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J. Biol. Chem., Vol. 276, Issue 50, 47361-47370, December 14, 2001
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From the Department of Molecular Biology and Genetics, Cornell
University, Ithaca, New York 14853
Received for publication, September 5, 2001
We report here that a broad spectrum of
phospholipase A2 (PLA2) antagonists
produce a concentration-dependent, differential block in
the endocytic recycling pathway of transferrin (Tf) and Tf receptors
(TfRs) but have no acute affect on Tf uptake from the cell surface. At
low concentrations of antagonists (~1 µM), Tf and TfR
accumulated in centrally located recycling endosomes, whereas at higher
concentrations (~10 µM), Tf-TfR accumulated in
peripheral sorting endosomes. Several independent lines of evidence
suggest that this inhibition of recycling may result from the
inhibition of tubule formation. First, BFA-stimulated endosome tubule
formation was similarly inhibited by PLA2 antagonists. Second, endocytosed tracers were found in larger spherical endosomes in
the presence of PLA2 antagonists. And third, endosome
tubule formation in a cell-free, cytosol-dependent
reconstitution system was equally sensitive PLA2
antagonists. These results are consistent with the conclusion that
endosome membrane tubules are formed by the action of a cytoplasmic
PLA2 and that PLA2-dependent
tubules are involved in intracellular recycling of Tf and TfR. When
taken together with previous studies on the Golgi complex, these
results also indicate that an intracellular PLA2 activity
provides a novel molecular mechanism for inducing tubule formation from
multiple organelles.
Endocytic compartments serve important sorting functions for the
segregation and delivery of membrane-bound receptors, their cognate
ligands, and soluble cargo to specific intracellular destinations (1,
2). Early light and electron microscopic studies revealed that some
membrane receptors become concentrated into endosomal tubules, 50-70
nm in diameter and extending from the main vesicular/vacuolar body of
endosomes, before recycling back to the plasma membrane (3-16). These
results suggested that tubules may serve to segregate membrane and
receptors away from the bulk fluid volume of the vacuolar part of the
endosome. From other studies, Maxfield and colleagues (2, 17, 18)
proposed that recycling of endocytosed TfRs1 occurs in part by the
iterative formation and budding of narrow 50-70-nm tubules from
endocytic compartments. These studies supported the idea that tubule
formation provides a mechanism for the separation of membrane from
soluble luminal contents based simply on differences in membrane
surface-to-volume ratios (19, 20).
In addition to membrane tubules, evidence has shown that cytoplasmic
coat proteins, such as clathrin, COPI coatomers, and the
Vps5-Vps17 complex in yeast cells, are also involved in
mediating recycling from endosomal and lysosomal compartments by
forming coated vesicles (15, 16, 21-28). Moreover, members of the
ADP-ribosylation factor family of GTP-binding proteins appear to
regulate endocytic trafficking, perhaps by mediating coat protein
binding (29-34). Thus, in a manner analogous to trafficking in the
secretory pathway, various types of ADP-ribosylation
factor-regulated coated vesicles may also package receptors and
membrane for recycling in the endocytic pathway. As with COPI and
clathrin-coated vesicles of the Golgi complex and
trans-Golgi network (TGN), respectively, binding of coat
proteins to endosomal membranes is sensitive to brefeldin A (BFA) (15,
23, 25), a potent inhibitor of trafficking through the secretory
pathway (35). As a result of BFA's action, some endosomes are
stimulated to form extensive membrane tubules, as do Golgi and TGN
membranes (36-41). However, unlike its effects on the secretory
pathway, BFA does not significantly inhibit endocytic uptake or
trafficking through endocytic compartments (37, 41, 42).
Our understanding of the exact roles that tubules and vesicles play in
endocytic recycling is incomplete because, as yet, no trafficking
intermediates have been definitively characterized. One possibility is
that endosome tubules provide a mechanism for bulk segregation of
membrane (including receptors) from fluid content, whereas coated
vesicles provide additional specificity for receptor packaging and
membrane fission (1, 2). Tubules and vesicles could work in concert
with coated vesicles budding from the tips of tubules, or they could
work independently (3, 15, 37, 39).
The mechanisms of endosome tubule formation are not known; however,
they are probably different from that of coated vesicle formation
because tubulation is enhanced in the presence of BFA, i.e.
when coat proteins are prevented from binding. By way of comparison,
recent studies on Golgi membranes have strongly suggested that
tubulation, both in vivo and in a cell-free reconstitution system, requires the action of a cytosolic Ca2+-independent
PLA2 activity (43-45). Moreover, we found that
constitutive and BFA-stimulated retrograde trafficking from the Golgi
complex to the endoplasmic reticulum was strongly inhibited by a
variety of compounds that antagonize cytoplasmic
Ca2+-independent PLA2 enzymes (43, 46).
In vitro studies suggest that the PLA2 acts
directly on the membranes to induce tubule formation (44, 45). These
results raise the possibility that endosomes and Golgi membranes
utilize the same mechanisms to induce tubules, which are involved in
different membrane trafficking events. Here we provide evidence that
tubulation of endosome membranes, both in vivo and in an
in vitro cell-free reconstitution system, was potently
inhibited by a variety of PLA2 antagonists, which also
significantly inhibited recycling of Tf-TfR complexes from various
endosomal compartments in vivo.
Materials and Cells
Stock solutions of the following reagents were prepared and
stored at Rat clone 9 hepatocytes and HeLa cells were grown in modified Eagle's
medium (MEM) with 10% Nu-Serum or Calf Serum-Supplemented and
1% penicillin/streptomycin. Mouse RAW 264.7 macrophages were grown in
Dulbecco's modified Eagle's medium with 10% fetal bovine serum and
1% penicillin/streptomycin. All cells were maintained at 37 °C in a
humidified atmosphere of 95% air, 5% CO2.
Binding, Endocytosis, and Trafficking of 125I-Tf,
Fluorescent Tf, and Horseradish Peroxidase (HRP)
HeLa cells were grown for 2 days to ~70-80% confluence in
12-well plates for 125I-Tf binding, uptake, and recycling
experiments or on glass coverslips for fluorescent Tf uptake and
recycling experiments. For all binding, uptake, and recycling
experiments, cells were first rinsed three times in MEM (without serum)
and incubated in the same for 30 min at 37 °C. Also, unless
otherwise indicated, MEM did not contain serum, which inhibits the
effects of PLA2 inhibitors. The subsequent procedures for
assaying binding, uptake, and recycling described below were modified
from previous studies (48).
Biochemical Determination of 125Tf
Trafficking--
To measure the effects of PLA2 inhibitors
on the relative amounts of TfRs found on the cell surface, cells were
incubated in the presence or absence of 10 µM BEL in MEM
for 45 min at 37 °C in a CO2 incubator. Plates were then
transferred to a cold room and placed on ice, and cells were rinsed
three times with ice-cold MEM containing a 1:10 dilution of the
normal sodium bicarbonate (MEM1/10). All subsequent surface binding
procedures were done at 4 °C. Cells were briefly rinsed with an acid
wash (500 mM NaCl, 0.2 N acetic acid, pH 2) to
remove any surface-bound Tf and then rinsed three times with MEM1/10,
which restored the pH to 7.4. Medium was removed and then replaced with
0.5 ml of MEM1/10 containing a saturating amount of 125I-Tf
(467 ng of Tf/well = 0.208 µCi/well). Cells were incubated for
2 h with gentle shaking. The medium was removed, and cells were
washed six times with MEM1/10 (by which time the last wash had only
background levels of 125I). Cells were extracted by the
addition of 1 N NaOH and gentle shaking for 30 min. The
extract was collected and counted in a
To measure the effects of PLA2 inhibitors on the endocytic
uptake of Tf from the cell surface, 12-well plates were placed on ice,
rinsed with ice-cold acid wash and then washed three times with
ice-cold MEM. The medium was removed, and cells were incubated in the
presence or absence of 10 µM BEL in ice-cold MEM for 10 min on ice. The medium was replaced with 0.5 ml of MEM plus 10 µM BEL (37 °C), as appropriate, which also contained
125I-Tf (amounts described above for surface binding).
Cells were allowed to internalize the bound 125I-Tf for 10 min at 37 °C. Following removal of the medium, cells were rinsed
with ice-cold acid wash, washed six times with ice-cold MEM, and then
extracted with 1 N NaOH as above.
To measure the effect of PLA2 inhibitors on the recycling
of Tf from internal compartments back to the cell surface, cells were
incubated with MEM containing 125I-Tf (0.17 µCi/ml) for
45 min at 37 °C in a CO2 incubator. The medium was
removed, and cells were washed six times in ice-cold MEM (while on ice)
and then incubated in the presence or absence of 10 µM
BEL in MEM for 5 min at 4 °C. The medium was removed; 0.5 ml of
37 °C efflux medium (MEM containing 0.1 mM
desferrioxamine, 3 µg/ml Tf) with or without 10 µM BEL
as appropriate was added; and cells were further incubated for various
periods of time at 37 °C. At the end of each time period,
medium was collected, and intracellular 125I-Tf was
harvested by extracting cells in 1 N NaOH. The amounts of
125I-Tf in both the medium and intracellular samples
were determined.
Morphological Analysis of Fluorescent Tf Trafficking--
To
visualize the endocytosis and trafficking of fluorescent Tf, cells were
treated essentially as above for biochemical labeling experiments. In
some cases, various specific pulse-chase protocols were conducted,
which will be described below. Cells were incubated in FITC-Tf (20 µg/ml) and/or Rhod-Tf (30 µg/ml) to label various endocytic
compartments. In many cases after labeling with fluorescent Tf, cells
were fixed and processed for immunofluorescence to localize TfR as
described (47) or the trans-Golgi/TGN membrane protein, galactosyltransferase (using polyclonal antibodies at a 1:600 dilution). In most cases, fluorescently labeled cells were viewed with
standard epifluorescence optics; however, in a few specific instances,
where noted, cells were viewed by scanning confocal microscopy with a
Bio-Rad 600 confocal microscope.
Endocytosis of soluble HRP and its visualization by
diaminobenzidine cytochemistry for electron microscopy were performed as previously described (47).
Preparation of Endosomes
A suspension of 12-nm colloidal gold particles
(Au12) was prepared as described (49), stabilized with
bovine serum albumin (BSA), resuspended in MEM (for clone 9 cells) or
Dulbecco's modified Eagle's medium (for RAW 264.7 cells), and stored
at 4 °C until used. Monolayers of clone 9 hepatocytes or RAW 264.7 macrophages were grown to confluence in 4-7 220-mm2
plastic tissue culture plates. To label endosomes with
Au12-BSA, cells were briefly rinsed once with MEM at
37 °C and incubated with prewarmed Au12-BSA suspension
(in MEM; 75 ml/dish) for 15 min (clone 9) or 10 min (RAW 264.7) at
37 °C. Labeling was terminated by rapidly pouring off the
Au12-BSA suspension and washing cells three times with
ice-cold MEM; plates were kept on ice as the cells were harvested with
a rubber policeman. All subsequent steps in the preparation procedure
were carried out at 4 °C. The cells were harvested in a table top
centrifuge (IEC) for 5 min, and the supernatant was discarded. The
pellet of packed cells was thoroughly resuspended in homogenization
buffer (250 mM sucrose, 10 mM Tris-HCl, 1 mM EDTA, pH 7.4) and centrifuged as above. This step was
repeated once, and the supernatant was discarded. The cells were
resuspended in homogenization buffer to yield a 20% (v/v) cell
suspension and homogenized by passage (2-4 times) through a
Balch/Rothman ball bearing homogenizer (50). The homogenate (~8 ml)
was centrifuged as above to yield a postnuclear supernatant. This
centrifugation step was repeated if the postnuclear supernatant contained any nuclei as detected by phase-contrast microscopy. The
postnuclear supernatant (4 ml) was layered on top of a sucrose step
gradient in Beckman SW28.1 centrifuge tubes containing the following
concentrations of sucrose at each step (3.4 ml/step): 0.5, 1.0, 1.5, and 2 M sucrose. Each sucrose solution was buffered with 1 mM Tris, 1 mM EDTA, pH 7.4. Additionally, the
0.5 M sucrose step contained 500 mM KCl, which
served to wash endosomes as they moved through the gradient and lowered
the background level of in vitro tubulation. The gradients
were centrifuged at 113,000 × g for 2 h in a
Beckman SW28.1 rotor. Material at the 1.0/1.5 M sucrose
interface contained the highest concentration of
Au12-BSA-loaded endosomes, was collected with a Pasteur
pipette, and was either used immediately or slowly frozen and stored at
Preparation of Bovine Brain Cytosol
Bovine brain cytosol used in these in vitro studies
was prepared as previously described (51). Two fractions of brain
cytosol from this preparation were used in these studies. The first,
called "crude cytosol" was the supernatant obtained immediately
after centrifuging the brain homogenate at 95,500 × g
for 2 h. The second fraction, called "bovine brain cytosol" or
BBC, was prepared from the above supernatant by precipitation with 60%
ammonium sulfate, reconstitution in buffer, and dialysis as described
(51). Unless otherwise indicated, all experiments used this BBC
preparation. Typically, 1.5 kg of brains yielded over 600 ml of cytosol
with a protein concentration of ~25 mg/ml.
In Vitro Tubulation Assay
The tubulation of isolated endosomes was visualized by a whole
mount EM-negative stain assay as used to document the tubulation of
Golgi membranes (52). In a typical assay, 12.5 µl of bovine brain
cytosol was mixed with 87.5 µl of tubulation buffer (final concentrations in the complete assay mixture: 50 mM KCl, 1 mM MgCl2, 25 mM Tris-HCl, 10 mM HEPES, pH 7.4) containing various combinations of other
reagents such as ATP and/or GTP. This buffer mixture and either fresh
or thawed endosome-enriched fractions were equilibrated to
37 °C for 15 min. Tubulation was initiated by slowly adding an equal
volume (20 µl) of the cytosol/buffer mixture to the endosome-enriched
fractions, followed by incubation at 37 °C for various periods of
time. Drops of the endosome suspension were placed on Formvar- and
carbon-coated EM grids for 15 min at room temperature, and grids were
negatively stained with 2.0% phosphotungstic acid, pH 7.2, and viewed
by electron microscopy (52). In an alternate method, drops of the
endosome suspension were placed on Formvar- and carbon-coated EM grids
for 5 min at room temperature, and glutaraldehyde (from 25% stock
solution) was added directly to the drops (to a final concentration of
0.1%) and allowed to fix the organelles for 5 min. The fixed
organelles were then stained with 2% phosphotungstic acid as described above.
Endosome tubulation was quantified by determining the
percentage of gold-loaded organelles that displayed at least one
membranous tubule or that were completely tubular. For each
experimental condition, at least 150 endosomes were counted from
triplicate samples. A tubule was defined as a membranous extension,
50-70 nm in diameter and at least twice as long, that was continuous with the lipid bilayer of the endosome. For practical reasons, however,
this assay can only provide a semiquantitative estimation of the
tubulation activity. Ideally, we would like to determine the number of
tubules/endosome. Unfortunately, determining this number slowed
analyses of the experiments to unacceptable levels. Therefore, an
endosome with one tubule was equal to an endosome with multiple
tubules. From our qualitative observations, however, we noticed a clear
positive correlation between the number of endosomes with tubules and
the number of tubules/endosome.
PLA2 Antagonists Inhibit Tf-TfR Recycling at Multiple
Steps--
Based on our previous studies, which revealed that
PLA2 antagonists were potent inhibitors of BFA-stimulated,
tubule-mediated retrograde trafficking from the Golgi complex (43), we
asked here if these same antagonists affected putative tubule-mediated trafficking steps in the endocytic recycling pathway. To investigate this question, the intracellular trafficking of TfR and its ligand, Tf,
were followed using various pulse-chase experiments and biochemical and
morphological assays. We found that incubation of cells with the
irreversible inhibitor of Ca2+-independent PLA2
enzymes, BEL, had little effect on a brief pulse (10 min) of
125I-Tf uptake (Fig.
1A). However, when cells were
first pretreated with BEL for 45 min at 37 °C and then assayed for
the presence of 125I-Tf binding sites, we found a
significant decrease on BEL-treated cells (Fig. 1A). These
results suggest that although BEL does not prevent uptake from the cell
surface, it does prevent recycling of TfR from internal compartments.
To directly test this idea, cells were incubated with
125I-Tf for 45 min to allow its internalization and
delivery to endosomal compartments, washed free of uninternalized
ligand, and then chased in 125I-Tf-free medium in
the presence or absence of BEL for various periods of time. The results
showed that the addition of 10 µM BEL substantially
inhibited the recycling of 125I-Tf to the extracellular
medium (Fig. 1B). Using a fixed chase time point (60 min), we found that increasing concentrations of BEL produced a linear
and saturable inhibition of 125I-Tf recycling with an
IC50 of ~4 µM (data not shown).
To determine where in the recycling pathway Tf trafficking
was inhibited, cells were pulse-labeled with FITC-Tf for 45 min and
then chased (in medium without FITC-Tf) in the presence or absence of various PLA2 inhibitors. Cells were then
prepared for immunofluorescence labeling of the TfR. In control cells
with no chase, Tf and the TfR were found to almost entirely colocalize in numerous punctate vesicles located throughout the cytoplasm (Fig.
2, A and B). When
cells were pulse-labeled and chased in medium without
antagonists for 60 min, FITC-Tf staining was significantly reduced,
consistent with its recycling and loss into the extracellular medium (Fig. 2, C and D). However, quite
different results were obtained when BEL was included in the chase
medium. In the presence of 10 µM BEL, FITC-Tf was
not lost from cells but was instead found to colocalize with TfRs in
punctate vesicles located throughout the cytoplasm (Fig. 2,
E and F), a pattern similar to that of untreated
cells. Interestingly, in 1 µM BEL, both FITC-Tf and TfR
were found to colocalize in a more compact, juxtanuclear staining pattern (Fig. 2, G and H). Similar
dose-dependent results were obtained with a structurally
different PLA2 inhibitors, ONO-RS-082, N-(p-amylcinnamoyl)anthranilic acid, and
aristolochic acid (data not shown). These results confirm the
biochemical studies above showing that PLA2 antagonists
inhibit Tf-TfR recycling and further suggest that BEL produced a
concentration-dependent block in recycling at two different
steps.
Previous studies have established that following endocytosis, Tf-TfR
complexes move sequentially through peripherally located early sorting
endosomes, then to a centrally located tubular recycling endocytic
compartment, and finally back to the cell surface for release into the
medium (for a review see Ref. 2). Thus, our above results suggest that
10 µM BEL may block trafficking of Tf-TfR out of
peripheral early endosomes and that 1 µM BEL allows transport to, but not out of, centrally located recycling endosomes. To
examine this issue further, we took advantage of the fact that ONO-RS-082 is a reversible inhibitor of Ca2+-independent
PLA2 enzymes (53) and displayed the same
concentration-dependent inhibition of Tf recycling as BEL
(data not shown). As with BEL, when cells were pulse-labeled with
FITC-Tf and then chased in medium without FITC-Tf but with high
(10 µM) concentrations of ONO-RS-082, FITC-Tf remained
located along with TfRs in vesicles throughout the cytoplasm (Fig.
3, A and B). When
similarly treated cells were then washed free of ONO-RS-082 and
incubated for an additional 15 min, both FITC-Tf and TfR moved to the
centrally located endocytic compartment (Fig. 3, C and
D). This central compartment was indeed the recycling
compartment, because incubation in ONO-RS-082-free medium for an
additional 45 min (60 min total) resulted in a significant loss of
FITC-Tf from the cells (Fig. 3, E and F).
Finally, the TfRs that recycled to the cell surface following recovery
from ONO-RS-082 were competent to re-endocytose another round of
FITC-Tf added to the medium (Fig. 3, G and
H).
Although our results are consistent with a
concentration-dependent block at two stages of the Tf/TfR
recycling pathway, the PLA2 antagonists might also be
causing a mistargeting of receptor-ligand complexes to or from other
compartments. For example, the compact juxtanuclear compartment in
which FITC-Tf and TfRs accumulate in cells treated with 1 µM ONO-RS-082, might instead be elements of the TGN,
given the close association between central recycling endosomes and the
TGN (2). To investigate this further, cells were incubated with FTIC-Tf
for 45 min in the presence of 1 µM ONO-RS-082 and then
chased in ligand-free medium for an additional 30 min in the
presence of ONO-RS-082. Cells were fixed; processed for
immunofluorescence to localize
Another possible explanation for the accumulation of FITC-Tf and TfR
within the central juxtanuclear compartment (when treated with 1 µM BEL or ONO-RS-082) is that peripheral sorting
endosomes redistribute to the center of the cell, rather than
receptor-ligand complexes exiting the peripheral sorting endosomes. To
distinguish between these possibilities, cells were pulse-labeled with
Rhod-Tf for 60 min, chased in ligand-free medium for 15 min, and
then incubated with a second pulse-chase of FITC-Tf (as for Rhod-Tf), all in the presence of 1 µM ONO-RS-082. The results
showed that there was significant overlap in staining of the two
endocytic markers within vesicles of the central compartment (Fig.
5). Because FITC-Tf was ultimately
delivered to Rhod-Tf-containing endosomes, we conclude that 1 µM ONO-RS-082 did not affect the function or distribution of early sorting endosomes, through which FITC-Tf must
have entered and exited on its way to the central sorting endosomes.
One possible mechanism by which PLA2 antagonists might
alter the trafficking of Tf-TfR is by changing the pH of acidified endosomal compartments. However, we found no difference in the ability of control or treated (10 µM ONO-RS-082 for 30 min at 37 °C) cells to take up the pH-sensitive dye acridine orange
into endosomal and lysosomal compartment (data not shown).
PLA2 Antagonists Inhibit BFA-stimulated Endosome Tubule
Formation--
Trafficking from the central recycling compartment has
been proposed to involve the formation of thin membrane tubules, whose presence is enhanced by BFA. Because we have previously shown that
BFA-induced Golgi tubulation was inhibited by PLA2
antagonists, we asked if BFA-stimulated tubulation of endosomes was
similarly affected. Cells were first incubated with FITC-Tf for
45 min to label all endocytic compartments and then treated with BFA in the presence or absence of BEL. In both untreated cells and cells treated with BEL alone (10 µM for 20 min), FITC-Tf and
TfR were primarily located in punctate vesicles throughout the
cytoplasm (Fig. 6, A-D).
Treatment of cells with BFA alone for 15 min resulted in the extensive
formation of membrane tubules that were labeled with both FITC-Tf and
TfR (Fig. 6, E and F). However, pretreatment of
cells for just 5 min with BEL (10 µM) before a 15-min
incubation in BFA significantly inhibited endosome tubule formation
(Fig. 6, G and H). This inhibition was confirmed
at the EM level in cells that had been incubated with soluble HRP to
identify endosomes. In control cells, HRP-labeled endosomes were
generally spherical, ~200-400 nm in diameter (Fig.
7A). Incubation of cells with
BFA for 10 min resulted in the formation of numerous tubular endosomes, ~50-70 nm in diameter and up to several µm in length (Fig.
7B). Pretreatment of cells with ONO-RS-082 for 5 min
inhibited this BFA-stimulated tubulation (Fig. 7C).
A broad spectrum of PLA2 antagonists were tested for their
ability to inhibit BFA-stimulated tubulation in cells whose endosomes were labeled by prior uptake with FITC-Tf and subsequently by immunofluorescence localization of the TfR. We found that all PLA2 antagonists tested were highly active inhibitors of
BFA-stimulated tubulation, with IC50 values in the low
micromolar range (Table I).
To more directly examine the ability of low concentrations of
PLA2 antagonists to inhibit tubulation of the recycling
compartment, cells were incubated with FITC-Tf in the continuous
presence of 1 µM ONO-RS-082 for 45 min. In these cells,
FITC-Tf was primarily located in the central recycling compartment
(Fig. 2, G and H). Similarly treated cells were
then either kept in 1 µM ONO-RS-082 or washed free of the
antagonist, BFA was immediately added, and the cells were further
incubated for various periods of time (Fig. 8). In cells washed free of ONO-RS-082,
BFA stimulated tubule formation within 5 min until, by 15 min, ~57%
of the cells exhibited tubules emanating from the central recycling
compartment. In contrast, cells kept in 1 µM ONO-RS-082
did not exhibit BFA-stimulated tubulation.
Formation of Endosome Tubules in Vitro--
To
complement these in vivo studies, we have developed a
cell-free system that reconstitutes tubule formation from isolated endosomes. Endosomes were unambiguously identified in subcellular fractions by first incubating cultured clone 9 hepatocytes with Au12-BSA gold particles for 15 min at 37 °C, to allow
fluid phase particle uptake into early and late endosomes prior to cell
homogenization (39). Cells were then homogenized, an endosome-enriched
fraction was obtained, and endosomes containing Au12-BSA
were visualized by EM-negative staining (arrowheads in
Fig. 9).
Endosomes incubated with an organelle-free extract of bovine brain
cytosol (BBC) alone (Fig. 9D), or BSA plus ATP (0.1 mM) (Fig. 9E), at 37 °C for 30 min were
primarily round vesicles and vacuoles, 0.25-1.5 µm in diameter
(average of 0.45 µm; 50 random measurements), and they rarely
exhibited tubular extensions. In contrast, incubation with BBC (1.5 mg/ml) and ATP (0.1 mM) induced the dramatic formation of
extensive membrane tubules (Fig. 9, F-H). These tubules
were generally uniform in diameter (average of 53 nm; 32 random
measurements), reached lengths up to 3.4 µm (average of 0.61 µm; 54 tubules measured), and the lipid bilayers of the tubules were
continuous with the bilayers of the main body of the endosomes.
Endosomes exhibited one or multiple tubules (Fig. 9, F and
H), which were morphologically similar to those seen
extending from endosomes in vivo (Fig. 9, A-C)
(in the latter case, endosomes that had been labeled by endocytic
uptake of soluble HRP). Also, endosomes fixed with glutaraldehyde after
incubation but before negative staining also exhibited a strict
dependence on cytosol for tubulation (data not shown).
Quantitation of these results showed that incubation with BBC or ATP
(0.1 mM) alone had little effect but that incubation with
both resulted in a 5-10-fold increase in the percentage of endosomes
with at least one tubule (Fig.
10A). In these experiments, ~50-75% of the endosomes were induced to form at least one tubule. Heating cytosol to 100 °C for 30 min before assaying greatly reduced the level of tubulation activity (Fig. 10A), and incubation
with BBC and GTP or GTP
The tubulation activity could be followed through the steps used to
prepare the BBC fraction from crude cytosol and characterized following
various treatments (Fig. 10D). The tubulation activity in
BBC was found to be insensitive to a number of agents including N-ethylmaleimide, N-ethylmaleimide in the
presence of dithiothreitol, or nocodazole, a compound the depolymerizes
microtubules (data not shown). Also, treating endosomes with BFA (10 µg/ml) alone or with BBC seemed to have little effect on the
tubulation activity. Finally, this tubulation activity appeared not be
a general perturbant of membrane structure because the active BBC
preparation used in these experiments had no effect on isolated
lysosomes.2
To investigate the origin of the membrane that forms tubules, we
determined the average diameter of the main bodies of randomly selected
nontubulated (50 selected) and tubulated (25 selected) endosomes. These
measurements revealed that tubulated endosomes have, on average, a
smaller diameter across their vacuolar body (0.31 µm) than their
nontubulated counterparts (0.45 µm). The decrease in body diameter is
consistent with the idea that the membrane for tubules originates from
the main body of the endosomes. In tubulated endosomes,
Au12-BSA particles were most commonly found in the tubular
extensions (Fig. 9, F-H), suggesting some weak interaction
with the membrane. However, with regard to tubulation, this interaction
is irrelevant, since endosome tubules formed without any gold particles
being present within the lumen of the tubule (Fig. 9F).
PLA2 Antagonists Inhibit in Vitro Endosome
Tubulation--
Cytosol-dependent endosome tubulation was
also examined for sensitivity to PLA2 antagonists and to
see if it was active on endosomes from another source. For these
experiments, endosomes were prepared from RAW 264.7 macrophages, and
BBC was incubated with a PLA2 antagonist before the
addition to isolated endosomes. The results showed that the
structurally distinct compounds BEL and ONO-RS-082 significantly
inhibited cytosol-dependent endosome tubulation in
vitro even at 1 µM (Fig.
11), concentrations similar to those
that inhibited endosome tubulation and receptor recycling in
vivo. In other studies, we have found that this BBC preparation contains both BEL- and ONO-RS-082-sensitive and -insensitive
PLA2 activities (43, 44). As with the Golgi complex (45),
we found that a nonspecific PLA2, snake venom
PLA2, did not increase endosome tubulation above background
levels. Quantitatively similar inhibition by PLA2
antagonists was obtained using endosomes from clone 9 hepatocytes (data
not shown).
This paper provides evidence for the role of a cytoplasmic
Ca2+-independent PLA2 in the recycling of
Tf-TfR from endosomal compartments to the cell surface and the
formation of endosome tubules both in vivo and in
vitro. These results are similar to those of recent studies that
demonstrated a nearly identical sensitivity of Golgi membrane
tubulation and retrograde trafficking to PLA2 antagonists (43, 45, 46). The fact that a broad spectrum of PLA2
antagonists were found to inhibit trafficking events, suspected or
known to involve membrane tubules, from both endosomes and the Golgi
complex, suggests that a cytoplasmic PLA2 activity plays a
general role in tubule formation from organellar membranes.
We found that higher concentrations of PLA2 antagonists
(~10 µM) inhibited trafficking from peripheral sorting
endosomes to central recycling endosomes, and lower concentrations (1 µM) inhibited trafficking from central recycling
endosomes to the cell surface. At either concentration, it appeared
that transport out of an endocytic compartment rather than fusion with
a target was the inhibited step. Although not shown directly, several
lines of evidence support this idea. First, uptake and trafficking of
endocytic tracers in the presence of low or high concentrations of BEL
did not appear to result in the accumulation of small transport
vesicles or tubules. Second, at the concentrations of inhibitors used
here or in previous studies on trafficking through the Golgi complex, no acute effect on any known coated vesicle-mediated transport step was
found, including endocytosis from the cell surface and anterograde
trafficking through the secretory pathway (43). Third, BFA-stimulated
tubulation of Tf-TfR-enriched endosomes was also potently inhibited
within the same concentration range for all PLA2
antagonists examined. And, fourth, cytosol-dependent endosome tubulation in the cell-free system was also potently inhibited
by the same PLA2 antagonists.
The conclusion that PLA2 antagonists inhibit the
tubule-mediated exit of Tf-TfRs from both early sorting and late
recycling endosomes is also consistent with our current understanding
of the Tf-TfR recycling pathway (1, 2). For example, morphological studies have shown that after endocytosis from the cell surface, Tf-TfR
complexes enter an early sorting endosome compartment located in the
periphery of the cell. The complexes segregate into tubular extensions
and are then packaged into vesicles and/or tubules that relocate to the
central recycling compartment (9, 17, 56). Based on various pulse-chase
experiments, Maxfield and colleagues (17, 18, 57) have suggested that
sorting within this compartment occurs by iterative fractionation,
yielding multiple rounds of tubule and vesicle formation and fusion. By
mechanisms that are not fully understood, these tubulo-vesicular
membranes, enriched in Tf-TfR, form an extensively interconnected
tubular recycling compartment (58-62), from which Tf-TfRs recycle back to the plasma membrane. Interestingly, BFA, which enhances endosome tubule formation (36, 37, 39, 40), has been reported to increase the
number of various receptors, including TfRs, on the cell surface (39,
42, 63), perhaps as a consequence of stimulating tubule-mediated
recycling. Thus, the observation that PLA2 antagonists inhibit Tf-TfR trafficking from sorting and recycling endosomes is
consistent with the idea that membrane tubules play an important role
in mediating these steps.
A role for PLA2 in endosome-to-endosome fusion has also
been suggested based on a similar pharmacological approach (64). However, the concentration of PLA2 antagonists required to
inhibit endosome fusion either in vivo or in
vitro was ~10-50-fold higher than that for inhibition of Tf-TfR
recycling or endosome tubulation. Thus, at the concentrations used
here, membrane fusion would not have been severely affected, a
conclusion that is supported by finding that delivery of Tf to
endosomal vesicles/vacuoles was not inhibited. These studies do,
however, raise the interesting possibility that
PLA2-dependent tubulation could be linked to a
PLA2-dependent fusion event.
The cell-free system that reconstitutes endosome tubulation in
vitro is an important adjunct to in vivo tubulation
assays. The endosome tubules produced in vitro were uniform
in diameter, 50-70 nm, matching their in vivo counterparts
(3, 4, 6-8, 12, 18, 65, 66), and they often possessed flared or
bulbous tips (see Fig. 7), which were also observed on endosome tubules in vivo (11). Interestingly, the requirements for endosome
tubulation in vitro were nearly identical to those for Golgi
membrane tubulation (51, 52), including 1) dependence on a heat-labile,
nondialyzable protein factor found in cytosol preparations; 2) the lack
of requirement for Ca2+; and 3) sensitivity to the same
spectrum of PLA2 inhibitors (44).
Although our in vivo and in vitro results suggest
that cytoplasmic PLA2 activity functions as a general
tubulation factor, this activity was not found to induce tubulation of
lysosomes, despite the well documented ability of lysosomes to become
tubular in certain cells (67). However, tubular lysosomes are larger in
diameter than endosome or Golgi tubules and may rely solely on
microtubules for their formation (68, 69). Thus, tubulation by a
cytoplasmic PLA2 may involve specific interactions with a subset of organelles on the cytoplasmic sides of their membranes.
In addition to PLA2-mediated mechanisms, intracellular
membrane tubules could also form in other ways. For example, several recent studies have shown that the clathrin-coated vesicle-associated GTPase, dynamin, can be induced to assemble onto membranes and form
tubules under conditions where the GTPase activity is inhibited (70-73). It is important to note, however, that the tubules in our
cytosol-dependent, PLA2 inhibitor-sensitive
in vitro assay were not affected by either GTP or GTP Taken together, these results suggest that a cytoplasmic
PLA2 activity is required for tubulation and membrane
trafficking from multiple organelles, including endosome tubule
formation and Tf-TfR recycling. One caveat is that we cannot
definitively rule out that a phospholipase A1,
lysophospholipase, or other lipase activity is responsible, because
some of the tested antagonists have been shown to inhibit these
activities as well (74, 75). However, our conclusion is strengthened by
finding that structurally unrelated compounds (BEL, ONO-RS-082,
quinacrine, etc.) all have in common the ability to inhibit
PLA2 activity. BEL is particularly interesting, because it
displays ~1000-fold more specificity for a cytoplasmic
Ca2+-independent versus
Ca2+-dependent PLA2 (74, 75), and
because our in vitro tubulation activity does not require
Ca2+, a Ca2+-independent PLA2 may
be the relevant enzyme. A variety of Ca2+-independent,
cytoplasmic PLA2 forms, with no definitive known function, have been characterized to varying degrees (76-79), and others are likely to be discovered. One of the most striking findings is that in tubulation, both endosomes and Golgi membranes
display virtually identical sensitivities to PLA2
antagonists. Thus, these results suggest similar mechanisms of tubule
formation on these organelles and a novel role for a cytoplasmic
Ca2+-independent PLA2 in tubule-mediated
intracellular trafficking.
We thank Esther Racoosin for critically
reviewing the manuscript and Dr. Ed Cluett for many helpful and
important suggestions early in this work. We also thank Dr. Ian
Trowbridge for generously supplying anti-TfR antibodies used early in
this work.
*
This work was supported by National Institutes of Health
Grant GM 60373 (to W. J. B.).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.
Published, JBC Papers in Press, October 3, 2001, DOI 10.1074/jbc.M108508200
2
A. van Vante and W. J. Brown, unpublished data.
The abbreviations used are:
Tf, transferrin;
TfR, transferrin receptor;
BBC, bovine brain cytosol;
BSA, bovine serum
albumin;
Au12-BSA, 12-nm colloidal gold particles
conjugated with BSA;
BEL, bromoenol lactone (or
(E)-6-(bromomethylene)tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one);
ONO-RS-082, 2-(p-amylcinnamoyl)amino-4-cholorbenzoic acid;
PLA2, phospholipase A2;
FITC-Tf, fluorescein-labeled transferrin;
Rhod-Tf, rhodamine-labeled
transferrin;
TGN, trans-Golgi network;
BFA, brefeldin A;
MEM, modified
Eagle's medium;
HRP, horseradish peroxidase;
AMP-PNP, adenosine 5'-(
Inhibition of Transferrin Recycling and Endosome
Tubulation by Phospholipase A2 Antagonists*
ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
20 °C: BFA (10 mg/ml in ethanol),
N-ethylmaleimide (0.1 M), nocodazole (6 mg/ml in
Me2SO), and AMP-PNP (0.1 M) were obtained from
Sigma; GTP
S (50 mM) was from Roche Molecular
Biochemicals. Nu-Serum was from Collaborative Research (Bedford, MA),
and Calf Serum-Supplemented was from Life Technologies, Inc.
125I-Tf was from Amersham Pharmacia Biotech. Phospholipase
and other inhibitors were obtained from the following sources:
N-(p-amylcinnamoyl)anthranilic acid,
2-(p-amylcinnamoyl)amino-4-cholorbenzoic acid (ONO-RS-082), and (E)-6-(bromomethylene)
tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one (or bromoenol lactone
(BEL)) were from Biomol Research Laboratories, Inc. (Plymouth Meeting,
PA); quinacrine, p-bromophenacyl bromide, and all other
common reagents were from Sigma. The following antibodies were
generously supplied: monoclonal anti-TfR antibodies (HTR-H68) by Dr.
Ian Trowbridge (Salk Institute, La Jolla, CA) and polyclonal antibodies
against 
1,4 galactosyltransferase by Dr. Eric Berger (University of
Zurich, Switzerland). Fluorescein-Tf (FITC-Tf) and rhodamine-Tf
(Rhod-Tf) were prepared exactly as described (47).
counter.
80 °C. Tubulation-competent endosomes had a useful shelf life of
~2 days at 80 °C.
RESULTS
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ABSTRACT
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RESULTS
DISCUSSION
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View larger version (20K):
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Fig. 1.
The PLA2 antagonist, BEL,
inhibits Tf-TfR recycling but not endocytosis. A,
measurements of the effects of BEL on 125I-Tf uptake from
the cell surface (two left bars) and on the
presence of TfR on the cell surface (two right
bars). To measure the direct effect of BEL on endocytosis
from the cell surface, cells were pretreated in the cold with (+) or
without ( ) 10 µM BEL and then shifted to 37 °C in
the presence of 125I-Tf for 10 min (still in the presence
or absence of BEL as appropriate). BEL had no appreciable effect on
125I-Tf uptake. To measure the long term effect of BEL on
the presence of TfRs on the cell surface, cells were incubated with (+)
or without () BEL for 45 min at 37 °C, and then cell surface TfRs
were detected by binding saturating amounts of 125I-Tf in
the cold for 2 h. Under these conditions, BEL caused a significant
decrease in 125I-Tf binding to the cell surface.
B, BEL inhibits the recycling of 125I-Tf from
intracellular compartments. Cells were incubated with
125I-Tf for 45 min at 37 °C to allow uptake and delivery
to endosomal compartments and then chased in ligand-free medium
in the presence or absence of 10 µM BEL for various
periods of time. At the time points indicated, medium samples and cell
extracts were collected, and the amounts of 125I-Tf were
determined in each. % Total Recycled represents
the amount of 125I-Tf found in the medium sample divided by
the total (medium plus cell extract) × 100. BEL significantly
inhibited the appearance of 125I-Tf in the medium. Results
in both A and B represent the average of two
experiments with triplicate determinations for each.
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Fig. 2.
PLA2 antagonists display a
dose-dependent inhibition of the intracellular recycling of
FITC-Tf and TfR at two discrete steps. In all experiments, HeLa
cells were pulse-labeled with FITC-Tf for 45 min to label all endocytic
compartments (left panels), subjected to various
chase protocols in the presence or absence of the PLA2
inhibitor, BEL, and then processed for immunofluorescence to localize
TfRs (right panels). A and
B, cells pulse-labeled for 45 min. C and
D, cells pulse-labeled and then chased in Tf-free
medium for 60 min. E and F, cells
pulse-labeled and chased in the presence of 10 µM BEL for
60 min. G and H, cells pulse-labeled and chased
in the presence of 1 µM BEL for 60 min.
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Fig. 3.
PLA2 antagonists inhibit
sequential steps in the recycling of FITC-Tf and TfR through early
(sorting) and late (recycling) endocytic compartments. In all
experiments, HeLa cells were incubated with FITC-Tf for 45 min at
37 °C to label all endocytic compartments (left
panels), subjected to various chase protocols in the
presence or absence of the reversible PLA2 inhibitor,
ONO-RS-082 (ONO), and then processed for immunofluorescence
to localize TfRs (right panels). A and
B, cells pulse-labeled and chased in the presence of 10 µM ONO-RS-082 for 60 min. C and D,
cells treated as in A and then washed and incubated in
ONO-free medium for 15 min. E and F, cells
treated as in A and then washed and incubated in ONO-free
medium for 60 min. G and H, cells treated as in
A, washed and incubated in ONO-free medium for 60 min, and
then pulse-labeled with FITC-Tf for 45 min.
1,4-galactosyltransferase, an enzyme
found in both trans-Golgi cisternae and the TGN of HeLa cells (54, 55); and then visualized by confocal microscopy. The results
from both individual and merged images show that FITC-Tf and
galactosyltransferase labeled separate compartments (Fig. 4).
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Fig. 4.
FITC-Tf endocytosed in the presence of 1 µM ONO-RS-082 accumulates in a
compartment that is distinct from the TGN. HeLa cells were
incubated with FITC-Tf for 45 min in the presence of 1 µM
ONO-RS-082 and then chased in ligand-free medium for an
additional 30 min in the presence of ONO-RS-082. Cells were then fixed,
processed for immunofluorescence to localize
1,4-galactosyltransferase (using rhodamine-labeled secondary
antibodies), and visualized by confocal microscopy. A,
merged image of FITC-Tf (green) and galactosyltransferase
(red); B, single image of FITC-Tf; C,
single image of galactosyltransferase.
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Fig. 5.
The centrally located endosomes that
accumulate Tf in the presence of 1 µM ONO-RS-082 are capable of receiving
another round of endocytic cargo. HeLa cells were pulse-labeled
with Rhod-Tf for 60 min, rinsed in ice-cold MEM (without Rhod-Tf), and
incubated in MEM containing 1 µM ONO-RS-082 for 5 min on
ice. Cells were then incubated in ligand-free medium with 1 µM ONO-RS-082 for 15 min at 37 °C and then with a
second pulse-chase of FITC-Tf for 60 min followed by a 15-min chase in
ligand-free medium, all in the presence of 1 µM
ONO-RS-082. The colocalization of both Rhod-Tf (A) and
FITC-Tf (B) within centrally located vesicles of a single
cell is indicated by the arrows.
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Fig. 6.
PLA2 antagonists inhibit
BFA-stimulated endosome tubulation. In all experiments, HeLa cells
were incubated with FITC-Tf for 45 min at 37 °C for label endocytic
compartments (left panels), subsequently
incubated in the presence or absence of BFA and/or BEL, and processed
for immunofluorescence of TfRs (right panels).
A and B, control cells; C and
D, cells subsequently treated with BEL alone (10 µM) for 20 min; E and F, cells
subsequently treated with BFA (10 µg/ml) for 15 min; G and
H, cells subsequently treated with BEL (10 µM)
and BFA (10 µg/ml) for 15 min.
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Fig. 7.
Electron microscopic observations of
PLA2 inhibitor effect on BFA-stimulated endosome
tubulation. Clone 9 cells were incubated with soluble HRP for 45 min at 37 °C to label endosomes, washed, and then incubated in
control medium for 15 min (A), BFA (10 µg/ml) for 10 min
at 37 °C (B), or ONO-RS-082 (25 µM) for 5 min followed by BFA plus ONO-RS-082 for 10 min (C). Cells
were then fixed and processed for DAB cytochemistry and
electron microscopy. In control cells (A),
HRP-labeled endosomes (endo with short
arrows) are primarily spherical, 200-300-nm diameter
organelles. Treatment with BFA (B) results in the formation
of numerous tubular endosomes, 50-70 nm in diameter and up to several
µm in length (endo with long
arrows). Treatment with ONO-RS-082 (C) inhibited
the formation of BFA-induced endosomal tubules. n, nucleus;
bars, 1 µm.
PLA2 antagonists inhibit BFA-stimulated endosome tubulation in
vivo
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Fig. 8.
Low concentrations of PLA2
antagonists inhibit tubulation of membrane in the central recycling
compartment. HeLa cells were incubated with FITC-Tf in the
continuous presence of 1 µM ONO-RS-082 for 45 min at
37 °C and then 1) rapidly washed free of the antagonist and
subsequently incubated in BFA (5 µg/ml) for various periods of time
at 37 °C or 2) incubated in BFA in the continuous presence of 1 µM ONO-RS-082 for various periods of time at 37 °C.
Cells were then fixed and viewed by fluorescence microscopy. % Tubulation represents the percentage of total cells with
tubules emanating from the central recycling compartment (mean
plus 1 S.D. from four independent samples).
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Fig. 9.
Morphological observations of endosome
tubules in vivo and in vitro.
A-C, rat clone 9 hepatocytes were incubated with HRP (15 min at 37 °C) and processed for DAB cytochemistry and thin section
EM. HRP-labeled endosomes display membrane tubules, 50-60 nm in
diameter and varying lengths. D-H, endosomes isolated from
clone 9 cells that had been allowed to endocytose Au12-BSA
particles were prepared and visualized by electron microscopy
negative staining. Endosomes were identified by the presence of
endocytosed Au12-BSA particles (arrowheads in
all panels). Due to the sparse surface density of
endosomes on the electron microscopy grids, representative
endosomes from various experimental conditions are shown. Endosomes
incubated in the presence of bovine brain cytosol alone (D)
or BSA plus ATP and GTP (E) were primarily vesicular or
vacuolar with few tubular extensions. Incubation of endosomes with
bovine brain cytosol plus ATP, however, resulted in the formation of
multiple, short tubules (F) or few long tubules
(G and H). In F, an endosome with
tubules of varying lengths is shown (arrows), and all have
the same morphology; as tubules protrude from the vacuolar membrane,
they taper into a narrow shaft, 50-70 nm in diameter, and terminate in
a bulbous end. The tubules maintain this morphology after reaching
lengths of >0.5 µm, which is similar to that of tubules seen
in vivo (A-C). Standard incubation conditions
were 30 min at 37 °C. Bars, 0.25 µm.
S (0.1 mM) did not support
tubulation (data not shown). Also, because the tubulation mixture
contained EDTA (0.5 mM), and the addition of excess
Ca2+ had no effect on tubulation (data not shown), we
conclude that Ca2+ is not required for this activity. A
time course of tubulation using the complete mixture of BBC (1.5 mg/ml)
and ATP (0.1 mM) at 37 °C showed that tubulation was
fairly rapid, with a t = 4 min (Fig.
10B). In addition, a cytosol concentration curve determined that tubulation was a saturable process at ~1.5 mg/ml, with
half-maximal activity at 0.15 mg/ml (Fig. 10C).
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Fig. 10.
Quantitation of in vitro
endosome tubule formation. A, isolated endosomes,
loaded by prior uptake with Au12-BSA, were incubated under
various conditions as indicated for 30 min at 37 °C and then
visualized by electron microscopy negative staining. The
concentrations of the various reagents present in the incubations were
as follows: 1.5 mg/ml bovine serum albumin (BSA), 1.5 mg/ml bovine
brain cytosol (BBC), 0.1 mM ATP. Heated refers
to BBC that was heated to 100 °C for 30 min before assaying.
B, time course of endosome tubulation in vitro.
Endosomes were incubated in the presence of bovine brain cytosol (1.5 mg/ml) and ATP (0.1 mM) for various times at 37 °C.
Endosome tubulation was fairly rapid, with t = 4 min under these conditions. C, cytosol concentration
dependence of endosome tubulation in vitro. Endosomes were
incubated with varying concentrations of bovine brain cytosol in the
presence of ATP (0.1 mM) for 30 min at 37 °C.
D, characterization of tubulation activity in bovine brain
cytosol. Isolated endosomes were incubated under various conditions as
indicated, in the presence of ATP (0.1 mM) for 30 min at
37 °C. Crude refers to the organelle free extract
resulting after bovine brains were homogenized and centrifuged at high
speed. BBC, our standard cytosol preparation of material
present after precipitation with 60% ammonium sulfate followed by
extensive dialysis in dialysis buffer. The concentrations of various
reagents present in the incubations were as follows: 1.5 mg/ml crude
cytosol or BBC, 0.1 mM ATP, 10 µg/ml BFA, 1 mM N-ethylmaleimide (NEM). The
percentage of tubulation on the y axis is the percentage of
gold-loaded endosomes with tubular extensions (determined as described
under "Experimental Procedures"). Error bars,
1 S.D. determined from at least three independent experiments.
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Fig. 11.
PLA2 antagonists inhibit
endosome tubulation in vitro. The effects of
various PLA2 antagonists were examined in the standard
in vitro tubulation assay using endosomes from RAW 264.7 macrophages. ONO, ONO-RS-082;
svPLA2, snake venom PLA2. The
concentrations of antagonists (in µM) are shown next to
each antagonist. Results are expressed as the percentage of control
(the level achieved with BBC alone) and represent the mean plus
1 S.D. from four samples.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
S
and did not appear to be coated by spirals of dynamin polymers.
Therefore, the two mechanisms appear to be quite different.
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence should be addressed. Tel.: 607-255-2444;
Fax: 607-255-2428; E-mail: wjb5@cornell.edu.
ABBREVIATIONS
,-imino)triphosphate;
GTPS, guanosine
5'-3-O-(thio)triphosphate.
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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