From the Departments of Pediatrics, § Cell
Biology, and ¶¶ Medicine, ¶ Division of Rheumatology,
Hospital for Sick Children, Institute of Medical Science, and
University Health Network, University of Toronto, Toronto, Ontario
M5G 1X8, Canada, ** Department of Immunology, University of
Washington, Seattle, Washington 98195,
Department of Medicine, University of
Pennsylvania School of Medicine, Philadelphia, Pennsylvania
19104-4283, and §§ Institute for Cancer
Research, The Norwegian Radium Hospital, Montebello, 0310 Oslo,
Norway
Received for publication, August 5, 2002, and in revised form, November 4, 2002
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ABSTRACT |
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Clustering of macrophage Fc Receptors on the surface of phagocytes are essential in the
recognition and elimination of microorganisms. Some phagocytic receptors interact directly with the pathogens, by recognizing motifs
that are conserved on the surface of various types of microorganisms (1, 2). A second group of receptors interact instead with opsonins,
host molecules such as antibodies, or complement components that coat
the surface of the microorganisms (3). The latter class includes Fc In phagocytes the mode of internalization of Fc Even though phagocytosis is thought to be driven by remodeling of
actin, assembly of clathrin and of the associated proteins dynamin and
amphiphysin has been reported to occur at sites of phagocytosis
(14-16). The purpose of this clathrin recruitment may be to drive the
budding and fission of vesicles from the nascent phagosomes,
contributing to their maturation process. In this regard, it is
noteworthy that phagosome maturation is akin to the progression of the
endocytic pathway and that clathrin assembles on endocytic vesicles and
is thought to contribute to the maturation of endosomes (17, 18). To
date, however, the role of clathrin-coated vesicles in phagosome
maturation has not been studied. The paucity of information likely
stems from the fact that, unexpectedly, interference with the formation
and/or detachment of the clathrin coat resulted in inhibition of
phagocytosis. Thus, impairment of the function of clathrin (19),
dynamin II (15), or amphiphysin II (16) all markedly depressed the
phagocytic efficiency.
Clathrin-dependent membrane budding and fission are also
essential for the secretory and recycling pathways (20, 21). This
raises the possibility that the seemingly paradoxical effects of
clathrin impairment on phagocytosis may be indirect, caused by
depletion of plasmalemmal components that are required for the
phagocytic process. To address this possibility and to analyze the role
of clathrin in phagosome maturation, we used an isoform of dynamin that
is predominantly localized to the plasma membrane. Three isoforms of
dynamin have been described. Dynamin I is abundant in the brain, and
dynamin III is abundant in testis, lung, and neurons (22), whereas
dynamin II is ubiquitous. Importantly, whereas dynamin II is found both
in endomembranes and at the plasma membrane, dynamin I is predominantly
plasmalemmal (23). We reasoned that transfection of cells with a
dominant-negative form of dynamin I was less likely to result in an
indirect inhibition of phagocytosis and would enable us to compare the
requirement of clathrin in endocytosis versus phagocytosis
and to assess the possible role of clathrin-dependent
budding in phagosomal maturation. In addition, we used antisense
technology to depress the level of clathrin. By controlling the
expression of the heavy chain using an inducible vector, we were able
to assess the role of clathrin in Fc Reagents and Antibodies--
Dulbecco's modified Eagle's
medium and fetal bovine serum were from Wisent. Sheep red blood cells
(RBC)1 and rabbit anti-sheep
RBC antibodies were from ICN Cappel (Aurora, OH). Rhodamine-conjugated
human transferrin (Tf) and rhodamine-conjugated dextran were from
Molecular Probes (Eugene, OR), and monoclonal anti-Tf receptor antibody
was from Zymed Laboratories Inc. (San Francisco, CA).
Cytochalasin D was from Calbiochem (San Diego, CA). Rat anti-mouse
(ID4B) LAMP1 antibodies were from the Developmental Studies Hybridoma
Bank, maintained by the University of Iowa and Johns Hopkins
University. Cy3-conjugated anti-mouse, anti-rat, and anti-human
antibodies were from Jackson ImmunoResearch Laboratories, Inc. (West
Grove, PA). Monoclonal anti-mouse CD18 (integrin Cell Culture and Transfection--
RAW 264.7 murine macrophages
obtained from the American Type Culture Collection (Manassas,
VA) were maintained in Dulbecco's modified Eagle's medium with
10% fetal bovine serum. Cells were grown at 37 °C in a humidified
atmosphere of 95% air and 5% carbon dioxide. Transient transfection
of RAW 264.7 cells with clathrin-GFP, dynamin (Dyn) I (K44A), Dyn II
(K44A), Dyn I (S45N), wild-type (WT) dynamin, and Rab5(S34N)-GFP, a
GFP-labeled dominant-negative (DN) form of Rab5, was performed
utilizing FuGENE 6 (Roche Molecular Biochemicals) according to the
manufacturer's protocol. Cells were used 24 h after transfection
of clathrin and DN-Rab5 and 48 h after dynamin transfection.
BHK21-tTA cells transfected with antisense clathrin heavy chain
(24-26) were grown in Dulbecco's modified Eagle's medium with 10%
fetal bovine serum, 2 mM L-glutamine, 1 µg/ml
puromycin, 0.2 mg/ml geneticin, and 2 µg/ml tetracycline. Cells were
used 48 h after inducing the expression of the antisense mRNA
by removing tetracycline from the medium.
EGFP-clathrin light chain cDNA (27) was kindly provided by Dr.
J. H. Keen (Kimmel Cancer Institute and Thomas Jefferson University, Philadelphia, PA). Plasmids encoding Dyn I K44A, Dyn S45N,
and Dyn WT were generously provided by Dr. S. L. Schmid (Department of Cell Biology, Scripps Research Institute, La Jolla, CA).
Dyn II K44A cDNA was prepared as described (16). Rab5(S34N)-GFP was
kindly provided by Dr. C. Bucci (Universita degli Studi de Lecce,
Lecce, Italy).
Receptor-mediated Endocytosis Assay--
Endocytosis was
assessed by quantifying the uptake of Tf or aggregated human IgG. Prior
to Tf uptake determinations RAW 264.7 cells grown on 25-mm coverslips
were pre-incubated in serum-free medium for 1 h. Rhodamine-Tf (50 µg/ml) was added, and the cells were incubated at 37 °C for 30 min. Tf bound extracellularly was removed by washing cells sequentially
in media of pH 5.5 and pH 7.4.
Human IgG (10 mg/ml) was aggregated by heating to 62 °C for 20 min.
Insoluble complexes were sedimented, and 1 mg/ml of soluble aggregated
IgG was added to the cells, which were incubated at 37 °C for 30 min. Subsequently, cells were washed in phosphate-buffered saline
(PBS), fixed in 4% paraformaldehyde for 1 h, and permeabilized with 0.1% Triton X-100 in PBS. Internalized IgG complexes were identified by incubating cells with anti-human Cy3-conjugated antibodies (1:1000).
Phagocytosis Assay--
Sheep RBC were opsonized with rabbit
anti-sheep RBC antibody (1:50) and latex beads with 1 mg/ml human IgG
at 37 °C for 1 h. Opsonized RBC or latex beads were added to
RAW 264.7 macrophages grown on coverslips (30-50 RBC or beads per RAW
264.7 cell) and incubated at 37 °C for 30 min. External RBC were
lysed by hypotonic shock with distilled water, lysis was terminated by
addition of 10× PBS, and the cells were washed and resuspended in
Hepes-buffered RPMI 1640 medium. Unbound particles were washed, and
external beads were labeled in the cold with Cy3-conjugated anti-human antibody. Cells were fixed and permeabilized, and the beads were labeled with Cy5-conjugated anti-human antibody. Those beads that were
Cy5-positive but Cy3-negative were considered to be internalized.
Fluid Phase Assay--
RAW 264.7 cells were incubated with
rhodamine-conjugated dextran (500 µg/ml) at 37 °C for 30 min,
washed with PBS, and either visualized immediately or fixed in 4%
paraformaldehyde for 1 h prior to visualization.
Assessment of Phagosomal Maturation--
Phagocytosis of
opsonized RBC by RAW 264.7 cells were induced as described above. After
5 min at 37 °C the external RBC were lysed, and maturation of the
phagosomes containing internalized RBC were allowed to proceed for
1 h. Cells were fixed in 4% paraformaldehyde for 1 h
followed by permeabilization and blocking in PBS with 0.1% Triton
X-100 and 5% milk. The cells were next immunostained with anti-human
Tf receptor (1:500), anti-mouse CD18 (1:500), or anti-mouse LAMP1 (1:4)
antibodies. Cy3-conjugated secondary anti-mouse and anti-rat antibodies
were used as appropriate, at 1:1500 dilution.
Samples were analyzed using a Leica fluorescence microscope (model
DMIRB) with a ×100 oil immersion objective and appropriate filter
sets. All cells and phagosomes were identified under DIC optics. Images
were acquired digitally utilizing a cooled charge-coupled device camera
(Micromax; Princeton Instruments, Trenton, NJ) controlled by the
Winview software. Confocal microscopy was performed with a Zeiss LSM
510 laser scanning confocal microscope. The presence of Tf receptor,
CD18, or LAMP1 in the phagosomal membrane was scored in individual
phagosomes in an all-or-none basis.
Measurements and Statistical Analysis--
All experiments were
performed at least in triplicate, and data are expressed as means ± S.E. The significance of differences between means was assessed
utilizing Student's t test.
EGFP-Clathrin Expressed in RAW 264.7 Cells Localizes to Coated Pits
and Phagocytic Cups--
Upon binding to Fc
Next, we assessed whether clathrin is associated with phagosomes during
engulfment of IgG-opsonized particles. RAW 264.7 cells transiently
transfected with EGFP-clathrin were incubated with IgG-opsonized RBC
for 5 min and analyzed by fluorescence microscopy. In agreement with
the report by Aggeler and Werb (14), we were able to detect clathrin on
the membrane of nascent phagosomes (Fig. 1, D and
E).
We also analyzed whether dynamin I associated with the phagosomal
membrane. RAW 264.7 cells were transfected with epitope-tagged wild-type dynamin I and analyzed by confocal microscopy following immunostaining. It is noteworthy that, prior to phagocytosis, dynamin I
was associated with the plasma membrane but not with endomembranes (not
shown). Upon addition of opsonized particles, dynamin I accumulated
markedly in the phagosomal cup (Fig. 1F), resembling the
distribution of dynamin II reported by Gold et al. (16).
Effect of Dyn I K44A on Tf Endocytosis--
Severing
of clathrin-coated pits to form intracellular vesicles requires
functional dynamin (29-31). To assess the role of clathrin-dependent vesiculation in Fc
Fig. 2 illustrates the effect of Dyn I
K44A on receptor-mediated endocytosis in RAW 264.7 cells. Uptake of Tf
was greatly diminished in cells expressing the dominant-negative
construct, identified by expression of co-transfected EGFP (Fig. 2,
A Effect of Dyn I K44A on Fc Effect of Dyn I K44A on Fc
These results contrast sharply with those obtained earlier by Gold
et al. (16) using the equivalent dominant-negative form of
dynamin II (Dyn II K44A). To ensure that these results reflect distinct
properties of the two isoforms and not differences in the experimental
system we tested the effect of transient transfection of Dyn II K44A on
RAW 264.7 cells. Under conditions like those of Fig. 4, transiently
transfected Dyn II K44A inhibited phagocytosis of IgG-coated sheep RBC
by Effect of Clathrin Antisense mRNA on Endocytosis and
Phagocytosis--
In view of the apparent discrepancy between the data
obtained with dynamin I and II, we sought alternative ways of
assessing the involvement of the clathrin complex in
phagocytosis. To this end, we used a cell line in which the expression
level of the heavy chain of clathrin can be controlled by inducing the
expression of antisense mRNA (24-26). The BHK cells into which the
tetracycline-regulated expression of antisense mRNA was engineered,
however, are normally unable to internalize IgG-coated particles. This
problem was circumvented by transiently expressing GFP-tagged human
Fc
The clathrin dependence of phagocytosis was tested next. As shown in
Fig. 5, E and F, engulfment of IgG-coated beads
persisted in cells expressing clathrin antisense mRNA, under
conditions where endocytosis was virtually obliterated. No significant
difference in the phagocytic efficiency was noted in control
versus antisense-expressing cells (Fig. 5G).
These results are consistent with the observations made in cells
expressing Dyn I K44A and indicate that, although essential for Fc Effect of Dyn I K44A and Cytochalasin D on Phagocytosis of Small
and Large Beads--
The diametrically opposed effects of Dyn I K44A
on Fc Effect of DN-Rab5 on Endocytosis and Phagocytosis--
Unlike most
receptor-associated solutes, extracellular fluids are taken up by cells
by a pinocytic process that is independent of clathrin, yet exquisitely
sensitive to inhibitory forms of Rab5 (35, 36). We considered the
possibility that an analogous Rab5-dependent process could
contribute to small particle uptake. We therefore tested the effects of
a dominant-negative form of Rab5 on Fc
Rab5 mediates the stimulation of epidermal growth factor receptor by
its ligand (37). It is noteworthy that Fc Effect of Dyn I K44A on Phagosome Maturation--
The ability of
RAW 264.7 cells to engulf IgG-opsonized particles despite the
expression of dominant-negative dynamin I enabled us to analyze the
role of clathrin-dependent fission in phagosome maturation.
The transition between the early and intermediate stages of maturation
is marked by the removal of Tf receptors from the phagosomal membrane,
a process that in RAW cells is completed ~30 min after completion of
phagocytosis (38). We therefore analyzed the rate of depletion of Tf
receptors from phagosomes formed in cells expressing Dyn I K44A.
Inhibition of Tf endocytosis by Dyn I K44A, as reported in Fig. 2,
results in a concomitant accumulation of Tf receptors on the plasma
membrane of the transfected cells (Fig.
8, A
Similar results were obtained when we measured the traffic of another
plasmalemmal protein, CD18, a subunit of leukocyte integrins. In
otherwise untreated cells, CD18 was found to be incorporated into newly
formed phagosomes and was subsequently removed during the course of
phagosomal maturation, becoming undetectable by 40 min. Essentially
identical results were obtained in cells transfected with Dyn I K44A
(data not shown).
Effect of Dyn I K44A on Acquisition of LAMP-1--
During the
course of maturation, phagosomes sequentially fuse with early and late
endosomes and eventually with lysosomes. The late stages of the
maturation process are characterized by the acquisition of LAMP-1, a
marker of late endosomes and lysosomes. Because clathrin assembles on
endosomes (18) and is also important in the delivery of proteins to
prelysosomes by the trans-Golgi (39), we studied whether
recruitment of LAMP-1 to late phagosomes requires functional dynamin.
In untreated cells, LAMP-1 acquisition by phagosomes becomes apparent
by 10 min and proceeds to reach a plateau by ~30 min (Fig.
9). In Dyn I K44A-transfected cells the
course of LAMP-1 recruitment to the phagosomes was virtually identical
(Fig. 9D). Thus, the acquisition of LAMP-1 by phagosomes appears to be a dynamin I-independent process.
Dynamin has been reported to be an important mediator of budding
and scission of clathrin-coated structures such as endocytic pits
(29-31, 33) and those derived from the trans-Golgi network (40, 41). We found that endocytosis of aggregated IgG was virtually
eliminated by Dyn I K44A (Fig. 3), adding Fc Recently, Gold et al. (15) found that expression of
dominant-negative dynamin II inhibited phagocytosis. Although we were able to reproduce the latter results, we also found that inhibitory forms of dynamin I were without effect on phagocytosis, under conditions where clathrin-dependent endocytosis was
obliterated. To the extent that the two isoforms share a high degree of
homology, it is unlikely that dynamin is required for pinching and
scission of the phagosomal membrane. Indeed, Gold et al.
(15) reported that in cells expressing dominant-negative dynamin II
phagocytosis was arrested at an early stage, seemingly because of the
inability of the cells to extend pseudopodia. This phenotype resembles
the appearance of cells treated with wortmannin (12) and also of cells
with impaired COP-I function (38), which are similarly defective in
phagocytosis. Both of these conditions depress the delivery of
endomembranes to the plasmalemma, whether from the secretory or
recycling compartments. We therefore speculate that in cells expressing
dominant-negative dynamin II, interference with the normal delivery of
endomembranes to the surface or to a reserve pool may result in the
depletion of components that are essential for pseudopod elongation.
This effect is not observed in cells expressing inhibitory forms of
dynamin I, likely because the latter are restricted to the surface
membrane. Alternatively, dynamin II (but not dynamin I) may contribute
to the assembly and remodeling of actin at the phagosomal cup.
The differential sensitivity of endocytosis and phagocytosis to
clathrin antisense mRNA and to inhibitory forms of dynamin emphasizes the divergence in the underlying molecular mechanisms and
may reflect the differential requirement for ubiquitination. It has
recently become apparent that receptor-initiated ubiquitination can
trigger the recruitment of clathrin and the associated coating and
scission machinery (44). The involvement of clathrin in endocytosis is
therefore not unexpected, inasmuch as Fc Although uptake of aggregated IgG and large IgG-opsonized particles
occur by clearly different mechanisms, the distinction is not as
obvious in the case of smaller particles, particularly those in the
size range of most bacteria ( Although clathrin may not be involved directly in the particle
engulfment process, it is nevertheless recruited to the phagocytic cup,
albeit in modest amounts. This may simply be the result of frustrated endocytosis, triggered by receptor cross-linking yet unable to proceed because of the physical size of the opsonized particle. However, the recruitment of clathrin to the cup may have an
ulterior purpose, namely, to participate in the fission of vesicles
from the phagosome during maturation. Although we could not identify a
role for dynamin I in the recycling of Tf receptors or of CD18, or in
the acquisition of LAMP-1, it is nevertheless likely that
clathrin-coated vesicles are responsible for some aspect of phagosomal
maturation, which is an elaborate phenomenon.
It is noteworthy that Tf receptors are removed from the phagosomal
membrane by a process that is insensitive to Dyn I K44A, whereas their
budding and pinching from the plasma membrane is exquisitely sensitive
to this dominant-negative mutant. This implies that the processes
underlying the remodeling of the phagosomal membrane differ drastically
from those of the plasma membrane wherefrom it originated. Little is
currently known about the fission events that remove constituents from
the early phagosome and that maintain near constant phagosomal size
despite continued fusion of endomembranes throughout the maturation
process. Earlier findings in
receptors by multimeric immunoglobulin complexes leads to their
internalization. Formation of small aggregates leads to endocytosis,
whereas large particulate complexes induce phagocytosis. In RAW-264.7
macrophages, Fc
receptor endocytosis was found to be dependent on
clathrin and dynamin and insensitive to cytochalasin. Clathrin also
associates with nascent phagosomes, and earlier observations suggested
that it plays an essential role in phagosome formation. However, we
find that phagocytosis of IgG-coated large (
3 µm) particles was
unaffected by inhibition of dynamin or by reducing the expression of
clathrin using antisense mRNA but was eliminated by cytochalasin,
implying a distinct mechanism dependent on actin assembly. The uptake
of smaller particles (
1 µm) was only partially blocked by
cytochalasin. Remarkably, the cytochalasin-resistant component was also
insensitive to dominant-negative dynamin I and to clathrin antisense
mRNA, implying the existence of a third internalization mechanism,
independent of actin, dynamin, and clathrin. The uptake of small
particles occurred by a process distinct from fluid phase pinocytosis,
because it was not inhibited by dominant-negative Rab5. The
insensitivity of phagocytosis to dominant-negative dynamin I enabled us
to test the role of dynamin in phagosomal maturation. Although
internalization of receptors from the plasma membrane was virtually
eliminated by the K44A and S45N mutants of dynamin I, clearance of
transferrin receptors and of CD18 from maturing phagosomes was
unaffected by these mutants. This implies that removal of receptors
from the phagosomal membrane occurs by a mechanism that is different from the one mediating internalization of the same receptors at the
plasma membrane. These results imply that, contrary to prevailing notions, normal dynamin and clathrin function is not required for
phagocytosis and reveal the existence of a component of phagocytosis that is independent of actin and Rab5.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
receptors, which bind to the constant region of IgG. Fc
receptors are involved in the recognition and elimination of IgG-coated
microbes, erythrocytes, and platelets and in the clearance of
IgG-containing immune complexes, thereby playing a role in antigen
presentation (4, 5). Abnormal function or regulation of Fc
receptors
may lead to the ineffective clearance of infectious organisms or can
cause immunohematologic and autoimmune disorders (4).
receptors depends on
the size of the opsonized complex. Small immune complexes and soluble
aggregated IgG are internalized through receptor-mediated endocytosis
(6-8). In contrast, the internalization of IgG-opsonized particles
occurs via phagocytosis (9). Although cross-linking of Fc
receptors
triggers both internalization processes, the molecular mechanisms
underlying phagocytosis and endocytosis differ markedly. Endocytosis
requires assembly of clathrin at the site of receptor clustering, yet
is not strictly dependent on the cytoskeleton and does not require
class I phosphatidylinositol 3-kinase activity (10). In
contrast, phagocytosis involves acute assembly of F-actin and is
blocked by inhibitors of class I phosphatidylinositol 3-kinase, such as
wortmannin (11, 12). A recent study further highlighted the mechanistic
differences between the two modes of Fc
receptor internalization
(13); endocytosis, but not phagocytosis, was found to require
receptor-induced ubiquitination.
receptor-mediated endocytosis
and phagocytosis with minimal nonspecific damage to the cells.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2 chain) antibody was from BD Biosciences. Human IgG was purchased from Sigma.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
receptors,
IgG-containing immune complexes are internalized and travel along the
endocytic pathway where they are targeted for degradation. This is part
of a key process involved in antigen processing and presentation (6,
7). Transient expression of a fusion of the light chain of clathrin with EGFP was used to examine whether clathrin is involved in the
receptor-mediated endocytosis of aggregated IgG by RAW 264.7 cells. As
illustrated in Fig. 1, A
C,
EGFP-clathrin was found to colocalize with some, but not all, of the
immune complexes undergoing internalization. The partial colocalization
may be attributed to asynchrony of the internalization process, or to the co-existence of clathrin-dependent and
clathrin-independent uptake pathways. It is noteworthy that another
immunoreceptor, the interleukin 2 receptor, is internalized primarily
via clathrin-independent endocytosis (28).
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Fig. 1.
Localization of EGFP-clathrin during
endocytosis and phagocytosis. RAW 264.7 macrophages were
transiently transfected with EGFP-clathrin 24 h before analysis.
A C, endocytosis was initiated by addition of aggregated
human IgG. After 10 min, the cells were fixed, permeabilized, and
stained with Cy3-conjugated anti-IgG and finally analyzed by confocal
microscopy. A, green (clathrin-EGFP) fluorescence;
B, red (Cy3-stained IgG) fluorescence; C, overlay
of A and B. Arrows indicate sites of
colocalization. D and E, phagocytosis
was initiated by addition of IgG-opsonized RBC. After 10 min, the cells
were fixed and analyzed by DIC (D) or by fluorescence
microscopy to detect clathrin-EGFP (E). Arrows
point to clathrin on phagocytic cups. Images are representative of
three similar experiments. F, localization of dynamin I
during phagocytosis was carried out in RAW 264.7 cells transfected with
hemagglutinin-tagged wild-type dynamin I. Following phagocytosis of
IgG-opsonized RBC for 5 min, the cells were fixed, permeabilized, and
stained with fluorescein isothiocyanate-conjugated anti-hemagglutinin
and analyzed by DIC (inset) or fluorescence confocal
microscopy.
receptor-mediated
endocytosis and phagocytosis, we transfected cells with an inactive
form of dynamin I (Dyn I K44A), which has been reported to exert a
dominant-negative effect (29, 31). To identify the transfected cells,
Dyn I K44A was cotransfected with soluble EGFP (10:1 cDNA ratio).
Because Dyn I K44A was tagged with the hemagglutinin epitope, we were able to verify the effectiveness of co-transfection (>90%) and, in
addition, confirmed that the dominant-negative protein was largely
associated with the plasma membrane (not illustrated).
C). For these and all subsequent experiments, cells were
incubated 48 h post-transfection, because inhibition at this time
was greater than after 24 h (data not shown). The inhibition was
not a consequence of the transfection process per se,
because expression of wild-type dynamin I was without effect. Moreover,
expression of a second dynamin I mutant (Dyn I S45N), also reported to
be inhibitory (32), similarly obliterated Tf uptake (Fig.
2D). These findings are in good agreement with the effects
of dynamin I mutants in other cell types (29-31, 33).
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Fig. 2.
Effect of dominant-negative dynamin I on Tf
endocytosis. RAW 264.7 cells were transiently co-transfected with
wild-type or mutant dynamin and EGFP (10:1 cDNA ratio). After
48 h, the cells were allowed to internalize rhodamine-labeled Tf
(Tf-rhod) for 30 min before analysis. A, DIC
image; B, green (EGFP) fluorescence; C, red (Tf)
fluorescence. Images are representative of three similar experiments.
D, quantification of Tf uptake in untransfected cells
(Control) and in cells transfected with WT or
dominant-negative forms of dynamin I (Dyn K44A and
S45N). Ordinate, percent Tf-positive cells. Data
are means ± S.E. of three experiments. A minimum of 100 cells
were counted per experiment.
Receptor-mediated
Endocytosis--
The effect of dominant-negative dynamin I
on Fc
receptor-mediated endocytosis was studied next (Fig.
3). RAW 264.7 cells were transfected with
Dyn I K44A, and their ability to internalize aggregated IgG was
quantified. As before, transfected cells were identified by
co-expression of EGFP (Fig. 3B), and uptake of IgG was
determined following permeabilization and addition of labeled anti-IgG
antibody (Fig. 3C). As summarized in Fig. 3D,
inhibition of dynamin function reduced IgG uptake by >80%.
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Fig. 3.
Effect of dominant-negative dynamin I on
endocytosis of aggregated IgG. RAW 264.7 cells were transiently
co-transfected with Dyn I K44A and EGFP (10:1 cDNA ratio). After
48 h, the cells were allowed to internalize aggregated IgG for 30 min before fixation. After permeabilization, IgG was stained using
Cy3-conjugated secondary antibodies. A, DIC image;
B, green (EGFP) fluorescence; C, red (IgG)
fluorescence. Images are representative of three similar experiments.
D, quantification of IgG uptake in untransfected cells
(Control) and in cells transfected with dominant-negative
dynamin I (Dyn K44A). Ordinate, percent
IgG-positive cells. Data are means ± S.E. of three experiments. A
minimum of 100 cells were counted per experiment.
Receptor-mediated Phagocytosis of Red
Cells--
We next tested the effect of Dyn I K44A on phagocytosis. We
anticipated that the preferential association of this isoform with the
plasma membrane would resolve whether the reported inhibitory effects
of dynamin II and other clathrin-disrupting maneuvers reflect a direct
role of plasmalemmal clathrin in phagocytosis or are indirect (see
Introduction). As shown in Fig.
4A, phagocytosis was scored by
DIC microscopy following hypotonic lysis of adherent extracellular RBC.
In the transfected cells, phagocytosis was also apparent from the
exclusion of EGFP from areas of the cytosol (Fig. 4B).
Remarkably, Dyn I K44A had only a modest, statistically insignificant
effect on phagocytosis, which was not different from that of the
wild-type protein (Fig. 4C). Similar negative results were
obtained using Dyn I S45N (Fig. 4C), under conditions where
IgG endocytosis was largely eliminated.
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Fig. 4.
Effect of dominant-negative dynamin I on
phagocytosis. RAW 264.7 cells were transiently co-transfected with
wild-type or mutant dynamin and EGFP (10:1 cDNA ratio). After
48 h, the cells were allowed to internalize IgG-opsonized RBC for
30 min before analysis. A, DIC image; B, green
(EGFP) fluorescence; C, quantification of the phagocytic
efficiency in untransfected cells (Control) and in cells
transfected with WT or dominant-negative forms of dynamin I (Dyn
K44A and S45N). Ordinate, phagocytic index
(RBC per 100 macrophages). Data are means ± S.E. of three
experiments. A minimum of 100 cells were counted per experiment.
NS, p > 0.05.
85%.
RIIA receptors in these cells, 24 h prior to the assay. Prior
to stimulation, these receptors are largely localized to the
plasmalemma, with a fraction found in juxtanuclear recycling endosomes
(not shown). As shown in Fig. 5,
A and B, addition of aggregated IgG displaced
most of the receptors to an intracellular compartment, where they
co-localized with the ligand. Partial depletion of the heavy chain of
clathrin by expression of antisense mRNA resulted in a pronounced
(
90%) inhibition of endocytosis of aggregated IgG and prevented the internalization of plasmalemmal receptors (Fig. 5, C,
D, and G).
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Fig. 5.
Effect of clathrin antisense mRNA on
endocytosis and phagocytosis. BHK21-tTA cells transfected with
antisense clathrin heavy chain were incubated in the presence or
absence of tetracycline, conditions that prevent (A and
B) or induce (C F) the expression of the
antisense mRNA, respectively. After 24 h, the cells were
transiently transfected with Fc
RIIA-GFP, and after a further 24 h they were assayed for endocytosis of aggregated IgG (A
D)
or phagocytosis of IgG-opsonized beads (E and F).
A
D, measurement of endocytosis of aggregated IgG. Cells
were allowed to internalize soluble aggregated IgG for 30 min and were
fixed. Surface-adherent IgG was labeled with Cy3-conjugated anti-IgG
antibodies in the cold. The cells were then permeabilized, and total
IgG was stained using Cy5-conjugated antibodies. In A and
C, total IgG (blue) and Fc
receptor
(green) are identified. In B and D,
extracellular adherent IgG (pink) and total IgG
(blue) are identified. Corresponding DIC images are shown in
the insets. E and F, measurement of
phagocytosis of 3.1 µm (E) and 0.8 µm (F)
beads. Cells were allowed to internalize opsonized beads for 30 min and
were fixed. Extracellularly exposed beads were labeled with
Cy3-conjugated anti-IgG antibodies in the cold (pink). The
cells were then permeabilized, and all the beads were stained using
Cy5-conjugated antibodies (blue). Corresponding DIC and
fluorescence images were overlaid. G, quantification of the
uptake of soluble aggregated IgG and IgG-coated polystyrene beads in
normal (open bars) or clathrin-depleted (solid
bars) cells. Ordinate, uptake of soluble aggregated IgG
or IgG-coated beads, normalized relative to clathrin-expressing
control. Data are means ± S.E. of three experiments. A minimum of
100 cells were counted per experiment. CHC, clathrin heavy
chain.
receptor endocytosis, normal clathrin/dynamin function is not required
for phagocytosis.
receptor-mediated endocytosis and phagocytosis suggests vastly
different underlying processes, a notion supported by the effects of
cytochalasin. This antagonist of actin assembly precludes phagocytosis
of opsonized RBC but has no discernible effect on endocytosis (not
illustrated). It is of interest, however, that the sensitivity of
phagocytosis to cytochalasins has been reported to vary directly with
the size of the particle (34). Particles
1 µm in diameter can be
internalized effectively in the presence of the actin antagonist. This
range is of particular importance, as it encompasses the size of most pathogenic bacteria. The sensitivity of endocytosis to Dyn I K44A suggests that the actin-independent uptake of small particles may be
mediated by clathrin/dynamin. To test this notion, we compared the
effects of cytochalasin D and Dyn I K44A on the uptake of small (0.8 µm) and large (3.1 µm) IgG-opsonized latex particles. Particle
internalization was scored by immunolabeling the opsonizing IgG before
and after permeabilization of the cells. In accordance with the results
obtained using RBC, phagocytosis of large beads was unaffected by Dyn I
K44A (Fig. 6, A and
E). Conversely, cytochalasin D inhibited the uptake of large
beads completely, and no further effect was seen by expression of Dyn I
K44A (Fig. 6, B and E). As reported earlier (34),
we found that cytochalasin had only a partial (~40%)
inhibitory effect on the phagocytosis of small (0.8 µm) beads (Fig.
6, D and E). Unexpectedly, expression of Dyn I
K44A had no significant effect on the uptake of small particles and
only marginally enhanced the inhibition induced by cytochalasin (Fig.
6, C
E). In accordance with this observation, we also found that the uptake of small beads was essentially unaffected by clathrin antisense mRNA (Fig. 5, F and G). Jointly,
these observations imply that small particles are ingested, in part, by
a mechanism that differs from that used by both large particles and
immune complexes.
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Fig. 6.
Effect of dominant-negative dynamin I and
cytochalasin D on the phagocytosis of small and large latex beads.
RAW 264.7 cells were transiently co-transfected with mutant dynamin and
EGFP (10:1 cDNA ratio). After 48 h, the cells were allowed to
internalize either 3.1 µm (A and B) or 0.8 µm
(C and D) IgG-opsonized beads for 30 min before analysis.
Exposed beads were labeled with Cy3-conjugated anti-IgG antibodies in
the cold. The cells were then permeabilized, and all the beads were
stained using Cy5-conjugated anti-IgG antibodies.
A-D, main panels are DIC images overlaid with
fluorescence identifying extracellular adherent beads (pink)
and intracellular beads (blue). Insets are green (EGFP)
fluorescence, identifying transfected cells. In B and
D cells were treated with 10 µM cytochalasin
D. E, quantification of the phagocytic efficiency in
untransfected cells (Control) and in cells transfected with
dominant-negative dynamin I (Dyn K44A) in the presence or
absence of cytochalasin D. Left ordinate, number of beads
ingested per cell (black bars). Right ordinate,
relative proportion of ingested beads, normalized to control
(open bars). Data are means ± S.E. of three
experiments. A minimum of 100 cells were counted per experiment.
receptor-mediated
phagocytosis of small beads. To ensure the effectiveness of the
construct we also assessed the pinocytic uptake of the fluid phase
marker rhodamine-dextran. As shown in Fig.
7, A, B, and
G, expression of DN-Rab5 obliterated fluid phase uptake in
macrophages. In contrast, DN-Rab5 had no discernible effect on the
ingestion of small particles (Fig. 7, E
G). Therefore, the
actin- and dynamin-resistant ingestion of small IgG-coated particles is
not akin to pinocytosis.
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Fig. 7.
Effect of dominant-negative Rab5 on
endocytosis and phagocytosis. RAW 264.7 cells were transiently
transfected with DN-Rab5-GFP. After 24 h, expression of
DN-Rab5-GFP was confirmed by fluorescence microscopy (main panels in
A, C, and E), and endocytosis or
phagocytosis were assayed (B, D, and
F). Insets show corresponding DIC images.
A and B, measurement of fluid phase uptake. Cells
were allowed to ingest rhodamine-dextran for 30 min before
visualization of red fluorescence (B). C and
D, measurement of endocytosis of aggregated IgG. Cells were
allowed to internalize soluble aggregated IgG for 30 min and were
fixed. Surface-adherent IgG was labeled with Cy3-conjugated anti-IgG
antibodies in the cold. The cells were then permeabilized, and total
IgG was stained using Cy5-conjugated antibodies. In D
extracellular adherent IgG (pink) and intracellular IgG
(blue) are identified. E and F,
measurement of phagocytosis of 0.8-µm beads. Cells were allowed to
internalize opsonized beads for 30 min and were fixed. Exposed beads
were labeled with Cy3-conjugated anti-IgG antibodies in the cold. The
cells were then permeabilized, and all the beads were stained using
Cy5-conjugated antibodies. F, DIC image overlaid with
fluorescence, identifying extracellular adherent beads
(pink) and intracellular beads (blue).
G, quantification of the uptake of rhodamine-dextran,
soluble aggregated IgG, and IgG-coated 0.8-µm polystyrene beads in
cells transfected with DN-Rab5. Ordinate, uptake of
rhodamine-dextran, soluble aggregated IgG, or IgG-coated 0.8-µm
beads, normalized relative to untransfected control. Where specified
the cells were transfected with DN-Rab5. Data are means ± S.E. of
three experiments. A minimum of 100 cells were counted per
experiment.
receptor endocytosis was
unaffected by DN-Rab5 (Fig. 7, C and D), implying a different mode of activation.
C), facilitating
detection of their rate of removal from the phagosomes. Despite the
overabundance of Tf receptors in the membrane of Dyn I K44A-transfected
cells, the receptors were rapidly cleared from the phagosomes shortly
after internalization (Fig. 8, G
I). Quantification of the
kinetics of removal from phagosomes showed that the half-life of Tf
receptors in the phagosomes of Dyn I K44A-expressing cells was 10 min
(Fig. 8J), which is similar to that we reported earlier for
untreated RAW 264.7 cells (38). Because the endocytosis of Tf was
demonstrably impaired in the plasma membrane of these cells, as
indicated by the accumulation of receptors at the plasmalemma, we
conclude that different processes mediate the budding and fission of Tf
receptor-containing vesicles from the surface and phagosomal
membranes.
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Fig. 8.
Effect of dominant-negative dynamin I on the
association of Tf receptors with phagosomes. RAW 264.7 cells were
transiently co-transfected with dynamin 1 K44A and EGFP (10:1 cDNA
ratio). After 48 h, the cells were either fixed (A C)
or were allowed to internalize IgG-opsonized RBC for 5 min and then
chased for either 5 (D-F) or 40 min
(G
I) before fixation. The cells were then permeabilized,
and the Tf receptors were immunostained as described under
"Experimental Procedures." A, D, and
G, DIC images; B, E, and H,
green (EGFP) fluorescence; C, F, and
I, red (Tf receptor) fluorescence. Images are representative
of three similar experiments. J, quantification of the
fraction of Tf receptor (TfR)-positive phagosomes as a
function of time after phagosome formation. Data are means ± S.E.
of three experiments. A minimum of 100 cells were counted for each time
point.
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Fig. 9.
Effect of dominant-negative dynamin I on
acquisition of LAMP-1 by phagosomes. RAW 264.7 cells were
transiently co-transfected with either WT or mutant (K44A) dynamin 1 and EGFP (10:1 cDNA ratio). After 48 h, the cells were allowed
to internalize IgG-opsonized RBC for 5 min and chased for 60 min. The
cells were then fixed, permeabilized, and immunostained for LAMP-1 as
described under "Experimental Procedures." A, DIC image;
B, green (EGFP) fluorescence; C, red (LAMP-1)
fluorescence. Images are representative of three similar experiments.
D, quantification of the fraction of LAMP-1-positive
phagosomes as a function of time after phagosome formation. Open
symbols and dotted line, wild-type dynamin. Solid
symbols and line, K44A dynamin. Data are means ± S.E. of three experiments. A minimum of 100 cells were counted for each
time point.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
receptor endocytosis to
the list of dynamin-dependent processes. Because dynamin is
intimately linked to clathrin-dependent endocytosis, this
process is also likely to mediate the internalization of immune
complexes. However, dynamin has also been implicated in the fission of
non-clathrin coated vesicles such as caveolae (42, 43). Two
observations argue against a role for caveolae in Fc
receptor
internalization. First, at least some of the sites of Fc
receptor
accumulation were decorated with clathrin (Fig. 1), and second,
extraction of plasmalemmal cholesterol using methylcyclodextrin (10 mM for 30 min) did not prevent the endocytosis of
aggregated IgG by RAW 264.7 cells (not illustrated). More importantly,
we found that partial depletion of clathrin using antisense mRNA virtually eliminated endocytosis of aggregated IgG. We therefore concluded that a clathrin- and dynamin-dependent process is
largely responsible for internalization of immune complexes.
receptor endocytosis
depends on ubiquitination. By contrast, phagocytosis of large particles
proceeds normally in ubiquitination-deficient cells (13). It remains to
be defined how Fc
receptor-induced endocytosis and phagocytosis, two
processes triggered by clustering of the same receptors, differ so
drastically at the molecular level. We speculate that the more intense
and prolonged stimulation elicited by large particles recruits
additional signaling pathways that are not activated during endocytosis.
1 µm). We confirmed earlier
observations (34) that such particles are only modestly sensitive to
cytochalasin but also found that, unlike immune complexes and soluble
aggregated IgG, small particle uptake was virtually unaffected by
inhibition of dynamin or by reduction of clathrin with antisense
mRNA. Of note, uptake of small particles also proceeded at near
normal rates in cells with impaired ubiquitination (not illustrated).
The existence of a cytochalasin- and clathrin/dynamin-insensitive fraction of ingestion implies that small particles can be internalized by a mechanism distinct from conventional clathrin-mediated endocytosis and actin-driven phagocytosis. We considered the possibility that a
process akin to pinocytosis may be involved. However, this mechanism appears unlikely, because the uptake of small beads was not affected by
DN-Rab5 under conditions where the uptake of a fluid phase marker was eliminated.
COP-deficient cells and in cells
treated with brefeldin A indicated that COP-1 plays a measurable yet
comparatively minor role in phagosome remodeling (38). To test the
cooperativity between the COP-I and clathrin pathways, we treated with
brefeldin A (2 µg/ml) cells that had been transfected with Dyn I
K44A. The expression of the dominant-negative dynamin did not
accentuate the effects of brefeldin on the removal of Tf receptors and
CD18, or on the acquisition of LAMP1 by the phagosomal membrane (data not shown). This implies that additional mechanisms operate during phagosome remodeling. Caveolae or other coating structures such as the
nexin-retromer complex (45) may be primarily responsible for vesicular
fission during phagosome maturation. Alternatively, clathrin-mediated
fission that is insensitive to Dyn I K44A may be taking place.
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ACKNOWLEDGEMENTS |
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We thank Dr. J. H. Keen (Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA) for advice and for providing clathrin light-chain cDNA. We also thank Dr. Otilia Vieira and Richard Collins for help.
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FOOTNOTES |
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* This work was supported in part by the Canadian Institutes of Health Research (CIHR), the Arthritis Society of Canada, the Arthritis Center of Excellence, and the Sanatorium Foundation.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 a CIHR fellowship, the Arthritis Society of
Canada, and the Arthritis Center of Excellence.
Current holder of the Pitblado Chair in Cell Biology at
The Hospital for Sick Children. Cross-appointed to the Department of
Biochemistry, University of Toronto. To whom correspondence should be
addressed: Dept. of Cell Biology, Hospital for Sick Children, 555 University Ave., Toronto, Ontario M5G 1X8, Canada. Tel.: 416-813-5727;
Fax: 416-813-5028; E-mail: sga@sickkids.ca.
Published, JBC Papers in Press, November 6, 2002, DOI 10.1074/jbc.M207966200
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ABBREVIATIONS |
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The abbreviations used are: RBC, red blood cells; DIC, differential interference contrast; Dyn, dynamin; PBS, phosphate-buffered saline; Tf, transferrin; GFP, green fluorescent protein; WT, wild-type; DN, dominant-negative; EGFP, enhanced GFP.
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REFERENCES |
---|
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---|
1. | Janeway, C. A., Jr. (1992) Immunol. Today 13, 11-16[CrossRef][Medline] [Order article via Infotrieve] |
2. | Stahl, P. D., and Ezekowitz, R. A. (1998) Curr. Opin. Immunol. 10, 50-55[CrossRef][Medline] [Order article via Infotrieve] |
3. | Daeron, M. (1997) Annu. Rev. Immunol. 15, 203-234[CrossRef][Medline] [Order article via Infotrieve] |
4. | Amigorena, S., and Bonnerot, C. (1999) Immunol. Rev. 172, 279-284[Medline] [Order article via Infotrieve] |
5. | Amigorena, S., and Bonnerot, C. (1999) Semin. Immunol. 11, 385-390[CrossRef][Medline] [Order article via Infotrieve] |
6. | Mellman, I. S., Plutner, H., Steinman, R. M., Unkeless, J. C., and Cohn, Z. A. (1983) J. Cell Biol. 96, 887-895[Abstract] |
7. | Mellman, I., and Plutner, H. (1984) J. Cell Biol. 98, 1170-1177[Abstract] |
8. | Hedin, U., Stenseth, K., and Thyberg, J. (1984) Eur. J. Cell Biol. 35, 41-48[Medline] [Order article via Infotrieve] |
9. | Aderem, A., and Underhill, D. M. (1999) Annu. Rev. Immunol. 17, 593-623[CrossRef][Medline] [Order article via Infotrieve] |
10. |
Mukherjee, S.,
Ghosh, R. N.,
and Maxfield, F. R.
(1997)
Physiol. Rev.
77,
759-803 |
11. | Araki, N., Johnson, M. T., and Swanson, J. A. (1996) J. Cell Biol. 135, 1249-1260[Abstract] |
12. |
Cox, D.,
Tseng, C. C.,
Bjekic, G.,
and Greenberg, S.
(1999)
J. Biol. Chem.
274,
1240-1247 |
13. |
Booth, J. W.,
Kim, M. K.,
Jankowski, A.,
Schreiber, A.,
and Grinstein, S.
(2002)
EMBO J.
21,
251-258 |
14. | Aggeler, J., and Werb, Z. (1982) J. Cell Biol. 94, 613-623[Abstract] |
15. | Gold, E. S., Morrissette, N. S., Underhill, D. M., Guo, J., Bassetti, M., and Aderem, A. (2000) Immunity 12, 285-292[Medline] [Order article via Infotrieve] |
16. |
Gold, E. S.,
Underhill, D. M.,
Morrissette, N. S.,
Guo, J.,
McNiven, M. A.,
and Aderem, A.
(1999)
J. Exp. Med.
190,
1849-1856 |
17. | Mellman, I. (1996) Annu. Rev. Cell Dev. Biol. 12, 575-625[CrossRef][Medline] [Order article via Infotrieve] |
18. | Stoorvogel, W., Oorschot, V., and Geuze, H. J. (1996) J. Cell Biol. 132, 21-33[Abstract] |
19. |
Perry, D. G.,
Daugherty, G. L.,
and Martin, W. J., II
(1999)
J. Immunol.
162,
380-386 |
20. | Schmid, S. L. (1997) Annu. Rev. Biochem. 66, 511-548[CrossRef][Medline] [Order article via Infotrieve] |
21. | Le Borgne, R., and Hoflack, B. (1998) Biochim Biophys Acta 1404, 195-209[Medline] [Order article via Infotrieve] |
22. |
Urrutia, R.,
Henley, J. R.,
Cook, T.,
and McNiven, M. A.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
377-384 |
23. |
Cao, H.,
Garcia, F.,
and McNiven, M. A.
(1998)
Mol. Biol. Cell
9,
2595-2609 |
24. |
Torgersen, M. L.,
Skretting, G.,
van Deurs, B.,
and Sandvig, K.
(2001)
J. Cell Sci.
114,
3737-3747 |
25. |
Llorente, A.,
Prydz, K.,
Sprangers, M.,
Skretting, G.,
Kolset, S. O.,
and Sandvig, K.
(2001)
J. Cell Sci.
114,
335-343 |
26. |
Iversen, T. G.,
Skretting, G.,
Llorente, A.,
Nicoziani, P.,
van Deurs, B.,
and Sandvig, K.
(2001)
Mol. Biol. Cell
12,
2099-2107 |
27. | Gaidarov, I., Santini, F., Warren, R. A., and Keen, J. H. (1999) Nat. Cell Biol. 1, 1-7[CrossRef][Medline] [Order article via Infotrieve] |
28. | Lamaze, C., Dujeancourt, A., Baba, T., Lo, C. G., Benmerah, A., and Dautry-Varsat, A. (2001) Mol. Cell 7, 661-671[Medline] [Order article via Infotrieve] |
29. | Damke, H., Baba, T., Warnock, D. E., and Schmid, S. L. (1994) J. Cell Biol. 127, 915-934[Abstract] |
30. | Herskovits, J. S., Burgess, C. C., Obar, R. A., and Vallee, R. B. (1993) J. Cell Biol. 122, 565-578[Abstract] |
31. | van der Bliek, A. M., Redelmeier, T. E., Damke, H., Tisdale, E. J., Meyerowitz, E. M., and Schmid, S. L. (1993) J. Cell Biol. 122, 553-563[Abstract] |
32. |
Damke, H.,
Binns, D. D.,
Ueda, H.,
Schmid, S. L.,
and Baba, T.
(2001)
Mol. Biol. Cell
12,
2578-2589 |
33. | Damke, H., Baba, T., van der Bliek, A. M., and Schmid, S. L. (1995) J. Cell Biol. 131, 69-80[Abstract] |
34. | Koval, M., Preiter, K., Adles, C., Stahl, P. D., and Steinberg, T. H. (1998) Exp. Cell Res. 242, 265-273[CrossRef][Medline] [Order article via Infotrieve] |
35. |
Xiao, G. H.,
Shoarinejad, F.,
Jin, F.,
Golemis, E. A.,
and Yeung, R. S.
(1997)
J. Biol. Chem.
272,
6097-6100 |
36. |
Mukhopadhyay, A.,
Barbieri, A. M.,
Funato, K.,
Roberts, R.,
and Stahl, P. D.
(1997)
J. Cell Biol.
136,
1227-1237 |
37. | Tall, G. G., Barbieri, M. A., Stahl, P. D., and Horazdovsky, B. F. (2001) Dev. Cell 1, 73-82[Medline] [Order article via Infotrieve] |
38. |
Botelho, R. J.,
Hackam, D. J.,
Schreiber, A. D.,
and Grinstein, S.
(2000)
J. Biol. Chem.
275,
15717-15727 |
39. | Traub, L. M., and Kornfeld, S. (1997) Curr. Opin. Cell Biol. 9, 527-533[CrossRef][Medline] [Order article via Infotrieve] |
40. |
Jones, S. M.,
Howell, K. E.,
Henley, J. R.,
Cao, H.,
and McNiven, M. A.
(1998)
Science
279,
573-577 |
41. |
Cao, H.,
Thompson, H. M.,
Krueger, E. W.,
and McNiven, M. A.
(2000)
J. Cell Sci.
113,
1993-2002 |
42. |
Oh, P.,
McIntosh, D. P.,
and Schnitzer, J. E.
(1998)
J. Cell Biol.
141,
101-114 |
43. |
Henley, J. R.,
Krueger, E. W.,
Oswald, B. J.,
and McNiven, M. A.
(1998)
J. Cell Biol.
141,
85-99 |
44. |
van Kerkhof, P.,
Sachse, M.,
Klumperman, J.,
and Strous, G. J.
(2001)
J. Biol. Chem.
276,
3778-3784 |
45. |
Sato, T. K.,
Overduin, M.,
and Emr, S. D.
(2001)
Science
294,
1881-1885 |