NSF are shown. All
axes show relative fluorescence, and plots are
representative of three separate experiments. b,
quantitation of particle adherence in cells expressing WT or E329Q-NSF.
Means ± S.E. from three experiments are shown, n >100
for each condition. c, actin cup formation in cells
transfected with WT and mutant NSF. RAW cells were transfected with WT
or E329Q-NSF for 8 h prior to phagocytosis. 5 min after exposure
to opsonized beads, cells were fixed and stained with anti-NSF antibody
and rhodamine-phalloidin to label actin structures. Arrows
point to fully formed actin cups. Scale bar is 10 µm.
-mannosidase II in cells expressing
E329Q-NSF indicated that Golgi morphology had been altered by
expression of the dominant-negative mutant (not illustrated). Ectopic
expression of WT-NSF had no apparent effect in either of these assays.
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Fig. 9.
E329Q-NSF blocks the accumulation of
VAMP3-GFP in Salmonella-induced membrane ruffles.
COS-1 cells were transfected with E329Q NSF together with either
VAMP3-GFP (a-c) or PM-GFP (e and f)
and then subjected to Salmonella infection followed by
immunofluorescent staining for LPS. a, DIC image
corresponding to the fluorescent micrographs shown in b and
c. VAMP3-GFP distribution in cells transfected with E329Q
and invaded by Salmonella is shown in b, and the
bacteria are shown in c. Arrowheads indicate lack of
VAMP3-GFP accumulation and corresponding sites of Salmonella
localization. d, cell transfected with PM-GFP alone, showing
resting distribution of PM-GFP. PM-GFP distribution in
E329Q-transfected cell during invasion is shown in e and
corresponding bacteria in f. Arrowheads in
e and f indicate overlapping bacteria and PM-GFP
accumulation. Scale bar represents 10 µm.
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Fig. 10.
Dominant-negative NSF does not inhibit
invasion by S. typhimurium. a,
quantitation of bacterial invasion in transfected COS-1 cells. In cells
transfected with EGFP alone (EGFP) or in combination with WT or
E329Q-NSF, the percentage of cells containing internalized bacteria was
determined. Means ± S.E. from four experiments are shown.
b, dominant-negative NSF inhibits phagocytosis in Fc
receptor-expressing COS-1 cells. Phagocytic indices (particles/100
cells) of COS-1 cells stably expressing Fc
receptor IIA were
determined, and the means ± S.E. of more than three experiments
are shown. The number of cells examined is indicated for each
condition. c, quantitation of cell surface Fc
receptor
IIA expression in NSF-expressing COS-2A cells. In parallel with
analyses of phagocytosis, transfected COS-2A cells were labeled with
monoclonal anti-human Fc receptor antibody. Experiments were done at
4 °C to prevent receptor internalization. Cells were then fixed and
counterstained with NSF antibody. Surface receptor levels in
transfected cells were then quantified using NIH Image. Shown are the
means ± S.E. from three experiments representing >100
cells.
receptor IIA. These cells, designated
COS-2A, have been characterized previously and shown to support
phagocytosis in a manner that closely resembles the behavior of
"professional" phagocytes such as macrophages (8, 17-19). COS-2A
cells were transiently cotransfected with EGFP and NSF constructs for
use in phagocytosis assays as described under "Experimental
Procedures." Cells transfected with WT-NSF ingested opsonized latex
beads efficiently, whereas their E329Q-transfected counterparts showed
significantly lower phagocytic indices (Fig. 10b). Cells
transfected with WT-NSF ingested particles as efficiently as
untransfected or mock-transfected COS-2A (data not shown). As was the
case for RAW cells, inhibition of phagocytosis was not attributable to
changes in receptor expression. Quantification of surface Fc
receptor IIA expression on transfected COS-2A cells, using indirect
immunofluorescence and NIH Image software, revealed no differences
between WT and E329Q-NSF-transfected samples (Fig. 10c).
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Fig. 11.
Maturation of S. typhimurium-containing vacuoles is impaired in COS-1 cells
expressing dominant-negative NSF. Bacterial invasion assays were
performed in COS-1 cells transfected with EGFP along with either WT-NSF
(a, b) or E329Q-NSF (c, d).
After invasion, cells were fixed and stained for Salmonella
LPS and endogenous LAMP-1. GFP expression revealed transfected cells in
which the distributions of SCVs and LAMP-1 were examined.
Arrowheads indicate SCVs in a and c
and corresponding locations in b and d,
respectively, showing LAMP-1 distributions. Scale bar is 10 µm. e, quantitation of LAMP-1-positive SCVs. In COS-1
cells transfected with EGFP alone or with EGFP together with WT or
dominant-negative NSF constructs, the percentage of SCVs that were
labeled with LAMP-1 antibody was determined. Means ± S.E. from
three independent experiments representing >100 cells for each
condition are shown (*, p < 0.0025).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENT |
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We thank Dr. Xiao-Rong Peng for providing VAMP3-GFP.
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FOOTNOTES |
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* This work was supported in part by the Arthritis Society of Canada, The Sanatorium Foundation, and the Canadian Institutes of Health Research.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.
§ Supported by Fellowships from the Canadian Institutes of Health Research.
** Honorary fellow of the Izaac Walton Killam Memorial Foundation.
Senior Scientist of the Canadian Institutes of Health Research
and a Howard Hughes International Investigator.
§§ International Scholar of the Howard Hughes Medical Institute, a recipient of a Canadian Institutes of Health Research Distinguished Scientist award, and the current holder of the Pitblado Chair in Cell Biology.
¶¶ Recipient of a Canadian Institutes of Health Research Scientist award. To whom correspondence should be addressed: Cell Biology Programme, Hospital for Sick Children, 555 University Avenue, Toronto, Ontario M5G 1X8, Canada. E-mail: wtrimble@sickkids.on.ca.
Published, JBC Papers in Press, November 22, 2000, DOI 10.1074/jbc.M007792200
2 M. G. Coppolino, S. Grinstein, and W. S. Trimble, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: SNARE, soluble NSF attachment protein receptor; NSF, N-ethylmaleimide-sensitive factor; SCVs, Salmonella-containing vacuoles; NEM, N-ethylmaleimide; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; Tfn, transferrin; pNPP, p-nitrophenyl phosphate; TRITC, tetramethylrhodamine isothiocyanate; FITC, Fluorescein isothiocyanate; WT, wild type; GFP, green fluorescent protein; EGFP, enhanced GFP; PBS, phosphate-buffered saline; SRBCs, sheep red blood cells; PE, phycoerythrin; TeTx, tetanus toxin; LPS, lipopolysaccharide.
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