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.

	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).

Biochemical 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.

	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.

View larger version (14K): [in this window] [in a new window]   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.

View larger version (32K): [in this window] [in a new window]   Fig. 2.   Taxol promotes accumulation of endosomal markers in early endosomes. Density gradient distributions of enzyme activities of Na+-K+-ATPase, acid phosphatase, and -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.

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).

View larger version (53K): [in this window] [in a new window]   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.

View larger version (63K): [in this window] [in a new window]   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.

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.

View larger version (35K): [in this window] [in a new window]   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).

View larger version (13K): [in this window] [in a new window]   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.

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.

View larger version (13K): [in this window] [in a new window]   Fig. 7.   Taxol does not alter tubulin association with cellular membranes. Distribution of -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.

View larger version (129K): [in this window] [in a new window]   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.

	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.

View larger version (12K): [in this window] [in a new window]   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 -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.