Cytoskeletal involvement in the cellular trafficking of plasmid/PEI derivative complexes
Résumé
We have studied the cytoskeletal involvement in the cellular trafficking of complexes made with plasmid/PEI or plasmid/lactosylated PEI in cystic fibrosis airway epithelial cells (SigmaCFTE29o- cells). Complexes were incubated in the presence of cytoskeletal inhibitors, and the number of transfected cells was determined by flow cytometry. Complexes were also generated with fluorescein-labeled PEI derivatives and the cell fluorescence intensity was determined by flow cytometry. In the presence of cytochalasin D to depolymerize actin filaments or nocodazole to disrupt microtubules, gene transfer efficiency with both PEI derivatives was decreased by 90%. The uptake of fluoresceinylated complexes studied by flow cytometry was decreased by 50% in the presence of cytochalasin D for both types of complexes (p<0.005) and unchanged in the presence of nocodazole. When cytoskeletal inhibitors were added to the cell culture after the complex uptake had occurred, gene transfer efficiency was decreased by 75% and 50% in the presence of nocodazole and cytochalasin D, respectively. Upon nocodazole-microtubule network disruption, the lysosomal localization of complexes was reduced, as assessed by confocal microscopy. Our results show a major cytoskeletal involvement in the cellular trafficking of complexes made with both PEI derivatives: actin filaments mainly in complex uptake, and microtubules in the trafficking of complexes towards the nucleus, probably through guided transport of complex-containing endosomal vesicles.