From the
In a recent study (Goltz, J. S., Wolkoff, A. W., Novikoff, P.
M., Stockert, R. J., and Satir, P.(1992) Proc. Natl. Acad. Sci. U.
S. A. 89, 7026-7030), we found that ligand- and
receptor-containing endocytic vesicles bind to endogenous microtubules in vitro after 60 min of receptor-mediated endocytosis of
asialo-orosomucoid. In the presence of ATP, ligand-containing endocytic
vesicles are released from microtubules, while those containing
receptor are not. We hypothesized that cytoplasmic dynein may associate
with ligand-containing, but not receptor-containing, domains of
endocytic vesicles and might be involved in the movement of
ligand-containing vesicles along microtubules during sorting of ligand
from receptor. Direct evidence in support of this hypothesis has been
obtained in the present study. Binding of ligand-containing vesicles to
microtubules correlates highly (p < 0.001) with binding of
dynein, but not kinesin, under a variety of conditions. Binding of
receptor-containing vesicles to microtubules is independent of both
cytoplasmic dynein and kinesin binding. Tight association of
cytoplasmic dynein with a population of ligand-containing vesicles is
seen directly by immunoprecipitation. These results support the view
that in receptor-mediated endocytosis, ligand-containing vesicles
become bound to microtubules by cytoplasmic dynein. While receptor
domains of endosomes remain attached to microtubules in an
ATP-independent manner, ligand-containing domains might be moved away
toward pericentrosomal lysosomes by this motor molecule.
Receptor-mediated endocytosis (RME)
In a recent study, we found that ligand- and
receptor-containing endocytic vesicles bind to stabilized endogenous
MTs in vitro after 60 min of RME of ASOR(11) . In the
presence of ATP, ligand-containing endocytic vesicles are released from
MTs, while those containing receptor are not(11) . Several
proteins, including cytoplasmic dynein, associate with MTs and are
released by ATP addition, as determined by SDS-PAGE analysis. Based on
this preliminary information, we hypothesized that cytoplasmic dynein
may associate with ligand-containing, but not receptor-containing,
domains of endocytic vesicles and might be involved in the movement of
ligand-containing vesicles along MTs during sorting of ligand from
receptor. Direct evidence in support of this hypothesis has been
obtained in the present study. Earlier methods (11) have been
modified to quantify endocytic vesicles and cytoplasmic dynein that
bind to MTs in the presence of nucleotides. In some experiments, we
have also quantified MT-bound kinesin, the plus-directed motor protein
that may mediate anterograde organelle
transport(18, 19, 22, 23, 24, 25, 26) .
Binding of ligand-containing vesicles correlates highly with binding of
dynein, but not kinesin, to MTs under a variety of conditions. Binding
of receptor-containing vesicles is independent of both cytoplasmic
dynein and kinesin binding. Most importantly, tight association of
cytoplasmic dynein with a population of ligand-containing vesicles
derived from a defined initiation of RME can now be demonstrated
directly by immunoprecipitation.
A number of investigators have described a requirement for
MTs in receptor-mediated
endocytosis(4, 7, 8, 9, 10) ,
but the molecular basis for this requirement has been unclear. In an
earlier study(11) , we presented evidence that ligand- and
receptor-containing endocytic vesicles bind to stabilized endogenous
MTs in vitro. In the presence of ATP, ligand-containing but
not receptor-containing vesicles were released from MTs. We suggested
that cytoplasmic dynein might functionally link the ligand-containing
vesicles to the MTs so as to produce the firm attachment in the absence
of ATP and release when ATP was added. Preliminary evidence was
presented that ATP simultaneously released cytoplasmic dynein and
ligand-containing vesicles from MTs. The present study quantitates and
extends our previous findings and, further, presents direct evidence
that cytoplasmic dynein binds to ligand-containing vesicles after RME.
As previously suggested, this motor molecule could provide the force
that is necessary for ligand-receptor segregation and/or for transport
of ligand-containing vesicles to lysosomes. MTs are polarized with a
plus (fast growing) end oriented toward the cell periphery and a minus
(slow growing) end oriented toward a MT-organizing center or
centrosome(36, 37, 38, 39) , which is
perinuclear in hepatocytes. Lysosomes are typically abundant near the
centrosome. Previous immunolocalization studies demonstrated
association of endosomes and lysosomes with MTs (40) and
colocalization of cytoplasmic dynein with lysosomes(27) . We
have confirmed by electron and confocal microscopy that
ligand-containing endocytic vesicles are associated with hepatocyte
MTs. We now demonstrate by co-immunolocalization that in the absence of
ATP, MTs in these hepatocytes become abundantly coated with cytoplasmic
dynein. Some minor, nonspecific background fluorescence was present,
but the procedure that we used for fixation and permeabilization of
cells removed cytosolic proteins, including virtually all dynein not
bound to MTs. The hepatocyte is known to have a high concentration of
cytoplasmic dynein. The abundant distribution of cytoplasmic dynein
along MTs in the absence of ATP before homogenization of the cells, the
demonstration of its presence in the supernatant after homogenization
and ATP addition, and the demonstration by immunoprecipitation that it
is also bound to ligand-containing endocytic vesicles strongly support
our hypothesis that cytoplasmic dynein is the ATP-sensitive link
between ligand-containing endosomes and MTs that could function as the
motor that moves endocytic vesicles toward lysosomes.
Several
different hypotheses of how sorting occurs have been proposed (11,
41-43). Maxfield and colleagues(41) , in studies of the
transferrin receptor performed in Chinese hamster ovary cells,
described a sorting endosome with which early endosomes fuse and from
which receptor is recycled. Eventually, this sorting endosome, enriched
in ligand and depleted of receptors, ``matures'' and fuses
with lysosomes. These findings are similar to those described in video
microscopic studies of A431 cells following endocytosis of gold-labeled
antibodies to the transferrin receptor(42) . Net centripetal
movement of endosomes toward the juxtanuclear area was seen. From this
area, small gold-containing vesicles were observed to pinch off and
migrate toward the cell periphery. Disruption of MTs with nocodazole
reduced both the juxtanuclear accumulation of endocytic vesicles and
separation of recycling vesicles. Alternatively, Hopkins and colleagues (43) have studied Hep-2 cells, where the sorting endosome forms
an extensive array of tubular cisternae. In this case, receptor may
remain tacked to microtubules, while multivesicular bodies containing
dissociated ligand move along the cisterns in a polar manner. We have
suggested that the early endosome itself may be a sorting compartment
in cultured hepatocytes from which ligand-containing vesicles bud in
succession, while receptor domains cap and are carried back to the cell
surface (11). Although the morphological details and perhaps the timing
of sorting vary with cell types, all of the models are consistent with
our current observations that suggest that endosomal maturation is
MT-based and that ligand movement toward lysosomes is driven by
cytoplasmic dynein.
The binding of dynein and ligand-containing
endosomes to MTs is reduced in parallel in the presence of ATP and
other nucleotides (). Consistent with these results are
previous observations of Collins and Vallee(31) , who compared
the rates of hydrolysis of several different nucleotides by cytoplasmic
dynein that had been prepared from rat liver and testis. CTP, TTP, and
UTP were the most rapidly hydrolyzed substrates, followed by GTP, ITP,
and ATP(31) , although the relative affinity of cytoplasmic
dynein for CTP was much lower than that for ATP (31). Only ATP,
however, supports force production(14) , and only ATP would be
expected to be present in cytosol in physiologically sufficient
concentrations. The persistence of ATP in our incubation mixtures for
up to 2 h is probably due to the presence of regenerating enzymes,
perhaps myokinase, in the hepatic cytosolic extract.
Although the
ligand-containing vesicles that have been released from MTs by ATP can
be immunoprecipitated from the supernatant using antibodies to
cytoplasmic dynein, these vesicles were not precipitated in control
studies using non-immune antibodies or antibodies against kinesin. As
seen in Fig. 6, although most dynein is immunoprecipitated, only
approximately 15% of ligand was recovered in the immunoprecipitate.
This may be due to the low probability that cytoplasmic dynein attached
to MTs is associated with a specific ligand-containing vesicle
population, to dissociation of dynein upon release from vesicles to
which it had been bound, or to the possibility that cytoplasmic dynein
associates with ligand-containing vesicles only through part of the
endocytic pathway. It would be interesting to know whether the
percentage of dynein associated with ligand-containing vesicles changes
with different assay times and conditions. We know little as yet
regarding the mechanism of binding of cytoplasmic dynein to
ligand-containing vesicles. The dynactin complex (44, 45, 46) copurifies with liver cytoplasmic
dynein and might serve as a ``bridge'' between vesicles and
dynein. This possibility is being investigated.
In our studies, only
a relatively small proportion (less than 10%) of available kinesin
bound to MTs. This binding was reduced significantly in the presence of
ATP or GTP. However, binding was slightly increased in the presence of
AMP-PNP, although both cytoplasmic dynein and ligand binding fell (). These results are consistent with previous reports
that kinesin has both ATPase and GTPase activities, is released from
MTs in the presence of ATP (47, 48) or
GTP(24, 25, 26) , but remains bound to MTs
strongly in the presence of AMP-PNP(49) . This suggests that the
kinesin binds to MTs independently of ligand-containing vesicles, such
that the vesicles do not attach to MTs by kinesin, a conclusion
supported by our immunoprecipitation results. Although it has been
hypothesized that kinesin may be involved in the recycling of receptor
to the cell surface after segregation, we found no association of
kinesin with receptor-containing endocytic vesicles. However, since the
amount of available kinesin associated with stabilized endogenous MTs
is so small, it may be that factors necessary for this binding (23) are lacking in the assay conditions used in our studies.
Our current observations are consistent with two models, as
diagrammed in Fig. 7. In these models, either before (a)
or after (b) segregation of receptor-containing domains, the
mature ligand-containing endosome is attached to and moves along a MT
toward the centrosomally placed lysosomes via cytoplasmic dynein.
Whether cytoplasmic dynein actually provides the force necessary for
fission of the segregating endosomal vesicle is an important issue that
is currently under investigation.
Data, corresponding to
Fig. 4, represent means ± S.E. (number of studies).
Following 30 min of incubation at 37
°C with 20 µM taxol to polymerize endogenous tubulin,
5 mM of each nucleotide or 50 µM vanadate was
added. After an additional 30 min of incubation at 37 °C, the
incubation mixture was centrifuged to sediment microtubules. Content of
ligand- or receptor-containing endocytic vesicles, cytoplasmic dynein,
kinesin, and tubulin in the microtubule-pellet was quantified as
described under ``Experimental Procedures.'' The values are
expressed as a percent of results obtained in the absence of
nucleotides. Data represent means ± S.E.
We thank Michael Cammer for expert assistance with the
confocal microscopy studies. The Bio-Rad MRC 600 confocal microscope
used in this study is part of the Image Analysis Facility of the
Department of Anatomy.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)of
asialo-orosomucoid (ASOR) in hepatocytes is initiated by binding of
ligand to specific cell-surface receptors. Following internalization,
the ligand-receptor complex dissociates within endosomes, and
segregation of ligand and receptor into distinct cellular compartments
occurs. While receptor returns to the cell surface where it is
reutilized, ligand is subsequently delivered to lysosomes for
degradation(1, 2, 3, 4, 5, 6) .
Treatment of cells with cytoskeletal inhibitors, such as nocodazole or
colchicine, inhibits segregation (4) and degradation of
ligand(7, 8, 9, 10) . In particular, it
was found that microtubule (MT) inhibitors markedly delayed segregation
of ligand and receptor into functionally discrete endosomal
compartments. Based on these initial observations, we hypothesized that
interaction of specific populations of late endocytic vesicles with a
polarized network of microtubules in hepatocytes might be important in
establishing their vectorial movement(11) . Although cytoplasmic
dynein, a MT-activated ATPase that has been implicated in centripetal
(minus end-directed) movement of vesicles and organelles along MTs
(12-21), was suggested as a possible linking protein that could
provide a vectorial force sufficient for ligand-receptor segregation,
evidence for its association with components of the endocytic pathway,
based largely on light microscopy immunofluorescent studies, was
tenuous.
Chemicals
ASOR was prepared from human
orosomucoid (Sigma) by neuraminidase digestion (4) and was
labeled with I by a chloramine-T method to a specific
activity of 10,000-13,000 cpm/ng(4) . Rabbit anti-chicken
embryo tubulin antibody, mouse monoclonal anti-74-kDa cytoplasmic
dynein intermediate chain (IC) antibody, mouse monoclonal anti-130-kDa
kinesin head chain (HC) antibody, mouse monoclonal anti-
-tubulin
antibody, goat anti-rabbit IgG conjugated to peroxidase, goat
anti-mouse IgM conjugated to peroxidase, goat anti-mouse IgM-agarose,
and mouse IgM (MOPC-104E) were purchased from Sigma. Goat anti-mouse
IgG
conjugated to either fluorescein isothiocyanate (FITC)
or Cy3 and goat anti-mouse IgM conjugated to FITC, Cy3, or Cy5 were
obtained from Jackson ImmunoResearch Lab. Inc. (West Grove, PA). Rabbit
anti-rat asialoglycoprotein receptor antibody (5) and rabbit
anti-calf brain cytoplasmic dynein antibody (27) were prepared
as previously described. ATP, ADP, AMP, AMP-PCP, AMP-PNP, GTP, CTP,
adenosine, and vanadate were purchased from Sigma or Boehringer
Mannheim. Taxol (Molecular Probes Inc., Eugene, OR) was dissolved in
dimethyl sulfoxide and stored as a 1 mM stock solution at
-20 °C.
Preparation of Short-term Cultured Rat
Hepatocytes
Rat hepatocytes were isolated from
200-250-g male Sprague-Dawley rats (Taconic Farms, Germantown,
NY) after perfusion of the liver with collagenase (Type IV, Sigma) and
were cultured overnight as we have previously
described(3, 4, 11, 28) .
Confocal Microscopy
For confocal
microscopy studies, hepatocytes were cultured on collagen-coated
coverslips as previously described(28) . At various times,
hepatocytes were permeabilized in MEPS buffer (5 mM
MgSO, 5 mM EGTA, 35 mM K
PIPES, and 0.2 M sucrose, pH 7.1), containing 0.1 M GTP and 0.1% Triton X-100 for 1 min at 37 °C, and subsequently
fixed in a 0.25% glutaraldehyde/MEPS mixture for 5 min at 37 °C.
After washing, the permeabilized cells were exposed either to mouse
monoclonal anti-
-tubulin antibody or anti-74-kDa dynein IC
overnight at 0 °C and then rinsed and exposed to either FITC- or
Cy3- labeled secondary antibody against mouse IgG
for
localization of microtubules or either FITC- or Cy3-labeled secondary
antibody against mouse IgM for localization of dynein. Controls for
nonspecific fluorescence binding of secondary antibodies were
performed. In some experiments, continuous wave endocytosis of Texas
Red-ASOR was established, and fluorescent endosomes and MTs were
colocalized. The preparations were examined with a confocal fluorescent
microscope (Bio-Rad 600) equipped with a krypton/argon laser at the
appropriate wavelengths. Several optical sections of permeabilized
cells were scanned for three-dimensional reconstruction.
Preparation of Endocytic Vesicles Bound to MTs during
RME of
Cells were washed with
serum-free medium (SFM), preincubated for 60 min at 37 °C, and then
incubated for 60 min at 4 °C with I-ASOR
I-ASOR (1.25
µg) in 3 ml of SFM to saturate cell surface
receptors(4, 11) . Cells were washed with SFM at 4
°C to remove unbound ligand and were incubated at 37 °C for 60
min to initiate a synchronous single wave of RME. Cells were harvested
into MEPS buffer (1.5 ml per 5 dishes) containing protease inhibitors
(2 mM phenylmethylsulfonyl fluoride, 20 µg/ml N-
-benzoyl-L-arginine methyl ester, 20 µg/ml
soybean trypsin inhibitor, 20 µg/ml p-tosyl-L-arginine methyl ester, 2 µg/ml
leupeptin) and 1 mM dithiothreitol(29) . After cells
were homogenized by 20 strokes in a tight Dounce on ice, the homogenate
was centrifuged at 1,000
g for 10 min at 4 °C.
This supernatant was centrifuged at 40,000
g for 20
min at 4 °C(11) . The supernatant (crude cytosolic extract)
was incubated at 37 °C for 30 min with 20 µM taxol to
polymerize endogenous tubulin. Following an additional timed incubation
at 37 °C with or without various concentrations of ATP, the
incubation mixture was centrifuged at 16,000
g for 30
min at 4 °C to sediment MTs and associated endocytic vesicles and
proteins. In some experiments, 40,000
g supernatants
were kept at 4 °C for 60 min without taxol addition prior to
centrifugation at 16,000
g. In other experiments,
various nucleotides, adenosine, and vanadate were used instead of ATP.
The pellet was dissolved in 0.5 ml of sample buffer, consisting of 2.3%
SDS, 10% glycerol, 5%
-mercaptoethanol, and 62.5 mM Tris,
pH 6.8(28) . All nucleotides and adenosine were dissolved in
MEPS buffer and adjusted to pH 7.1 with NaOH just before use. A small
aliquot of the supernatant was used for protein determination according
to Lowry et al.(30) .
Quantitative Analysis of Ligand-containing Vesicles
Bound to MTs
The pellet dissolved in sample buffer was
subjected to 10% SDS-PAGE. The dried gel was exposed to X-OMAT AR film
(Eastman Kodak Co.) for 6-18 h at -70 °C. The amount of I-ASOR bound to MTs was estimated by densitometric
analysis of autoradiograms using an UltraScan XL scanner (Pharmacia LKB
Biotechnology, Uppsala, Sweden).
Quantitative Immunoblot Detection of Tubulin,
Receptor, Cytoplasmic Dynein, and Kinesin in the MT
Pellet
Following 5, 7.5, or 10% SDS-PAGE, proteins were
transferred to 0.45-µm pore size nitrocellulose paper (Micron
Separations Inc., Westboro, MA) in a Trans-Blot SD Cell (Bio-Rad) at
200 mA for 1 h. After transfer, the nitrocellulose sheet was soaked for
1 h in 3% nonfat dry milk in 0.15 M NaCl containing 50
mM Tris, pH 7.4 (TBS), followed by 1 h of shaking in rabbit
anti-chicken embryo tubulin antibody (diluted 1:50 in 3.5% bovine serum
albumin and 0.1% NaN in TBS), rabbit anti-rat
asialoglycoprotein receptor antibody (diluted 1:100), rabbit anti-calf
brain cytoplasmic dynein antibody (diluted 1:500), or mouse
anti-130-kDa kinesin HC antibody (diluted 1:500). Each sheet was washed
for 1 h in 1% Tween 20 in TBS and was agitated gently for 1 h in goat
anti-rabbit IgG conjugated to peroxidase (diluted 1:1,000 in TBS) or
anti-mouse IgM conjugated to peroxidase (diluted 1:1,000), as
appropriate, followed by washing three times in 1% Tween 20 in TBS. IBI
Enzygraphic
Web (International Biotechnologies, Inc., New
Haven, CT) was used for colorimetric detection of peroxidase-linked
probes. All procedures were performed at room temperature. The amount
of tubulin, receptor, cytoplasmic dynein, or kinesin in the MT pellet
was estimated by densitometric analysis of each immunoblot using an
UltraScan XL scanner.
Immunoprecipitation of Endocytic Vesicles with Dynein
Antibody
Following a 60-min single wave of RME of I-ASOR, the 40,000
g supernatant of
hepatocytes was incubated at 37 °C for 30 min with 20 µM taxol for MT polymerization. After an additional 30-min incubation
at 37 °C with 10 mM ATP to release cytoplasmic dynein and
endocytic vesicles from MTs, the incubation mixture was centrifuged at
16,000
g for 30 min at 4 °C. This supernatant
(tubulin free) was collected and incubated overnight at 4 °C with
monoclonal anti-74-kDa cytoplasmic dynein IC antibody, 130-kDa
anti-kinesin HC antibody, or non-immune IgM. After addition of
secondary antibody (anti-mouse IgM)-agarose beads, each mixture was
incubated at 4 °C for 4 h. After low speed centrifugation, the
pellet was washed four times in 50 mM Tris, pH 8.0, containing
150 mM NaCl and 2.5 mM EDTA. The pellet was heated at
90 °C for 10 min in 200 µl of sample buffer and was subjected
to SDS-PAGE and radioautography to detect ligand or immunoblot to
detect receptor.
Statistical Methods
Results are expressed
as means ± S.E. Significance was evaluated by one-way analysis
of variance. Correlations between either cytoplasmic dynein or kinesin
and endocytic vesicles containing either ligand or receptor bound to
MTs in the presence of various nucleotides were tested by linear
regression analysis.
Immunolocalization of Dynein, Tubulin, and
Ligand
Using confocal microscopy, we have confirmed earlier
observations (11) that following RME, ligand-containing
endosomes are found in linear arrays that lie in close association
parallel to hepatocyte MTs in situ (data not shown). We have
now shown that cytoplasmic dynein colocalizes with MTs in these cells (Fig. 1). Cytoplasmic dynein, as seen in Fig. 1b,
is abundant in hepatocytes. Under the conditions of permeabilization in
the absence of ATP, the major localization is clearly coincident (Fig. 1c) with the microtubular cytoskeleton (Fig. 1a). When primary antibody against cytoplasmic
dynein is omitted, only background fluorescence that is not coincident (Fig. 1f) with MTs (Fig. 1d) is still
seen (Fig. 1e).
Figure 1:
Colocalization by confocal microscopy
of cytoplasmic dynein and MTs in short-term cultured rat hepatocytes.
Cultured rat hepatocytes were prepared, permeabilized, and fixed as
described under ``Experimental Procedures.'' Panelsa-c represent cells incubated with antibodies to
tubulin and dynein visualized with FITC- (a) or Cy3- (b) conjugated second antibodies, respectively. Overlay of
both channels is seen in panelc and demonstrates
coincidence of cytoplasmic dynein with microtubules (yellow). Panelsd-f represent another group of cells
exposed to antibody to tubulin but not to cytoplasmic dynein. These
cells were exposed to both secondary antibodies and serve as a control.
When primary antibody against cytoplasmic dynein is omitted, a small
amount of background fluorescence is seen (e), but this is not
coincident with MTs (f).
Time Dependence of in Vitro Polymerization of
Endogenous Tubulin
We have modified our previous
qualitative assay (11) to develop an assay to quantify the
association of cytoplasmic dynein and ligand-containing endosomes in vitro. The zero time points in each panel in Fig. 2represent aliquots of 40,000 g supernatant, which were kept at 4 °C for 60 min without taxol
addition prior to centrifugation at 16,000
g. Under
this condition, there was no recovery of tubulin, dynein, ligand,
receptor, or kinesin (Fig. 2). This indicates that in the absence
of microtubules, there is no sedimentation of endocytic vesicles at
16,000
g. Although GTP hydrolysis is needed to promote
the assembly of tubulin(32, 33) , there was little
difference in tubulin recovery in the MT pellets from crude cytosolic
extracts incubated in 20 µM taxol at 37 °C (34) with
or without 1 mM GTP (data not shown). We therefore polymerized
MTs in the presence of taxol but not GTP as described above. Formation
of polymerized MTs from endogenous tubulin during incubation at 37
°C with 20 µM taxol was linear for 30 min, after which
a plateau was reached (Fig. 2a). Virtually all initially
soluble tubulin was recovered in this pellet. In a preliminary
experiment, immediate addition of 5 mM ATP to the incubation
mixture reduced the amount of tubulin in the MT pellet (data not
shown). However, addition of ATP after a 30-min preincubation with
taxol had no effect on the recovery of MTs in the pellet (Fig. 2a). The amount of tubulin in the MT pellet
increased linearly with the protein concentration of hepatic crude
cytosolic extract from 1.8 to 7.2 mg/ml. We therefore chose 30 min of
preincubation with 20 µM taxol, 30 min of additional
incubation with or without 5 mM ATP, and 4-6 mg/ml of
hepatic crude extract protein concentration as the optimal assay
conditions.
Figure 2:
Effects of incubation time on the
polymerization of tubulin and on binding to MTs of cytoplasmic dynein,
kinesin, and endocytic vesicles containing ligand or receptor. Rat
hepatocytes were cultured overnight as previously described (3, 4, 11,
28). Cells were then washed with SFM and incubated for 60 min at 4
°C with I-ASOR (1.25 µg/3 ml) to saturate surface
receptors. Unbound ligand was removed, and cells were incubated for 60
min at 37 °C to initiate a synchronous single wave of RME. Cells
were then harvested into MEPS buffer containing protease inhibitors.
They were homogenized by 20 strokes in a tight Dounce on ice, and the
homogenate was centrifuged at 1000
g for 10 min at 4
°C. The supernatant was centrifuged at 40,000
g for 20 min at 4 °C. This supernatant was incubated at 37
°C for various times with 20 µM taxol to polymerize
endogenous tubulin. After 30 min of incubation, some tubes were made 5
mM in ATP, as indicated. For all samples at the indicated
time,, the mixture was centrifuged at 16,000
g for 30
min at 4 °C to sediment MTs and associated endocytic vesicles and
proteins. The zero time point in these studies represents 40,000
g supernatants that were incubated at 4 °C for 60
min without taxol addition prior to centrifugation at 16,000
g. The 16,000
g pellets were analyzed for
contents of tubulin (a), cytoplasmic dynein (b),
ligand (c), receptor (d), and kinesin (e).
Values are expressed as a percent of the total amount of the
corresponding molecule quantified after SDS-PAGE of the 40,000
g supernatant. It should be emphasized that in the absence of
MT polymerization (zero time), none of these constituents are pelleted
at 16,000
g.
Time Dependence of in Vitro Binding of Cytoplasmic
Dynein and Endocytic Vesicles to Polymerized MTs
During
incubation at 37 °C, binding of cytoplasmic dynein to MTs increased
for 90 min and then plateaued when approximately 50% of initially
soluble dynein pelleted with MTs (Fig. 2b). Binding of
ligand-containing endocytic vesicles to MTs paralleled that of
cytoplasmic dynein (Fig. 2c). In contrast, binding of
receptor-containing vesicles to MTs reached a plateau within 30 min
when 80% or more of receptor pelleted with MTs (Fig. 2d), similar to results for MT polymerization.
Only a small percent of total soluble kinesin associated with MTs
prepared under these conditions (Fig. 2e). Addition of 5
mM ATP after a 30-min preincubation with taxol resulted in
inhibition of binding of cytoplasmic dynein and kinesin to MTs. Binding
of ligand-containing endocytic vesicles was also inhibited, but there
was no effect of ATP on binding of receptor-containing vesicles (Fig. 2, b-e).
ATP Concentration Dependence of Binding of
Cytoplasmic Dynein, Kinesin, and Endocytic Vesicles to
MTs
Upon addition for 30 min of increasing concentrations
of ATP (0.5-10 mM), binding of ligand-containing
vesicles to MTs was reduced in parallel to binding of cytoplasmic
dynein and also to kinesin, while binding of receptor-containing
vesicles to MTs was unchanged (Fig. 3a). Under these
conditions, there was no effect on recovery of tubulin in the MT pellet (Fig. 3a). Double-reciprocal plots of the fractional
inhibition of binding of cytoplasmic dynein, kinesin, and
ligand-containing vesicles versus [ATP] to MTs were
linear (Fig. 4). The apparent values for K and maximal inhibition of binding to MTs are shown in .
Figure 3:
Relative content of tubulin (),
cytoplasmic dynein (▾), kinesin (
), ligand (
), and
receptor (
) in the MT pellet as a function of nucleotide
concentration. Taxol-treated supernatants, prepared as in the legend to
Fig. 2, were incubated at 37 °C for 30 min prior to addition of
various concentrations of ATP (a) or AMP-PNP (b) for
30 min before a 16,000
g MT pellet was prepared. The
amount of tubulin (n = 6), cytoplasmic dynein (n = 5), ligand (n = 6), receptor (n = 6), and kinesin (n = 1) are expressed as
a percentage of control values (incubation without nucleotide). Data
represent means ± S.E.
Figure 4:
Inhibition by ATP of binding of
cytoplasmic dynein, kinesin, and ligand-containing vesicles to MTs.
Data here correspond to data in Fig. 3 and are presented as a double
reciprocal plot of the percent inhibition of MT binding versus [ATP]. The apparent values for K and maximal
inhibition of binding to MTs are shown in Table
I.
Effect of AMP-PNP on Binding of Cytoplasmic Dynein,
Kinesin, and Endocytic Vesicles to MTs
The non-hydrolyzable
ATP analog AMP-PNP was substituted for ATP in the previous experiment (Fig. 3b). With increasing concentrations of AMP-PNP,
binding of cytoplasmic dynein and ligand-containing vesicles to MTs
still decreased in parallel, while binding of receptor-containing
vesicles to MTs and recovery of tubulin in the MT pellet remained
unchanged. In contrast to results with ATP, binding of kinesin to MTs
did not change in the presence of AMP-PNP.
Nucleotide Specificity
The effects of 5
mM concentrations of nucleotides and adenosine and 50
µM vanadate on binding of cytoplasmic dynein, kinesin, and
endocytic vesicles to MTs are summarized in . None of the
agents affected either receptor-containing vesicles bound to MTs or MT
polymerization as measured by percent recovered in the pellet. GTP
reduced binding of cytoplasmic dynein, kinesin, and ligand-containing
vesicles to MTs only slightly less effectively than did ATP. CTP also
reduced binding of cytoplasmic dynein and ligand-containing vesicles to
MTs but had no significant effect on binding of kinesin. ADP, and to a
lesser extent AMP, reduced binding of cytoplasmic dynein and
ligand-containing vesicles to MTs. However, utilizing P
nuclear magnetic resonance(35) , we found that ADP was converted
rapidly to ATP in the hepatic extract, probably by myokinase activity.
AMP-PCP, adenosine, and vanadate had no effect on binding of
ligand-containing vesicles or cytoplasmic dynein.
Correlation between MT Binding of Cytoplasmic Dynein
or Kinesin and Endocytic Vesicles
When data for all
nucleotides were considered, high correlation was found between MT
binding of ligand-containing vesicles and cytoplasmic dynein (r = 0.77, n = 62, p < 0.001) (Fig. 5a). In contrast, MT binding of cytoplasmic dynein
did not correlate with binding of receptor-containing vesicles (r = 0.06, n = 62, p > 0.6) (Fig. 5b). There was no correlation between MT binding
of kinesin and that of endocytic vesicles containing either ligand (r = 0.25, n = 13, p > 0.4)
or receptor (r = 0.15, n = 13, p > 0.6) (Fig. 5, c and d).
Figure 5:
Correlation between cytoplasmic dynein or
kinesin and ligand or receptor in the MT pellet. Individual data points
used to generate the means in Table II are shown here. Significant
correlation is seen only for ligand-containing vesicles and dynein (r = 0.77, n = 62, p <
0.001). MT binding of cytoplasmic dynein did not correlate with binding
of receptor-containing vesicles (p > 0.6). There was no
correlation between MT binding of kinesin and that of endocytic
vesicles containing either ligand or receptor. ATP, ; GTP,
;
CTP,
; AMP-PNP,
; AMP-PCP,
; ADP,
; AMP,
; adenosine,
; and vanadate,
.
Immunoprecipitation of Endocytic Vesicles with Dynein
Antibody
To demonstrate directly that cytoplasmic dynein is
bound to ligand-containing vesicles after RME, MTs were polymerized,
and 10 mM ATP was added for 30 min as above. Following
centrifugation, the supernatant was subjected to immunoprecipitation
using monoclonal antibodies directed against the 74-kDa cytoplasmic
dynein intermediate chain coupled to anti-IgM-agarose beads. Controls
utilized isotype-specific non-immune IgM. In additional studies,
immunoprecipitation was performed utilizing a monoclonal antibody to
the 130-kDa kinesin heavy chain. Immunoprecipitates were analyzed for
content of receptor and dynein by immunoblot. Ligand in the
immunoprecipitate was quantified by radioautography. As seen in the toppanel of Fig. 6, cytoplasmic dynein, which
is present in the initial ATP-containing supernatant, is not
immunoprecipitated with non-immune IgM. However, dynein is
quantitatively immunoprecipitated by the anti-dynein antibody. Both
heavy chains and intermediate chains of cytoplasmic dynein have been
identified in the immunoprecipitate. As anticipated, ligand-containing
vesicles are also found in the supernatant. Immunoprecipitation with
anti-dynein, but not control antibodies, precipitated approximately 15%
of the total ligand from the supernatant. Immunoprecipitation of
ligand-containing vesicles by kinesin antibodies was <1%. In
confirmation of our results showing that receptor is not released from
the MT pellet in the presence of ATP, there was no detectable receptor
in the initial ATP-containing supernatant, nor was any seen in the
dynein or kinesin immunoprecipitates.
Figure 6:
Immunoprecipitation of ligand-containing
vesicles by antibody to cytoplasmic dynein. Following a 60-min single
wave of RME of I-ASOR, taxol-treated 40,000
g supernatants were incubated at 37 °C for 30 min with 10 mM ATP. The mixture was centrifuged at 16,000
g. The
16,000
g supernatant (tubulin free) was subjected to
immunoprecipitation with IgM (non-immune (Non-Imm.),
anti-dynein, or anti-kinesin) coupled to anti-IgM-agarose beads. Upper panel, immunoblot of 74-kDa cytoplasmic dynein
intermediate chain. Lower panel,
I-labeled
ligand visualized by autoradiography. Lane 1, supernatant
before immunoprecipitation. Lane 2, precipitate with
isotype-specific non-immune IgM. Lane 3, precipitate with
anti-74-kDa dynein IC antibody. Lane 4, precipitate with
anti-130-kDa kinesin HC antibody.
Figure 7:
Hypothetical association of endocytic
vesicles and cytoplasmic dynein with MTs during RME of ASOR in
hepatocytes. Two potential models of the role of cytoplasmic dynein in
endosomal sorting are shown. Both models are consistent with the data
presented. a, cytoplasmic dynein becomes attached to the
sorting endosome, pulling the endosome apart and moving the mature
ligand-containing endosome toward the lysosome. b, cytoplasmic
dynein attaches only to the mature ligand-containing endosome after
segregation of receptor and ligand. Ligand, ; receptor, ;
receptor-MT attachment molecule,
; vesicle-dynein attachment
complex, possibly dynactin,
; dynein,
.
Table: Analysis of
inhibition by ATP of binding of cytoplasmic dynein and
ligand-containing endocytic vesicles to MTs
Table: The effects of nucleotides on binding to
microtubules of cytoplasmic dynein, kinesin, and ligand- or
receptor-containing endocytic vesicles and on the recovery of tubulin
in the microtubule pellet
,
-methylene)triphosphate; AMP-PNP, adenosine
-5`(
,
-imino)triphosphate; PAGE, polyacrylamide gel
electrophoresis; PIPES, 1,4-piperazinediethanesulfonic acid; TBS,
Tris-buffered saline; SFM, serum-free medium.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.