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

Although taxol inhibits membrane trafficking, the nature of this inhibition has not been well defined. In this study, we define the effects of taxol o n endocytosis in CV-1 cells using density gradient centrifugation of membra nes over sorbitol density gradients. After taxol treatment, resident endoso mal enzymes and the epidermal growth factor (EGF) receptor (EGFR) showed si gnificant (P less than or equal to 0.05) enrichment in membranes with prope rties of early endosomes (fractions 4 and 5); the EGFR and Na+-K+-ATPase we re also significantly (P less than or equal to 0.05) depleted in lysosomal fractions (fractions 10 and 11). The suggestion that taxol specifically red uces movement of endosomal constituents to lysosomes was supported by fluor escence microscopy studies revealing restriction of EGF to the peripheries of taxol-treated cells, in contrast to the perinuclear lysosomal-like distr ibution of EGF seen in controls. Kinetic studies with I-125-labeled EGF wer e also consistent with a taxol-induced block in traffic from endosomes and lysosomes after 15 min of uptake but also suggested an additional taxol-sen sitive step in trafficking that involved redistribution of I-125-EGF within high-density compartments after 150 min. Related changes in cytoplasmic dy nein 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 compa rtment that we propose is a subdomain of the lysosomes.