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Paclitaxel and nocodazole differentially alter endocytosis in cultured cells
S F Hamm-Alvarez1, M Sonee, K Loran-Goss
1Department of Pharmaceutical Sciences, USC School of Pharmacy, Los Angeles 90033, USA.
Purpose:
Microtubule-based transport facilitates the endocytosis of exogenous macromolecules. We have determined how microtubule accumulation and disassembly alter endocytosis.
Methods:
The effects of paclitaxel, which promotes microtubule assembly, and nocodazole, which promotes microtubule disassembly, on fluid-phase and receptor-mediated endocytosis were measured using uptake of horseradish peroxidase and 125I-transferrin, respectively. Changes in membrane and microtubule organization were examined by fluorescence microscopy.
Results:
Neither paclitaxel (4 microM, 60 min pretreatment) nor nocodazole (1 microgram/ml, 60 min pretreatment) significantly inhibited fluid-phase endocytosis. However, paclitaxel caused a redistribution of fluorescent fluid-phase marker to the periphery. Both paclitaxel and nocodazole treatment significantly (p < or = 0.05) reduced the initial uptake of 125I-transferrin at 5 min to approximately 50% of control. Despite the similarity of the effects on initial endocytic uptake, the effects on steady state accumulation of 125I-transferrin were quite distinct. Exposure of CV-1 cells to paclitaxel for an additional 30, 60 or 90 min also showed reduced accumulation of 125I-transferrin up to a maximum significant (p < or = 0.05) inhibition of 48% +/- 10% of control at 90 min. In contrast, nocodazole caused an initial significant (p < or = 0.05) increase in 125I-transferrin accumulation after 30 min (159% +/- 13% of control), while by 90 min 125I-transferrin accumulation had returned to control levels. Microtubule content, particularly of stable microtubules, was increased in CV-1 cells by paclitaxel, but abolished by nocodazole treatment.
Conclusions:
Our data show that changes in the microtubule array can alter the dynamics of receptor movement through the endosomal pathway. However, microtubule assembly versus disassembly have different effects.
Insights
Altering microtubule dynamics impacts endocytosis. Microtubule assembly and disassembly differently affect receptor-mediated endocytosis, influencing macromolecule uptake pathways.
Area of Science:
- Cell Biology
- Molecular Biology
Background:
- Microtubule-based transport is crucial for endocytosis of exogenous macromolecules.
- Understanding how microtubule organization affects endocytic pathways is essential for cellular processes.
Purpose of the Study:
- To investigate the impact of microtubule accumulation and disassembly on endocytosis.
- To determine the differential effects of microtubule assembly versus disassembly on cellular uptake mechanisms.
Main Methods:
- Utilized paclitaxel to promote microtubule assembly and nocodazole for disassembly.
- Measured fluid-phase endocytosis via horseradish peroxidase uptake and receptor-mediated endocytosis using 125I-transferrin.
- Examined cellular organization using fluorescence microscopy.
Main Results:
- Neither drug significantly altered fluid-phase endocytosis, though paclitaxel caused peripheral redistribution.
- Both paclitaxel and nocodazole reduced initial 125I-transferrin uptake by approximately 50%.
- Paclitaxel decreased steady-state 125I-transferrin accumulation, while nocodazole initially increased it before returning to control levels.
Conclusions:
- Changes in the microtubule array significantly alter endosomal pathway dynamics.
- Microtubule assembly and disassembly exert distinct effects on receptor-mediated endocytosis.