1 Marion Bessin Liver Research Center
2 Department of Anatomy and Structural Biology, Albert Einstein College of
Medicine, Bronx, NY 10461, USA
* Author for correspondence (e-mail: wolkoff{at}aecom.yu.edu)
Accepted 14 March 2003
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Summary |
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Key words: Endocytosis, Microtubules, Rabs, Kinesin, Motility
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Introduction |
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Studies by other laboratories in different endocytic systems have
implicated several Rab proteins as regulatory factors
(Mohrmann and van der Sluijs,
1999; Rodman and
Wandinger-Ness, 2000
; Zerial
and McBride, 2001
). In the present study, using immunofluorescence
microscopy, image analysis, and biochemical assays, we provide evidence that
Rab4 in the hepatocyte ASOR/asialoglycoprotein receptor (ASGPR) system is a
regulator of early endocytic vesicle processing, serving as a `molecular
switch' (Zerial and McBride,
2001
) that, in its GTP-bound state, interacts with
vesicle-associated proteins to inhibit vesicle motility. Addition of GDP
converts Rab4 to its GDP-bound state and results in activation of motility and
vesicle fission, corresponding to increased ligand-receptor vesicular
sorting.
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Materials and Methods |
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Assay of early endocytic vesicle motility and fission in vitro
Texas-Red-labeled early endocytic vesicles were prepared from livers that
were removed from 200-250 g male Sprague-Dawley rats (Taconic Farms,
Germantown, NY) 5 minutes after portal venous injection of Texas-Red-labeled
ASOR (Bananis et al., 2000;
Murray et al., 2000
). All
animal procedures were approved by the University Animal Use Committee. After
Dounce homogenization, a postnuclear supernatant was prepared and
chromatographed on a Sephacryl S200 column. Vesicle-enriched peaks were pooled
and subjected to centrifugation (200,000 g for 135 minutes) on
a sucrose step gradient consisting of 1.4, 1.2 and 0.25 M sucrose. Vesicles
were harvested from the 1.2 M/0.25 M sucrose interface and stored at -80°C
until used. Details of these procedures have been published recently
(Murray et al., 2000
). Three
vesicle isolations were used for the majority of the data presented.
Significant variation between isolations was not observed. Motility assays
were performed in a chamber consisting of two pieces of double-sided tape
sandwiched between optical glass; the internal volume was approximately 3
µl. The chamber was coated with 0.02 mg/ml DEAE-dextran (Amersham Pharmacia
Biotech, Piscataway, NJ) and rhodamine-labeled, Taxol-stabilized microtubules
(MTs) were added and incubated for 3 minutes
(Bananis et al., 2000
).
Following three washes with PMEE motility buffer (35 mM Pipes, 5 mM
MgCl2, 1 mM EGTA, 0.5 mM EDTA, 4 mM DTT, 20 µM Taxol, 2 mg/ml
BSA, pH 7.4, containing an oxygen scavenging system) containing 5 mg/ml
casein, endocytic vesicles were added to the chamber, incubated for 10
minutes, and washed. Motility was initiated by the addition of 50 µM ATP in
the absence of a regenerating system. In some experiments, polarity-marked
rhodamine-labeled microtubules were prepared to determine directionality of
moving Texas-RedASOR vesicles
(Murray et al., 2000
). In
other experiments, MT-bound vesicles were incubated with 4 mM guanine
nucleotide analogues, 2 µM (100 mg/ml) purified GST-Rab4 or GST-Rab5
proteins, or 0.5 µM (0.030 mg/ml) purified rat liver Rab-GDI for 5 minutes
at room temperature, prior to ATP addition.
Image analysis
Imaging was performed at the Analytical Imaging Facility of the Albert
Einstein College of Medicine. A 60x, 1.4 numerical aperture planapo
objective was used on an Olympus 1X70 inverted microscope containing automatic
excitation and emission filter wheels connected to a Photometrics
charge-coupled device camera run by IPLab Spectrum software (Scanalitics,
Fairfax, VA) running on a Power Macintosh. IPLab Spectrum scripting software
was used to collect images rapidly and to switch between fluorescence
channels. Images were also recorded directly onto videotape. In all
experiments the microscope stage was maintained at 35°C. Videos were
digitized with the use of the Scion Image (Scion Corporation, Frederick, MD)
movie-making macro (1 frame/second) and saved as tiff files. Corresponding
frames indicating the staining in the different fluorescence channels were
merged using Adobe Photoshop 5.0.
Purification of Rab-GDI from rat liver
Rab-GDI was isolated as previously described
(Sasaki et al., 1990) with
modifications. Rat liver was used as the source material. Volumes were scaled
down 15-fold, and Rab-GDI was released from the final Mono Q column with 33 mM
KCl (in appropriate buffer) rather than extensive washing. Rab-GDI was
detected as a doublet at the appropriate molecular weight by immunoblot and
Coomassie-blue SDS-Page. The purified Rab-GDI did not contain Rab4 as
demonstrated by immunoblotting with anti-Rab4.
Immunoblot analysis
Appropriate protein samples (15 µg total protein) were subjected to
10-20% gradient SDS-Page under reducing conditions. Following transfer to
nitrocellulose, the blot was blocked in PBS containing 0.1% Tween 20 and 10%
nonfat dried milk. Immunoblot analysis was then performed using appropriate
primary and HRP-conjugated secondary antibodies as we have described
previously (Hilgard et al.,
2002
).
Preparation of Rab4 and Rab5 GST-fusion proteins
Rab4 and Rab5 cDNAs in pBlueScriptKS+ were a gift from Ira
Mellman (Yale University School of Medicine, New Haven, CT). Rab4 and Rab5
cDNAs were digested with EcoRI and inserted in frame into pGEX-6P-2
and pGEX-6P-1 vectors, respectively (Amersham Pharmacia Biotech). Bacterial
expression of GST fusion proteins was induced with 0.5 mM IPTG for 3.5 hours
at 37°C. Bacteria were lysed by sonication, and recombinant fusion
proteins were subjected to affinity chromatography on GSH-agarose from which
they were eluted with 5 mM GSH. Recovery of GST-Rab4 and GST-Rab5 was
determined by immunoblotting with goat anti-GST antibody, as well as with
specific Rab4 and Rab5 monoclonal antibodies.
Immunocolocalization of vesicle-associated proteins with fluorescent
early endocytic vesicles
Endocytic vesicles were attached to motility chamber-bound microtubules
(Bananis et al., 2000). Primary
antibodies diluted as appropriate in 5 mg/ml casein-containing PMEE buffer
were perfused through the vesicle-containing motility chamber, incubated for 6
minutes, and blocked with motility buffer containing 5 mg/ml casein. Cy2- or
FITC-labeled affinity-purified secondary antibody was added and incubation was
continued for an additional 6 minutes. After extensive washing with PMEE
buffer containing 5 mg/ml casein, immunofluorescence microscopy was performed.
In some experiments, prior to primary antibody perfusion, MT-bound vesicles
were incubated with guanosine nucleotide analogues with or without 2 µM
GST-Rab4, 2 µM GST-Rab5, or 0.5 µM Rab-GDI for 5 minutes and washed with
motility buffer.
Assay of dissociation of Rab4-GDP from endocytic vesicles in vitro
mediated by Rab-GDI or monoclonal antibody to Rab4
Endocytic vesicles (40 µg total protein in 50 µl) were
reconstituted with 120 µl PMEE buffer containing 2 mg/ml BSA and were
centrifuged at 15 psi for 5 minutes at room temperature in a Beckman Airfuge
(Palo Alto, CA). Pellets were reconstituted in 50 µl PMEE buffer or in PMEE
buffer containing 4 mM GDP or 4 mM GTP-
-S and were incubated at
37°C for 15 minutes. Following an additional centrifugation step, vesicle
pellets were reconstituted in 50 µl buffer containing 1 µM Rab-GDI
and/or 4 mM GDP or 4 mM GTP-
-S. Vesicles were incubated at 37°C for
15 minutes and pelleted at 15 psi for 5 minutes. Supernatants and pellets were
subjected to 4-20% gradient SDS-Page under non-reducing conditions and
immunoblot analysis with monoclonal antibody to Rab4. Densitometric analysis
was performed using a Fluorchem imaging system (Alpha Innotech, San Leandro,
CA). Similar conditions were used to examine Rab4 monoclonal antibody-mediated
dissociation of Rab4-GDP from endocytic vesicles. In these studies, vesicles
were incubated with buffer containing 0.05 µM (0.75 mg/ml) Rab4 IgG and/or
4 mM GDP following the second centrifugation step.
Binding of GST-Rab4 to endocytic vesicles in vitro
Vesicles (60 µg total protein in 70 µl) were reconstituted with
120 µl PMEE buffer containing 2 mg/ml BSA and were centrifuged at 15 psi
for 5 minutes at room temperature. Pellets were reconstituted in 70 µl
buffer containing 0.5 µM GST-Rab4 and were incubated at 37°C for 15
minutes. Following three additional centrifugation and wash steps with 150
µl buffer, vesicle pellets were subjected to 4-20% gradient SDS-Page and
immunoblot analysis with Rab4 monoclonal antibody.
Surface plasmon resonance studies of ligand-antibody binding
An IAsys manual biosensor (Affinity Sensors, Cambridge, UK) using surface
plasmon resonance technology was employed to quantify the binding of
antibodies to their ligands at 25°C
(Duffy et al., 2001). GST-Rab4
and GST-Rab5 as well as Rab4 and Rab5 monoclonal antibodies were dialyzed into
PBS-T (10 mM sodium phosphate, 2.7 mM potassium chloride, 138 mM sodium
chloride, 0.05% Tween 20). GST-Rab4 and GST-Rab5 were crosslinked to
carboxymethyl dextran cuvettes using NHS/EDC activation according to the
manufacturer's protocols. Antibody was added to GST-Rab4 or GST-Rab5 coated
cuvettes at the indicated concentrations and instrument response (in arc
seconds) was recorded for 10 minutes. After this time, cuvettes were rinsed
and the surface regenerated (bound protein removed) by incubating in 50 mM
HCl.
The equilibrium constant for antibody-ligand interaction was estimated by
plotting the maximal binding response (extrapolated to infinite time) versus
the total concentration of antibody and fitting this to the quadratic
equation:
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Alternatively, the free concentration of antibody was calculated by
subtracting the bound from total and plotting free versus bound to determine
the apparent Kd in standard Langmuir binding isotherm
fashion. A calibration constant of 50 arc s ng-1 mm2 was
used to convert response into nanograms of protein bound
(Hall and Winzor, 1999).
Molecular weights of 52 kDa and 150 kDa were used to convert nanograms to
molar concentrations of GST-Rab4/5 and antibody, respectively.
Immunofluorescence localization of Rab4 in cultured rat
hepatocytes
Isolated rat hepatocytes were prepared by collagenase digestion and
cultured onto glass coverslips coated with 1 mg/ml calf skin collagen as we
have described previously (Novikoff et
al., 1996). After overnight culture, cells were washed in
Waymouth's 752/1 medium (Life Technologies) and incubated at 37°C for 60
minutes prior to incubation in Texas-RedASOR (5 µg/ml in Waymouth
medium) for 20 minutes followed by plasma membrane lysis in 100 units/ml
Streptolysin O (Sigma catalogue # S5265) in MEPS (35 mM
piperazine-N,N'-bis(2-ethanesulfonic acid) (Pipes), 5 mM EGTA, 5 mM
MgCl2, 0.2 M sucrose, pH 7.1), 4 mM DTT, protease inhibitor
cocktail (Sigma cat. #p8340 used 1:50 dilution) for 5 minutes. Cells were then
blocked in PMEE + 2 mg/ml BSA + 5 mg/ml casein for 5 minutes, incubated in
anti-Rab4 monoclonal antibody (30 nM) for 30 minutes, washed, incubated in
Cy-2 anti-mouse IgG (15 mg/ml) for 30 minutes, washed, mounted for microscopy
in 50% glycerol/PMEE and photographed immediately. Cells lacking
Texas-RedASOR and primary antibody were completely black under these
conditions. Rab4 immunofluorescence of fixed cells was unsuccessful as
paraformaldehyde fixative reduced Rab4 staining. Images were captured on a
Biorad Radiance 2000 confocal microscope at the analytical imaging facility of
the Albert Einstein College of Medicine.
Statistical analysis
Statistical analysis was performed using Chi-square or Student's
t-tests as appropriate (SigmaStat v 2.0, SPSS, Chicago, IL).
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Results |
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Approximately 23% of vesicles colocalized with dynamin II, a `pinchase'
that is thought to mediate budding of clathrin-coated vesicles from plasma
membrane and Golgi (McNiven et al.,
2000). Although Rab7, clathrin, or Rab5 antibodies were found
localized to MT-attached vesicles, few of these vesicles contained
Texas-RedASOR (Table 1,
Fig. 1D-F). In contrast, Rab4
immunolocalized to approximately 80% of MT-bound ASOR-containing vesicles
(Table 1,
Fig. 1A-C). There was no
association of these vesicles with Rab-GDI, and we were unable to find this
protein in the vesicle preparation by immunoblot (data not shown). That Rab4
co-localized with ASOR-containing endocytic vesicles in vivo was demonstrated
in overnight cultured rat hepatocytes that were incubated for 20 minutes at
37°C with Texas-RedASOR. As seen in
Fig. 2, there was frequent
association of Rab4 with Texas-RedASOR-containing vesicles. We were
unable to demonstrate association of these vesicles with Rab5.
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Antibody affinities for Rab4 and Rab5
Differences in vesicle association of antibodies against Rabs 4 and 5
(Fig. 1, 30 nM antibody) could
be due to differential affinities of these antibodies for their respective
ligands. Several studies were conducted to examine this possibility. As seen
in Fig. 3, colocalization of
these antibodies with early endocytic vesicles did not change over a 10-fold
range of monoclonal antibody concentration (15-150 nM). In addition,
affinities of these antibodies for their respective ligands were determined
directly by surface plasmon resonance (SPR). Cuvettes were coated with
GST-Rab4 or GST-Rab5 fusion proteins and probed with the corresponding
antibody. As seen in Fig. 4A,B,
each antibody interacted specifically with the protein to which it was raised.
There was no interaction of anti-Rab4 with GST-Rab5, anti-Rab5 with GST-Rab4,
or either protein with a connexin 43 C-terminal peptide. Analysis of these
data (Fig. 4C) revealed high
affinity binding (Kd of 2 nM and 22 nM for Rab4 and Rab5,
respectively). Although there was a 10-fold difference in the affinities of
these antibodies for their respective Rab's, the results in
Fig. 3 indicate that
experiments were performed under conditions where this made no important
difference in the amount of colocalization that was found. Rab5 was therefore
used in our experiments as a control for the actions of Rab4.
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Interaction of recombinant Rab4 and Rab5 with early endocytic
vesicles
Rab4 GST fusion protein was purified from transformed E. coli.
Initially, binding of GST-Rab4 to endocytic vesicles was examined
(Fig. 4D). Endocytic vesicles
were incubated with 0.5 µM GST-Rab4 for 15 minutes at 37°C and after
multiple washes vesicle pellets were analyzed by immunoblot for GST-Rab4 and
endogenous Rab4. As seen in Fig.
4D, the amount of GST-Rab4 bound is equivalent to the endogenous
levels on endocytic vesicles. GST-Rab fusion proteins also bound the
nucleotides [3H-GDP] and GTP--[35S] as confirmed
by filtration assays (data not shown). Additionally, the concentration of Rab4
in liver cells was determined to be 30 nM based upon GST-Rab4 standards and a
total cell protein concentration of 65 mg/ml
(Hiller and Weber, 1978
).
Interaction of endosome-bound endogenous Rab4 with Rab-GDI
Rabs are known to be prenylated in vivo and require Rab-GDI to dissociate
from membranes (Dirac-Svejstrup et al.,
1994; Pfeffer et al.,
1995
). As seen by western blot in
Fig. 5A, when endocytic
vesicles were centrifuged at high speed, Rab4 partitioned into the vesicle
pellet following treatment with buffer alone, GDP, or GTP-
-S,
indicating that Rab4 was bound to the vesicles. However, Rab4 appeared in
vesicle supernatants after incubation with Rab-GDI
(Fig. 5B). Treatment of
vesicles with GDP prior to Rab-GDI addition increased the amount of Rab4 in
the supernatants (40% by gel densitometry) and concomitantly decreased the
amount in the pellet. Treatment with GTP-
-S prior to Rab-GDI addition
reduced Rab4 in the supernatant by 86% and increased the amount in the pellet.
The data demonstrate that GDP and GTP-
-S exchange into Rab4 present on
the vesicle under these experimental conditions. Unexpectedly, as seen in
Fig. 5C, we found that
monoclonal antibody to Rab4 also released Rab4 into the supernatants, and that
the amount of release was also increased following GDP treatment. Vesicles
were incubated with Rab4 monoclonal antibody, centrifuged and immunoblots were
performed on the supernatant and pellets as in the GDI experiments. Rab4 was
visible as a faint band running just beneath the 25 kDa band of the monoclonal
antibody IgG. Preincubation of the vesicles with GDP lead to an increase in
the amount of Rab4 in the supernatants and a corresponding decrease in the
amount of Rab4 in the pellets. Similar studies could not be performed with
GTP-
-S, as GTP-
-S treatment caused reduction of the Rab4 mAb
light chain, yielding a large, obscuring band at the 25 kDa position (data not
shown).
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Release of Rab4-GDP from endocytic vesicles via the anti-Rab4 mAb was also
seen by immunofluorescence. Pre-treatment of vesicles with GDP or
GDP--S decreased the number of vesicles that stained positive for Rab4
(Fig. 6A-C,
Table 2), as compared to
untreated controls (Fig. 1A-C,
Table 2). Pre-treatment of
vesicles with GTP-
-S did not reduce Rab4 immunofluorescence
(Table 2). Dot-blot analysis
confirmed that the antibody recognized both GDP and GTP-
-S forms of
Rab4 equally well (data not shown). To confirm that GDP by itself was not
responsible for loss of immunofluorescence, we pre-incubated vesicles with
GDP, then added 4 mM GTP-
-S, and finally immunostained for Rab4. This
scenario gave bright Rab4 fluorescence similar to control Rab4 staining
(Fig. 6D-F,
Table 2). No change in dynamin
II, ASGPR, or kinesin staining was observed upon pre-incubation with GDP (data
not shown).
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Effect of nucleotide treatment on motility and fission of endocytic
vesicles
Upon pre-treatment with 4 mM GDP, the number of motile vesicles and the
number of fission events in preparations of ASOR-containing MT-bound endosomes
appeared to be substantially increased.
Fig. 7 shows examples of two
microscope fields of endocytic vesicles bound to microtubules, the lower of
which has been pretreated with GDP. The small arrows point to examples of
moving vesicles while the large arrows show the original vesicle position.
Arrow heads mark vesicle fission events, and these are only seen in the GDP
case (lower panels). Quantification of over 1600 MT-bound endocytic vesicles
in control conditions showed that 22% became motile and of these, 12%
underwent fission, in agreement with previous results
(Bananis et al., 2000).
Pre-incubation with GDP or its non-hydrolysable derivative GDP-ß-S,
resulted in significantly increased numbers of motile vesicles (32% and 34%
respectively, P<0.001). The proportion of motile vesicles
undergoing fission was also increased significantly following exposure of
vesicles to GDP or GDP-ß-S (22% and 21% respectively,
P<0.001), so that any moving vesicle was almost twice as likely to
undergo fission after GDP addition. Pre-treatment of vesicles with
GTP-
-S, resulted in a significant decrease of motile vesicles as
compared to control (15% versus 22%, respectively, P<0.001).
However, the percentage of motile vesicles undergoing fission following
GTP-
-S treatment did not differ from control (P>0.50).
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Vesicles were also treated with GDP followed by Rab-GDI. These conditions released Rab4 from the vesicles (Fig. 5, Table 2). However, the number of motile vesicles and fission events were not substantially different from GDP treatment without Rab-GDI (Fig. 8). We did observe that microtubule `bending' or `gliding' was reduced by Rab-GDI. The microtubules are attached to glass via pre-coating the glass with DEAE-dextran; vesicles are then incubated with the microtubules. Upon ATP addition, some of the microtubules bend or glide small distances across the glass due to motors present in the vesicle preparation. This activity appeared reduced after Rab-GDI treatment. However, as stated above, the number of moving and fissioning ASOR-containing vesicles was not changed.
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Rab4-GTP--S and Rab4GDP were assayed for their effect on
motility. GST-Rab4 was incubated with either GDP or GTP-
-S, resulting
in nucleotide incorporation, and these were then incubated with microtubule
bound vesicles, washed, and motility and fission were scored.
GST-Rab4-GTP-
-S substantially reduced motility and fission compared to
treatment with GTP-
-S alone (percent moving: 5% versus 15%, fission
rate: 4% versus 12%), whereas GST-Rab4-GDP was similar to GDP treatment alone
(percent moving: 29% versus 32%, fission rate: 17% versus 22%). These data
indicate that the GTP-
-S form of Rab4 is inhibitory toward
motility.
GDP enhances minus-end-directed movement of early endocytic
vesicles
The direction of movement of endocytic vesicles bound to polarity marked
microtubules with or without pretreatment with 4 mM GDP was also determined.
As seen in Fig. 9, following
pre-incubation with buffer alone, 52% of motile MT-bound early endocytic
vesicles moved towards the plus end while 48% moved towards the minus end.
These results are consistent with our previous observations
(Bananis et al., 2000;
Murray et al., 2000
). In
contrast, following pre-incubation with buffer containing 4 mM GDP, only 33%
of motile MT-bound early endocytic vesicles moved towards the plus end of
polarity marked microtubules while 67% moved towards the minus end
(P<0.01).
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Antibody to Rab4 inhibits motility of early endocytic vesicles
Studies were also performed to examine the effect of antibodies to Rab4 and
Rab5 on vesicle motility and fission (Table
3). Using similar methodology, we previously showed that
antibodies towards kinesin, but not dynein, inhibited early endocytic vesicle
motility and fission (Bananis et al.,
2000). This was also demonstrated for endocytic vesicles derived
from HeLa cells (Nielsen et al.,
1999
). In the current experiments, endocytic vesicles were bound
to MTs, incubated for 6 minutes with 30 nM anti-Rab4 or anti-Rab5 IgG, washed,
and motility and fission of ASOR-containing vesicles were quantified upon
addition of 50 µM ATP. The Rab5 antibody did not alter motility. In
contrast, motility was markedly reduced by anti-Rab4 antibody. Addition of
fluorescent secondary antibody prior to ATP addition revealed that the small
number of ASOR-containing vesicles that moved in the presence of Rab4 antibody
did not stain for Rab4 (data not shown). Neither antibody reduced the rate of
vesicle fission. There were no changes in motility or fission of vesicles with
addition of anti-dynamin II IgG (data not shown) or anti-ASGPR IgG
(Bananis et al., 2000
).
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To determine whether guanine nucleotides alter the effect of anti-Rab4 on
motility, vesicles were pre-incubated with GTP--S, GDP, or
GDP-
-S, followed by anti-Rab4 and ATP. GTP-
-S followed by
anti-Rab4 produced no further inhibition of motility than anti-Rab4 alone.
However, GDP or GDP-
-S pre-incubation released the inhibition of the
Rab4 antibody (Table 3). Under
this condition, motility was slightly lower than GDP treatment without anti
Rab4 antibody. However, fission activity increased to the high levels seen
with GDP (Table 3,
Fig. 8). These results suggest
that antibody interaction with GTP-Rab4 is responsible for inhibition of
motility. Addition of GDP results in formation of Rab4-GDP, allowing
solubilization of Rab4 by anti-Rab4 antibody (Figs
5,
6), and preventing the antibody
from inhibiting motility. GTP-
-S treatment did not induce further
inhibition by Rab4 antibody as motility was already suppressed to near
completion.
The mechanism by which Rab4 antibody inhibits motility is not yet known,
but we favor the interpretation that crosslinking of vesicle-associated
endogenous Rab4-GTP by antibody potentiates its inhibitory effect on motility.
Inhibition of microtubule-based motility by specific antibodies has been used
by many investigators (e.g. McDonald et
al., 2002; Nielsen et al.,
1999
), and blockage of enzymatic activity (ATP hydrolysis) does
not appear necessary for this effect. Instead steric interference or
structural crosslinking has been offered as an explanation
(Brady et al., 1990
).
Functional association of KIFC2 on early endocytic vesicles
Our previous studies demonstrated that ASOR-containing early endosomes did
not show immunofluorescence for cytoplasmic dynein, nor did they respond to
dynein inhibitors (antibody and vanadate), yet these endosomes moved
bi-directionally on microtubules (Bananis
et al., 2000). Fig.
9 demonstrates that GDP increases minus-end movement of the
endosomes. We therefore predicted that minus-end-directed kinesins are present
on the endosomes and that these kinesins are somehow stimulated by
pre-incubation with GDP. Three minus-end kinesins are known to exist in
mammals, the C-terminal motor domain proteins, KIFC1, 2, and 3
(Miki et al., 2001
). KIFC2 and
KIFC3 are known to be associated with intracellular organelles
(Hanlon et al., 1997
;
Noda et al., 2001
), and a
commercial available antibody to KIFC2 was available to probe our vesicle
fractions for this minus-end-directed kinesin.
Because KIFC2 has been described as neural specific and was not detected by
northern and western blot in liver (Saito
et al., 1997), this issue was reinvestigated using immunoblot
analysis to liver subcellular fractions. As seen in
Fig. 10A and in agreement with
previous studies (Saito et al.,
1997
), KIFC2 is not detected in liver homogenate. However, KIFC2
is detected in the final endocytic vesicle fraction when assayed at same total
protein concentration. This fraction is heavily purified from total cell
protein and devoid of soluble protein and most liver membrane components
(Murray et al., 2000
). The
same antibody was used to confirm the absence of KIFC2 in a mouse knockout
strain (Yang et al., 2001
). We
therefore conclude that KIFC2 is weakly expressed in liver (and possibly other
tissues) and concentrates in the fraction containing ASOR-containing early
endocytic vesicles.
|
The effect of the KIFC2 antibody on motility and direction of movement of early endocytic vesicles on MTs was also examined. In the presence of KIFC2 antibody, overall motility of vesicles was inhibited by over 60% as compared to control (Fig. 10B), due largely to a decrease in minus-end-directed motility (19% vs. 47%, P<0.002, Fig. 10C).
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Discussion |
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The Rab family of small GTPases is composed of a large number of similar
but biologically distinct proteins. They have been described as functioning as
molecular switches that oscillate between GTP- and GDP-bound forms
(Zerial and McBride, 2001). In
the GTP, or activated forms, Rabs associate with membranes and form a scaffold
with specific cytosolic proteins
(Nagelkerken et al., 2000
;
Segev, 2001
;
Zerial and McBride, 2001
).
Although the best established function of Rabs is in promoting membrane fusion
(Rodman and Wandinger-Ness,
2000
; Takai et al.,
2001
; Zerial and McBride,
2001
), additional functions of specific Rabs have recently been
elucidated (Segev, 2001
;
Takai et al., 2001
;
Zerial and McBride, 2001
).
These include Rab6 serving as an adaptor for a Golgi-associated kinesin-like
protein termed Rabkinesin-6 (KIF20A)
(Echard et al., 1998
).
Rabkinesin-6 associates specifically with the GTP form of Rab6, serving as a
putative adaptor complex to link Golgi membranes with MTs
(Echard et al., 1998
). Other
studies have shown binding of myosin Va to Rab27a
(Hume et al., 2001
;
Strom et al., 2002
). It has
been suggested that Rab27a is necessary for the recruitment of myosin Va to
melanosomes, resulting in their distribution at the periphery of melanocytes
(Hume et al., 2001
;
Strom et al., 2002
). It was
also found that Rab4 can bind to the light intermediate chain-1 of cytoplasmic
dynein (Bielli et al.,
2001
).
Other studies indicated that populations of early endocytic vesicles are
associated with Rab4 and Rab5 (Chavrier et
al., 1997; Di Fiore and de
Camilli, 2001
; Nielsen et al.,
1999
; Sonnichsen et al.,
2000
; Trischler et al.,
1999
) and it has been suggested that Rab5 regulates motility of
transferrin-containing early endocytic vesicles on MTs
(Nielsen et al., 1999
). In
this system, in vitro bi-directional motility of endosomes is also mediated by
kinesins rather than dynein. However these investigators found that treatment
with Rab-GDI abolishes motility and that treatment with Rab-GDI-Rab5,
resulting in Rab5 delivery, increases motility. In our system Rab-GDI has
minimal effect on the motility of ASOR vesicles
(Fig. 8). However we do see a
decrease in microtubule gliding, indicating that the activity of motors
associated with other vesicles in the preparation may be decreased.
Furthermore, we find that Rab5 is associated with only 9% of the
ASOR-containing vesicles (Table
1, Fig. 1). Among
the differences between the two systems, the earlier study
(Nielsen et al., 1999
) used
transferrin vesicles from cultured HeLa cells whereas the ASOR vesicles are
isolated from rat liver, and the ASOR vesicles do not require cytosol for
motility.
Under normal conditions, transferrin does not dissociate from its receptor,
and recycles shortly after internalization. It is possible that early
endocytic vesicles from a receptor-ligand system (e.g. ASGPR/ASOR) in which
ligand dissociates from receptor and targets to lysosomes requires distinct
regulators. It must also be considered that the ASOR vesicles have already
passed through a Rab5 compartment
(Sonnichsen et al., 2000).
Similarly, other proteins such as Rab7, EEA1, clathrin, or dynactin that are
known to interact with the endosome at different processing steps are found
only as minor fractions on the ASOR vesicles. Although previous studies of
Rab4 in endocytic processing suggested a role in formation of recycling
endocytic vesicles (Chavrier et al.,
1997
; Daro et al.,
1996
; McCaffrey et al.,
2001
; Nagelkerken et al.,
2000
; Rodman and
Wandinger-Ness, 2000
; van der
Sluijs et al., 1992
), evidence has also been provided for two
populations of early endosomes, with Rab4 being enriched in one of these
populations that was distinct from the transferrin receptor
(Sheff et al., 1999
), and a
role for Rab4 in fission of vesicles from a Rab5-mediated fusion compartment
has also been reported (Chavrier et al.,
1997
).
Our studies provide several lines of evidence that Rab4 is a regulator of
early endocytic vesicle processing. These include inhibition of vesicle
motility by addition of recombinant Rab4-GTP--S. Similar to our
findings, others have found that specific Rab proteins in their GTP-bound form
inhibit specific vesicle transport pathways. A mutant Rab2 in its GTP-bound
form blocked ER-to Golgi transport of VSV-G
(Tisdale, 1999
). Rab3A in its
GTP-bound form is a negative modulator of cholecystokinin secretion from STC-1
cells (Gevrey et al., 2001
).
Overexpression of Rab7 specifically inhibited late endocytic vesicle motility
in HeLa cells (Lebrand et al.,
2002
).
Addition of 4 mM GDP to the endocytic vesicles converts Rab4 into its GDP state (Fig. 5) and enhances minus-end-directed motility (Fig. 9) and fission (Fig. 8). Our data suggest that conversion of Rab4-GTP to Rab4-GDP is sufficient for this effect, as results are identical whether or not Rab4-GDP is removed from vesicles by Rab-GDI. In the absence of added GDP, both plus- and minus-end-directed motility are equally active. Following GDP addition, plus-end-directed motility is unaffected but minus-end-directed motility is doubled (Table 4).
|
Because dynein is neither present nor functional on the vesicles
(Bananis et al., 2000) we
propose that the GDP pretreatment leads to activation of minus-end-directed
kinesins that are then responsible for the increased minus-end movement and
increased fission rate. Consistent with this suggestion, we have shown that a
minus-end-directed kinesin, KIFC2, is present on these vesicles and that
antibody against KIFC2 inhibits minus-end motility
(Fig. 10).
Our hypothesis for how sorting of the asialoglycoprotein receptor and its ligand is regulated within endocytic vesicles is shown in Fig. 11. Ligand (ASOR) binds to its receptor (ASGPR) at the cell surface where it is internalized into an endocytic vesicle. The vesicle binds to microtubules through plus- and minus-end-directed kinesins. GTP-Rab4 is present on the vesicle and inhibits minus-end kinesins (possibly KIFC2). Hydrolysis of GTP on Rab4 releases the minus-end kinesin inhibition, allowing minus-end-directed movement, resulting in fission and sorting of the vesicle contents along the microtubule.
|
We have previously demonstrated sorting of receptor and ligand by vesicle
fission events along microtubules, and we proposed that multiple rounds of
fission could yield segregated receptor and ligand
(Bananis et al., 2000). In the
current report we show: (1) Rab4 is associated with the vesicles; (2) purified
GTP-
-S-Rab4 but not GDP-Rab4 inhibits vesicle motility; (3) GDP
exchanges into endogenous Rab4; and (4) GDP addition results in increased
minus-end-directed motility. Furthermore we show that the minus-end-directed
kinesin, KIFC2 is present on the vesicles.
We propose that the addition of GDP in vitro mimics the endogenous event of
GTP hydrolysis on Rab4, both yielding GDP-Rab4. In the cell, endogenous
Rab-GDI could remove Rab4-GDP from the vesicle, allowing Rab4 to be recycled.
Currently we are investigating whether Rab4-GTP interacts directly
(Bielli et al., 2001;
Echard et al., 1998
;
Hume et al., 2001
;
Strom et al., 2002
) or through
a protein scaffold (Setou et al.,
2000
; Verhey et al.,
2001
) with KIFC2. Identification of Rab4-interacting proteins on
early endocytic vesicles will provide an important key to unraveling further
the complexities of endosome trafficking, segregation and processing.
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Acknowledgments |
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References |
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