Taxol inhibits endosomal-lysosomal membrane trafficking at two distinct steps in CV-1 cells

Manisha Sonee1, Ernesto Barrón2, Francie A. Yarber1, and Sarah F. Hamm-Alvarez1

1 Department of Pharmaceutical Sciences and 2 Electron Microscopy Core Facility, Doheny Eye Institute, University of Southern California, Los Angeles, California 90033

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 alpha -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), and beta -hexosaminidase (lysosomal). Statistical differences in the recovery of markers in each fraction from control vs. taxol-treated cells were analyzed with a Student's t-test; values were considered significant at P <=  0.05.

Biochemical assays. beta -Hexosaminidase activity was determined with 4-methylumbelliferyl-N-acetyl-beta -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 (beta -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.

We used total rather than specific 125I-EGF binding, since several different types of control experiments revealed that 125I-EGF nonspecific binding was minimal. First, after exposure to 125I-EGF with and without excess (100-fold) unlabeled EGF for 90 min, nonspecific binding in control cells and taxol-treated cells was 9 and 6%, respectively, of total cell-associated binding (average from n = 2 separate experiments). Also, nonspecific association of 125I-EGF with membranes across density gradient fractions from cells incubated with 125I-EGF with and without excess (100-fold) unlabeled EGF for 90 min was also low, accounting for only 26 and 21% of total in membranes from control and taxol-treated cells, respectively. Finally, difference plots (taxol minus control) of the 125I-EGF composition of density gradient fractions were comparable when 125I-EGF total binding and specific binding were compared (data not shown). According to each of these measurements, nonspecific binding was low and also unchanged following taxol treatments.

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 alpha -tubulin, a mouse monoclonal alpha -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.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 beta -hexosaminidase.

EGFR is known to be transported to the early endosome before sorting and transport to the lysosomes (5). As shown in Fig. 1, the EGFR in density gradient samples from control CV-1 cells was distributed broadly across the density gradient in fractions 4-12. After taxol treatment, the EGFR content of fractions 4 and 5 was significantly (P <=  0.05) increased. A significant (P <=  0.05) decrease in EGFR content of fraction 10 was also seen following taxol treatment. We interpreted this redistribution as reflecting a block in the movement of EGFR from an early endosomal pool (fractions 4 and 5), possibly in transit to a lysosomal pool represented by fractions 10 and 11. However, it was also possible that this redistribution reflected inhibition of EGFR movement from plasma membrane to early endosomes, consistent with some reports suggesting a role for MTs in facilitating transport from plasma membrane to endosomes (11, 14).


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Fig. 1.   Taxol alters traffic of epidermal growth factor (EGF) receptor (EGFR) through endosomal pathway. Membranes from control (A) and taxol-treated (B; 4 µM, 150 min, 37°C) CV-1 cells were subjected to subcellular fractionation and isolation of membranes by isopycnic gradient centrifugation as described in MATERIALS AND METHODS. EGFR in density gradient fractions was analyzed by Western blot analysis, and percentage in each fraction was determined by densitometry. P, pellet. Values (means ± SE; n = 4 preparations) are percentages of totals recovered in summed density gradient fractions. C: "change" plot represents taxol-induced change in density gradient distribution of EGFR. * Significance at P <=  0.05.

To distinguish between these alternatives, we examined the taxol-induced changes in the recovery of several other endosomal and lysosomal markers across the density gradient fractions. Figure 2 shows the density gradient distributions of Na+-K+-ATPase, acid phosphatase, and beta -hexosaminidase in membranes from control and taxol-treated cells. Na+-K+-ATPase has been shown to be enriched in the endosomal pathway of epithelial cells (6), although it is also found in plasma membranes. The relative distribution of this marker in control membranes showed two populations: one broadly distributed in the lower density pool in fractions 3-6 and the other distributed over the higher density pool in fractions 10-12. Taxol treatment caused a significant (P <=  0.05) increase in the lower density pool of Na+-K+-ATPase (fractions 4 and 5) and a significant (P <=  0.05) decrease (fraction 11) in the higher density pool.


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Fig. 2.   Taxol promotes accumulation of endosomal markers in early endosomes. Density gradient distributions of enzyme activities of Na+-K+-ATPase, acid phosphatase, and beta -hexosaminidase in membranes from control and taxol-treated cells (4 µM, 150 min, 37°C) are shown here. Enzymatic activities (means ± SE; n = 5 preparations) were calculated in standard units (nmol/h). * Significance at P <=  0.05.

The density gradient distribution of acid phosphatase catalytic activity in Fig. 2 for both control and taxol-treated CV-1 cell membranes closely followed the distribution pattern of Na+-K+-ATPase. Analysis of this activity showed a peak in the higher density fractions (fractions 10-12) as well as distribution throughout the lower density fractions of the gradient. After taxol treatment, the recovery of acid phosphatase activity was significantly (P <=  0.05) increased in the lower density fractions (fractions 4 and 5). As for Na+-K+-ATPase, a trend toward decreased acid phosphatase content of the higher density pool (fractions 10 and 11) was seen, although this change was not significant at P <=  0.05.

The similarity of these changes, particularly for acid phosphatase, which does not label plasma membranes, to those observed for EGFR strongly suggested that the effects on membrane traffic by taxol involved accumulation of EGFR, acid phosphatase, and Na+-K+-ATPase in an early endosomal compartment, rather than in plasma membranes. That the significant depletion in EGFR and Na+-K+-ATPase activities in fractions 10 and 11 involved lysosomal membranes was confirmed by examination of the distribution of the lysosomal marker, beta -hexosaminidase. Previous work has reported that enrichment of beta -hexosaminidase activity can be used to identify lysosomal membranes (24). beta -Hexosaminidase activity was enriched in fractions 10-12 from control cells, supporting our hypothesis that these fractions contained lysosomal membranes. However, no significant changes in the distribution of beta -hexosaminidase activity were observed after taxol treatment.

No changes in the sizes of the membrane-associated pools of any of these markers were observed following taxol treatment (data not shown), so these differences are not attributable to entry of additional marker enzymes into the total pool. Also, no significant changes in protein distribution across density gradients were observed between taxol-treated and control samples (data not shown). Although other biochemical activities were assayed, including alpha -glucosidase (endoplasmic reticulum) and galactosyltransferase (Golgi), no major taxol-induced changes were observed with these marker enzymes, suggesting that the effects of taxol were concentrated within the endosomal pathway (data not shown).

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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Fig. 3.   Lysosomal morphology is not substantially altered in CV-1 cells after taxol treatment. CV-1 cells were incubated with Lyso-tracker red as described in MATERIALS AND METHODS to label lysosomal membranes. A: control CV-1 cells. B: taxol-treated (4 µM, 150 min, 37°C) CV-1 cells. In both cases, staining is recovered in a perinuclear distribution with limited extension into cell periphery. Scale bar, 15 µm.


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Fig. 4.   EGF distribution in taxol-treated CV-1 cells is more peripheral than in controls. CV-1 cells were incubated with Texas red-EGF. A: control CV-1 cells. B: taxol-treated (4 µM, 150 min, 37°C) CV-1 cells. In controls fluorescent label is primarily found in perinuclear region, whereas in taxol-treated cells EGF is predominantly distributed at cell periphery. Scale bar, 10 µm.

When the distribution of Texas red-EGF was examined under comparable conditions, we found that, after 60 min of uptake, the label was recovered in a punctate perinuclear pattern, similar to the staining pattern seen for lysosomal membranes (Fig. 4A). This staining was inhibitable by incubation with excess unlabeled EGF (data not shown), demonstrating that it arose specifically via EGF binding to EGFR. However, EGF distribution in taxol-treated cells was predominantly recovered in the cell periphery (Fig. 4B). When EGF distribution was quantified in controls, 63% of cells revealed EGF labeling in a perinuclear distribution after 60 min of uptake (n = 356 cells analyzed from 4 separate preparations); in contrast, only 39% of taxol-treated cells revealed perinuclear EGF labeling under these conditions (n = 284 cells analyzed from 4 separate preparations), with the remaining 61% of cells showing EGF distribution in the cell periphery. Because we clearly see taxol-induced accumulation of labeled EGF in peripheral vesicles, rather than at the plasma membrane, these data further support our hypothesis that the taxol-induced accumulation of EGFR (Fig. 1) occurs at early endosomes.

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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Fig. 5.   Taxol alters 125I-labeled EGF accumulation in membrane compartments. Distribution of 125I-EGF across density gradient fractions of membranes from control and taxol-treated (4 µM, 150 min, 37°C) CV-1 cells is shown here after 15 min (A), 30 min (B), 60 min (C), or 150 min (D) of exposure to 125I-EGF. Values (means ± SE; n = 3 preparations) are percentages of totals recovered in summed density gradient fractions. Change plots represent taxol-induced changes in density gradient distributions of 125I-EGF. * Significance at P <=  0.05.

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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Fig. 6.   Taxol treatment causes a redistribution of cytoplasmic dynein across higher density membrane fractions. Distribution of cytoplasmic dynein in density gradient fractions of membranes from control and taxol-treated (4 µM, 150 min, 37°C) CV-1 cells was detected by Western blotting and analyzed by densitometry (means ± SE; n = 5 preparations). * Significance at P <=  0.05.

Although cytoplasmic dynein was not enriched in the lower density endosomal fractions in either control or taxol-treated cells, taxol treatment resulted in an overall change in the pattern of cytoplasmic dynein association with the higher density membranes. Although an even distribution was typical among fractions 9-12 on membranes from untreated CV-1 cells, taxol treatment resulted in a significant (P <=  0.05) depletion in the cytoplasmic dynein associated with the lysosomal pool in fraction 10. These findings suggest that cytoplasmic dynein normally associates with the lysosomal rather than the early endosomal membrane pool and that taxol treatment results in changes in cytoplasmic dynein association with high-density membranes. These changes are strikingly similar to the changes in 125I-EGF observed after 150 min of uptake (Fig. 5D).

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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Fig. 7.   Taxol does not alter tubulin association with cellular membranes. Distribution of alpha -tubulin in density gradient fractions of membranes from control and taxol-treated (4 µM, 150 min, 37°C) CV-1 cells was detected by Western blotting and analyzed by densitometry (means ± SE; n = 3 preparations).

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.

As shown in Fig. 8A, fraction 12 membranes from control CV-1 cells were characterized by the appearance of numerous large vesicles (~500 nm in diameter), many of which exhibited properties of multivesicular bodies, including membrane bifurcations and additional compartmentation. Also, some smaller vesicles were seen. In contrast, Fig. 8B reveals fraction 12 membranes from taxol-treated CV-1 cells, which were characterized by a preponderance of smaller vesicles. Many of these smaller vesicles were multivesicular in appearance; however, almost no vesicles with the larger diameter typical of the control membranes were observed. A quantitative summary of a representative experiment analyzed by blind scoring is shown in Table 1. These findings show that taxol treatment (150 min) is associated with a fourfold increase in the percentage of vesicles that were categorized as small (<500 nm in diameter) and multivesicular; a corresponding decrease in the percentage of large (>500 nm in diameter) vesicles was seen. Treatment of cells with taxol for 1 min followed by density gradient separation of membranes and electron microscopy analysis of fraction 12 revealed membranes with properties that were indistinguishable from those of control membranes (Table 1). These findings demonstrate that taxol itself does not directly alter the density and appearance of membrane organelles, but that prolonged exposure of cells to taxol resulted in pronounced morphological changes in the high-density vesicles recovered in fraction 12.


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Fig. 8.   Transmission electron microscopy images of fraction 12 membranes from control and taxol-treated CV-1 cells show increased recovery of small dense vesicles and depletion of large vesicles after taxol. A: fraction 12 membranes from control CV-1 cells. B: fraction 12 membranes from taxol-treated (4 µM, 150 min, 37°C) CV-1 cells. Fraction 12 membranes were pelleted and processed for electron microscopy as described in MATERIALS AND METHODS.

                              
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Table 1.   Fraction 12 vesicle morphology as determined by EM

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 beta -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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Fig. 9.   Hypothetical model for taxol-induced changes in trafficking within endosomal pathway. A: vesicles normally move along microtubules from endosomes to lysosomes. Lysosomal constituents are sequestered into subdomains of lysosomes. Cytoplasmic dynein is also enriched on lysosomal membranes. B: taxol impedes movement of material from endosomes to lysosomes, possibly by preventing either formation of endocytic transport vesicles or their movement along microtubules to lysosomes. Once at lysosomes, taxol also elicits apparent changes in sorting that result in redistribution of 125I-EGF. Changes in cytoplasmic dynein distribution across lysosomal membranes suggest that this motor may participate in or power taxol-sensitive sorting events.

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 beta -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.

    ACKNOWLEDGEMENTS

We thank Dr. Austin Mircheff for many helpful discussions. We are also grateful to the members of the Hamm-Alvarez lab for feedback.

    FOOTNOTES

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.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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