1 Department of Pharmaceutical Sciences and 2 Electron Microscopy Core Facility, Doheny Eye Institute, University of Southern California, Los Angeles, California 90033
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ABSTRACT |
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Although taxol inhibits membrane trafficking, the nature of this
inhibition has not been well defined. In this study, we define the
effects of taxol on endocytosis in CV-1 cells using density gradient
centrifugation of membranes over sorbitol density gradients. After
taxol treatment, resident endosomal enzymes and the epidermal growth
factor (EGF) receptor (EGFR) showed significant
(P 0.05) enrichment in membranes
with properties of early endosomes
(fractions 4 and
5); the EGFR and
Na+-K+-ATPase
were also significantly (P
0.05)
depleted in lysosomal fractions
(fractions
10 and
11). The suggestion that taxol
specifically reduces movement of endosomal constituents to lysosomes
was supported by fluorescence microscopy studies revealing restriction
of EGF to the peripheries of taxol-treated cells, in contrast to the perinuclear lysosomal-like distribution of EGF seen in controls. Kinetic studies with 125I-labeled
EGF were also consistent with a taxol-induced block in traffic from
endosomes and lysosomes after 15 min of uptake but also suggested an
additional taxol-sensitive step in trafficking that involved
redistribution of 125I-EGF within
high-density compartments after 150 min. Related changes in cytoplasmic
dynein distribution were observed within high-density compartments from
taxol-treated cells, suggesting that this motor might participate in
this later taxol-sensitive trafficking event. Electron microscopic
examination of high-density membranes
(fraction
12) showed that taxol increased the
numbers of small (<500 nm) dense vesicles, with a relative depletion
of the larger (>500 nm) vesicles found in controls. These data
demonstrate that disruption of endocytic events by taxol includes the
early accumulation of protein and endocytic markers in endosomes and the later accumulation in a dense compartment that we propose is a
subdomain of the lysosomes.
cytoplasmic dynein; endosome; lysosome; epidermal growth factor receptor
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INTRODUCTION |
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A MAJOR ROLE FOR INTERPHASE microtubules (MTs) is the support of membrane vesicle movements involved in cellular membrane trafficking (reviewed in Ref. 4). Two MT-dependent cytoplasmic motor proteins, kinesin and cytoplasmic dynein (reviewed in Ref. 26), are known to power the MT-dependent vesicle movements that play an integral role in events ranging from endocytosis to exocytosis. Although a role for these motor proteins has been proposed for some specific trafficking events, it is likely that the roles assigned to these motors thus far represent only a small sampling of their membrane trafficking repertoire. The use of several MT-targeted drugs, including taxol, has been helpful in assigning a role for MT-dependent vesicle transport and the motor proteins in different membrane trafficking events.
Taxol is an MT-targeted drug that promotes MT assembly and stabilization even in the absence of factors usually essential for polymerization (GTP, MT-associated proteins) and in the presence of conditions that normally promote disassembly (Ca2+, cold) (23). The cellular effects of taxol are diverse and include reorganization of the MT array (13), disruption of membrane organization and membrane trafficking (9, 11, 27), changes in mitotic spindle dynamics (15), and, demonstrated most recently, changes in signal transduction (3, 17, 25) and induction of apoptosis (3, 17).
Our previous work showed that taxol treatment significantly reduced several parameters of MT-dependent vesicle movement in CV-1 cells (10) and that this inhibition was correlated with reduced receptor-mediated endocytosis, possibly by eliciting effects within the endocytic pathway in CV-1 cells (11). Also, other groups have utilized microscopy to demonstrate taxol-induced inhibition of endocytosed material to the perinuclear region (12, 19). Several steps in receptor-mediated endocytosis may be influenced by taxol-induced changes in MTs, including receptor internalization (11, 14), endosomal sorting (7), and movement and maturation of endosomal vesicles from early endosomes to the perinuclear region (1, 19, 20).
The current study utilizes biochemical methods (density gradient centrifugation) and immunofluorescence and electron microscopy to identify taxol-sensitive steps in endocytosis and, furthermore, to investigate the role played by cytoplasmic dynein, the MT-based motor protein implicated in movement of endosomal material to the cell interior (1). Our findings reveal that taxol impedes traffic from endosomes to lysosomes and also alters the trafficking of lysosomal constituents between subdomains of the lysosome.
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MATERIALS AND METHODS |
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Reagents.
Taxol was obtained from LC Laboratories (Woburn, MA). Sheep polyclonal
anti-human epidermal growth factor (EGF) receptor (EGFR) antibody was
obtained from Upstate Biotechnology, and mouse anti-cytoplasmic dynein
monoclonal antibody (74.1) was purchased from Chemicon International
(Temucula, CA). The monoclonal antibody to -tubulin (clone B-5-1-2)
was obtained from Sigma. The horseradish peroxidase-conjugated goat
anti-mouse antibody used as a secondary antibody was from Pierce.
125I-labeled protein G and
125I-labeled EGF were obtained
from ICN. All cell culture supplies were obtained from GIBCO BRL, and
other chemicals were obtained from standard suppliers.
Cell culture and treatment. CV-1 (African green monkey kidney epithelial) cells were obtained from the American Type Culture Collection. They were maintained in MEM (Earle's salts) containing 10% fetal bovine serum and penicillin-streptomycin at 37°C and 5% CO2. Cells were split at confluence using trypsin-EDTA. For membrane fractionation studies, CV-1 cells were grown in 175-cm2 flasks before isolation. For fluorescence microscopy, CV-1 cells were grown on glass coverslips in 60-mm petri dishes before processing. Taxol treatment was at 4 µM for 150 min at 37°C for all experiments unless otherwise indicated. Control treatments utilized an equivalent amount of vehicle (DMSO).
Immunofluorescence microscopy. For labeling of lysosomal membranes, CV-1 cells were incubated with Lyso-tracker red (Molecular Probes) according to the manufacturer's instructions. Labeling of cells with Texas red-EGF (Molecular Probes) was also according to the manufacturer's instructions. Samples were analyzed with a Zeiss Axioskop equipped with ×40, ×63, and ×100 objectives attached to an MC-100 spot camera.
Subcellular fractionation. The procedures for subcellular fractionation were previously described in detail (6, 18). Briefly, CV-1 cells were incubated with and without taxol and then isolated by trypsinization and centrifugation in a clinical centrifuge. After a washing in Dulbecco's PBS, the cell pellet was resuspended in 5 mM histidine-imidazole (pH 7.5) with 0.2 mM phenylmethylsulfonyl fluoride and containing 5% sorbitol. The cells were lysed in a cell homogenizer (H&Y Enterprises, Redwood City, CA) to minimize the disruption of membranes and the release of markers from the endosomal system that can occur during cell lysis. The lysed suspension was centrifuged at 3,000 rpm for 10 min in a clinical centrifuge. The resulting low-speed supernatant fraction was adjusted to 55% sorbitol before insertion into a continuous sorbitol density gradient. To form the continuous density gradients, we utilized a three-chambered gradient maker containing 26.5, 55, and 70% sorbitol in the chambers. An 80% sorbitol cushion was positioned below the highest density region (70%) of the formed gradient. Density profiles of representative gradients were measured using an oscillating capillary rheometer and densitometer (data not shown) and were comparable to those previously described (28).
Density gradients were equilibrated overnight at 4°C before sample loading. After the loading, density gradients were subjected to centrifugation for 5 h at 55,000 g to separate the membrane fractions. Membrane fractions were removed from the gradient and concentrated by further ultracentrifugation at 250,000 g for 90 min. Membrane compartment markers were analyzed by biochemical assays and Western blot analysis as described below. Analysis of the following markers is considered in this study: Na+-K+-ATPase (endosomal), acid phosphatase (endosomal), EGFR (endosomal and lysosomal), andBiochemical assays.
-Hexosaminidase activity was determined with
4-methylumbelliferyl-N-acetyl-
-D-glucosaminide
as the substrate (2). Acid phosphatase and
Na+-K+-ATPase
activities were measured as previously described (18). Protein levels
in subcellular fractions were determined with the Bio-Rad assay kit
(Bio-Rad, Richmond, CA).
Analysis of 125I-EGF uptake.
For uptake studies, CV-1 cells were treated with 4 µM taxol for a
total of 150 min. 125I-EGF (0.5 µCi/106 cells) was added with
taxol (150-min uptake), or after 90, 120, or 135 min of taxol treatment
(60-, 30-, and 15-min uptake, respectively) at 37°C. Cells were
processed, and membranes were inserted into sorbitol density gradients
as described above. 125I-EGF
content was determined by scintillation counting. That the separation
over these gradients paralleled earlier separations was determined by
analysis of endosomal (acid phosphatase,
Na+-K+-ATPase)
and lysosomal (-hexosaminidase) marker composition. To minimize the
amount of 125I-EGF that was used,
less material was loaded on these gradients; EGFR and cytoplasmic
dynein contents of the fractions could not be assayed because of
limiting amounts of sample. Also, samples were pooled and analyzed in
pairs (fractions
1 and
2,
fractions 3 and
4, and so forth) because of limiting signal.
Gel electrophoresis and Western blotting.
Membranes from density gradient fractions were suspended in sample
buffer, and aliquots of equal volume were analyzed by SDS-PAGE on 7.5%
gels. The proteins were transferred to nitrocellulose and probed with
the appropriate primary and secondary antibodies. For measurement of
cytoplasmic dynein, a mouse monoclonal anti-cytoplasmic dynein
intermediate chain antibody (74.1) was utilized with a secondary
antibody conjugated to horseradish peroxidase for development with the
enhanced chemiluminescence (ECL) detection kit. For measurement of
-tubulin, a mouse monoclonal
-tubulin antibody was utilized (clone B-5-1-2) with a secondary antibody conjugated to horseradish peroxidase for development with the ECL detection kit. For measurement of EGFR, the sheep polyclonal anti-human EGFR antibody was first exposed to nitrocellulose, followed by incubation with
125I-protein G and exposure to
film overnight. Developed blots were scanned with a Bio-Rad GS-670
imaging densitometer, and the values for each density gradient fraction
were expressed as a percentage of the total recovered signal. All
values considered were within the linear range as determined by
generation of a standard curve using purified tubulin. The linear range
was determined by plotting optical density against protein
concentration at different dilutions of purified tubulin and after
different exposure times.
Electron microscopy. The fraction 12 membranes from the control and taxol-treated density gradient fractions were centrifuged at 12,000 rpm in an Eppendorf microcentrifuge. The supernatant was removed, and the membranes in the pellet were incubated in half-strength Karnovsky's fix and postfixed in 1% osmium tetroxide in 0.1 M cacodylate (pH 7.4). They were next infiltrated with epon resin, embedded, thin sectioned, and stained with 3% uranyl acetate and counterstained with Reynolds lead stain. Sectioning was in the same direction as the vector of centripetal force. Photomicrographs were taken on a Zeiss EM 10 electron microscope.
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RESULTS |
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Taxol alters the distribution of endosomal compartment markers in
membranes from CV-1 cells.
Control and taxol-treated (4 µM, 150 min) CV-1 cells were subjected
to subcellular membrane fractionation by isopycnic density gradient
centrifugation. Resulting changes in membrane trafficking were
discerned by analysis of the distribution of various biochemical markers, indicating changes in communication of the membrane
populations that they mark. Many of these markers have diverse
subcellular distributions; however, a peak on a gradient reveals the
modal density of a population of membranes bearing that marker. We
focused on the distributions of biochemical markers known to be
enriched in endosomal-lysosomal pathways: EGFR,
Na+-K+-ATPase,
acid phosphatase, and -hexosaminidase.
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Taxol treatment prevents movement of EGF-containing vesicles to the perinuclear region. To further understand the potential disruptive effects of taxol on endosome-to-lysosome traffic, we examined the distribution of lysosomal membranes (Fig. 3) and fluorescently labeled EGF (Fig. 4) in control and taxol-treated (4 µM, 150 min) cells. Lysosomal membranes are organized in a primarily perinuclear distribution with some extension into the periphery (Fig. 3A); no major changes in lysosomal distribution or perinuclear locale were observed in taxol-treated cells (Fig. 3B).
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Kinetic studies suggest taxol promotes an early accumulation of
125I-EGF in endosomes and a later
redistribution of 125I-EGF across higher
density membrane pools.
Because major changes in EGFR localization in isolated membranes were
caused by taxol, we further explored the effects of taxol on EGF
trafficking using kinetic studies. Control and taxol-treated CV-1 cells
were incubated with 125I-EGF for
15, 30, 60, and 150 min to compare the patterns of
125I-EGF accumulation over time.
The results are shown in Fig. 5. Like the
EGFR, 125I-EGF displayed a broad
distribution across fractions
3-12
of the density gradient under each condition. However, examination of
difference plots revealed some marked differences in the patterns of
125I-EGF in taxol-treated cells
relative to controls. After 15 min of exposure to
125I-EGF, difference plots
revealed a trend toward accumulation of 125I-EGF in
fractions
3 and
4 of the taxol-treated cells, with a corresponding depletion of
125I-EGF in
fractions
9 and
10, consistent with the changes
previously observed for EGFR trafficking in Fig. 1. By 30 min of
exposure, the accumulation of
125I-EGF in
fractions
3 and
4 observed at 15 min was no longer
present in the taxol-treated cells; this bolus of
125I-EGF may have moved to the
later components of the endosomal pathway, as suggested by the trend
toward increased recovery of 125I-EGF seen in
fractions
9 and
10. After 60 min of exposure, very little difference in 125I-EGF
distribution across cellular membranes was seen between taxol-treated
and control cells. However, by 150 min of exposure, some evidence for a
later inhibitory event in EGF processing was seen; taxol-treated cells
displayed significantly (P 0.05)
increased recovery of 125I-EGF in
the highest density fractions
(fractions
11 and
12) and a relative depletion in
fractions
9 and
10, relative to controls.
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Taxol causes a redistribution of cytoplasmic dynein on membranes.
Our findings showing significant (P 0.05) accumulation of endosomal markers in a low-density pool and
depletion in a high-density pool (lysosomes; Figs. 1, 2, and
5A) and the observation that fluorescently labeled EGF was restricted to the cell periphery (Fig. 4)
were consistent with the existence of a taxol-induced block in vesicle
transport from endosomes to lysosomes. We therefore examined the
distribution of the MT-dependent motor cytoplasmic dynein, which is
known to drive the movement of membranes along MTs from the early
endosomes to the lysosomes (1, 7, 19, 20). The distribution of
cytoplasmic dynein on membranes from control and taxol-treated CV-1
cells separated over density gradients was examined by Western blotting
(Fig. 6).
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Taxol-induced changes in endosomal marker and cytoplasmic dynein distribution are not due to increased MT content of fractions. To determine whether any of the taxol-induced changes in endosomal marker or cytoplasmic dynein distribution could be due to taxol-induced bundling of MTs and the resulting cosedimentation of MT bundles and cytoplasmic dynein-containing membranes at different densities, we examined the association of tubulin with membranes from the density gradient. These results are shown in Fig. 7. Although tubulin was recovered with some of the higher density membrane fractions (fractions 6-12), no evidence for increased accumulation of tubulin in higher density fractions from taxol-treated cells that could account for any redistributions of the endosomal markers or cytoplasmic dynein was observed.
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Transmission electron microscopy reveals differences in the
morphology of fraction
12 membranes following taxol treatment.
Although the density gradient data obtained in Figs. 1 and 2 clearly
revealed a significant (P 0.05)
taxol-induced accumulation of markers in the endosomes as well as a
significant (P
0.05) depletion in
some endosomal markers in fraction
10 or
11, interpretation of events at the
highest density regions of the gradient
(fraction 12) was more difficult because of
the lack of statistical significance associated with most of the
changes in these regions. This could be partly attributed to the
proximity of these fractions to the membrane pellet that formed beneath
the gradient; trace contamination by the pellet might explain some of
the variability in these higher density gradient fractions. Although
they were not statistically significant, we were intrigued by the
apparent increases in endosomal markers and cytoplasmic dynein caused
by taxol treatment in fraction 12. Also, the findings from the
kinetic studies with 125I-EGF
reinforced this interest in the identity of
fraction
12, since taxol induced a significant
(P
0.05) increase in the
125I-EGF contents in
fractions
11 and
12 after 150 min of uptake. The
fraction
12 membranes from control and
taxol-treated cells were examined by transmission electron microscopy
to obtain additional information about the nature of these membranes.
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DISCUSSION |
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The goal of our study was to identify the step or steps within the
endocytic pathway that are altered by taxol treatment. Using membrane
fractionation techniques, we show that taxol treatment leads to
significant (P 0.05) accumulation
of endosomal markers (EGFR, acid phosphatase, and
Na+-K+-ATPase)
in a pool of low-density membranes with properties of early endosomes.
A corresponding significant (P
0.05) depletion in
Na+-K+-ATPase
and EGFR was observed in a pool of high-density membranes with
properties of lysosomes, as evidenced by the enrichment of
-hexosaminidase in these same membranes. Support for the idea that
the accumulation of early endosomal constituents reflected an
inhibition of traffic to the lysosomes was also provided by immunofluorescence microscopy studies; taxol inhibited the normal perinuclear accumulation of internalized fluorescently labeled EGF and
instead restricted the labeled EGF to a punctate peripheral distribution. Finally, analysis of
125I-EGF distribution in control
and taxol-treated cells after 15 min of exposure supports our
hypothesis of a block in the trafficking of
125I-EGF from endosomes to lysosomes.
Vesicle transport from endosomes to lysosomes has previously been shown
to utilize cytoplasmic dynein-driven vesicle transport along MTs.
Surprisingly, our data from density gradient analyses did not reveal
evidence for association of cytoplasmic dynein with endosomes in either
control or taxol-treated cells, revealing instead that cytoplasmic
dynein was enriched primarily in lysosomal membranes from both control
and taxol-treated cells. This finding is consistent with previous
immunofluorescence investigations in cultured cells showing that
cytoplasmic dynein is associated primarily with lysosomes (16).
However, taxol treatment did cause a redistribution of cytoplasmic
dynein across high-density lysosomal membranes, including a significant
(P 0.05) depletion in the
cytoplasmic dynein content of fraction
10.
Evidence for an additional taxol-sensitive step involved in the
trafficking between high-density lysosomal membranes was provided by
kinetic studies with 125I-EGF.
Although a marked trend toward accumulation of some endosomal-lysosomal markers in fractions
11 and
12 was observed in the density
gradients shown in Figs. 1, 2, and 6, these changes were not
statistically significant. However, the kinetic studies revealed that
taxol significantly (P 0.05)
increased accumulation of 125I-EGF
in fractions
11 and
12 after 150 min of exposure,
supporting our conjecture that changes in trafficking were elicited
between high-density membrane compartments. Furthermore, the kinetic
analysis clearly distinguished this later change in trafficking from
the initial effects of taxol on endosome-to-lysosome traffic that occurred after 15 min of exposure. This reorganization of high-density membranes is similar to the effects of taxol on cytoplasmic dynein in
fractions
9-12,
suggesting that cytoplasmic dynein may participate in the
rearrangement. Finally, examination of the properties of the
fraction
12 membranes that accumulate in the
presence of taxol by electron microscopy reveals marked morphological
differences, again supporting the hypothesis that taxol may alter
lysosomal sorting or trafficking.
In the model shown in Fig. 9, we propose that taxol elicits at least two actions on the endocytic pathway. First, our findings suggest that taxol impedes endosome-to-lysosome traffic. The origins of this taxol-induced defect in endosome-to-lysosome traffic are as yet unclear. Taxol may somehow reduce or inhibit the MT-dependent formation of endosomal transport vesicles (21). Alternatively, taxol could inhibit endosomal traffic by reducing the ability of cytoplasmic dynein to propel formed endosomal transport vesicles to the lysosomes. If this latter mechanism were operational, we would expect to observe the accumulation of cytoplasmic dynein with the endosomal markers in fractions 4 and 5; however, no evidence for cytoplasmic dynein association on endosomal membranes was found (Fig. 6). Our inability to detect cytoplasmic dynein on endosomal membranes in control or taxol-treated cells does not eliminate the possibility that cytoplasmic dynein participates in this trafficking event in our system. Lack of signal might simply reflect the low amounts of cytoplasmic dynein recruited to this membrane population, which could be below our limits of detection. Changes in cytoplasmic dynein content in the higher density membranes (for instance, fraction 10) might be relatively easier to detect, since there is a large resting pool of cytoplasmic dynein recovered in these membranes, increasing the baseline levels to above detection limits.
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Second, Fig. 9 suggests that taxol elicits an effect on the sorting of
material within the lysosomal compartment. Sequestering of materials in
membrane subdomains (as well as differential sorting of the contents of
membrane compartments) is a major theme underlying membrane
trafficking. We propose that the changes in
125I-EGF and cytoplasmic dynein
contents of fractions
9-12,
which are each enriched in the lysosomal marker -hexosaminidase,
reflect altered lysosomal trafficking. Conceivably, these changes
originate via taxol-induced changes in cytoplasmic dynein-driven
lysosomal sorting events.
What would be the mechanism of the proposed inhibition by taxol of cytoplasmic dynein-driven vesicular transport? Although taxol-induced inhibition of intracellular MT-based vesicle transport (10) and receptor-mediated endocytosis (9, 11, 19) is correlated with increased MT accumulation and bundling, MT tracks can still be detected in the taxol-treated cells that extend from the periphery to the perinuclear region. More importantly, membrane vesicles are able to move along taxol-stabilized MTs formed from phosphocellulose affinity-purified tubulin in vitro (10), suggesting that association of taxol with MTs formed from purified tubulin is not sufficient to inhibit vesicle movement. However, taxol treatment does increase the cellular formation of more "stable" MTs with much longer half-lives (8, 11, 22). These stable MTs may be unable to sustain certain kinds of motor-driven vesicle movements, such as the movement of transport vesicles driven by cytoplasmic dynein. Alternatively, these stable MTs may not support the binding of cytoplasmic linker proteins (CLIPs) and other factors implicated in the formation of endosomal transport vesicles (21).
These studies provide the first detailed biochemical demonstration that taxol induces inhibition of endocytic processing and that it does so at two distinct steps within the endocytic pathway. This characterization of the specific effects of taxol on the endosomal pathway will enhance further studies aimed at characterization of the effects of taxol on the individual components required for vesicle formation to further delineate the exact actions of this intriguing drug.
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ACKNOWLEDGEMENTS |
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We thank Dr. Austin Mircheff for many helpful discussions. We are also grateful to the members of the Hamm-Alvarez lab for feedback.
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FOOTNOTES |
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This work was supported by National Cancer Institute Grant CA-63387 to S. F. Hamm-Alvarez.
Address for reprint requests: S. F. Hamm-Alvarez, Dept. of Pharmaceutical Sciences, 1985 Zonal Ave., University of Southern California, Los Angeles, CA 90033.
Received 24 October 1997; accepted in final form 9 September 1998.
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