Cytoskeletal involvement in the cellular trafficking of plasmid/PEI derivative complexes.
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.
Grosse
Stéphanie
S
Université Paris-Descartes, Faculté de Médecine, AP-HP, Hôpital Cochin, Paris, France.
Aron
Yolande
Y
Thévenot
Guiti
G
Monsigny
Michel
M
Fajac
Isabelle
I
D000199:Actins / Q000378:metabolism; D002460:Cell Line; D003550:Cystic Fibrosis / Q000473:pathology; D015638:Cytochalasin D / Q000494:pharmacology; D003599:Cytoskeleton / Q000187:drug effects / Q000378:metabolism*; D011992:Endosomes / Q000378:metabolism; D004847:Epithelial Cells / Q000378:metabolism / Q000502:physiology; D005434:Flow Cytometry; D019793:Fluorescein / Q000737:chemistry / Q000493:pharmacokinetics; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000737:chemistry / Q000235:genetics / Q000493:pharmacokinetics; D006801:Humans; D008247:Lysosomes / Q000378:metabolism; D008870:Microtubules / Q000187:drug effects / Q000378:metabolism; D015739:Nocodazole / Q000494:pharmacology; D010957:Plasmids / Q000737:chemistry / Q000235:genetics / Q000493:pharmacokinetics*; D011094:Polyethyleneimine / Q000737:chemistry / Q000493:pharmacokinetics*; D014132:Trachea / Q000473:pathology; D014162:Transfection
D000199:Actins; D015638:Cytochalasin D; D011094:Polyethyleneimine; D015739:Nocodazole; D019793:Fluorescein