Microtubule involvement in the intracellular dynamics for gene transfection mediated by cationic liposomes.
The effects of microtubule polymerization on liposome-mediated gene transfection were investigated by confocal laser scanning microscopy in target living cells. Both nocodazole and taxol apparently increased the efficiency of gene transfection. Lipofection with fluorescence-labeled cationic liposomes in a COS-7 cell expressing yellow fluorescent protein (YFP)-tagged tubulin revealed that the liposomes were transported along microtubules to lysosomes which are colocalized with the microtubule organizing center (MTOC). Nocodazole disrupted microtubules and produced a uniform distribution of YFP-tagged tubulin in the cytoplasm. Under these conditions, both liposomes and lysosomes were scattered throughout the cytoplasm and they did not colocalize. In the presence of taxol, microtubules were stabilized and several focal regions, like the MTOC, were formed. Lysosomes resided around the nucleus, while liposomes were trapped in microtubules. Under these conditions, neither liposomes nor DNA colocalized with lysosomes. These results demonstrated that the liposome-DNA complexes are transported to lysosomes by a microtubule-mediated pathway, and the effects of nocodazole and taxol on transfection efficiency can be explained by failure of the transport of the liposome-DNA complexes to lysosomes where DNAs are degraded.
Hasegawa
S
S
Graduate School of Pharmaceutical Sciences, Nagoya City University, Japan.
Hirashima
N
N
Nakanishi
M
M
D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D019556:COS Cells / Q000378:metabolism* / Q000648:ultrastructure; D002412:Cations; D002782:Cholestenes / Q000378:metabolism*; D002784:Cholesterol; D015870:Gene Expression; D008081:Liposomes; D008156:Luciferases / Q000235:genetics; D008247:Lysosomes / Q000378:metabolism; D018613:Microscopy, Confocal; D008870:Microtubules / Q000378:metabolism*; D015739:Nocodazole / Q000494:pharmacology; D017239:Paclitaxel / Q000494:pharmacology; D014162:Transfection / Q000379:methods*
D000970:Antineoplastic Agents; D002412:Cations; D002782:Cholestenes; C452320:HyC-Chol; D008081:Liposomes; D002784:Cholesterol; D008156:Luciferases; D017239:Paclitaxel; D015739:Nocodazole