The density of GM1-enriched lipid rafts correlates inversely with the efficiency of transfection mediated by cationic liposomes.
Although cationic liposome-mediated transfection has become a standard procedure, the mechanistic details of the process are unknown. It has been suggested that endocytic uptake of lipoplexes is efficient, and transfectability is largely determined by later steps. In this article, we stained GM1-enriched membrane microdomains, a subclass of lipid rafts, with subunit B of cholera toxin and correlated transfection efficiency with their density by quantitatively evaluating microscopic images. We found a strong anticorrelation between the density of GM1-enriched membrane microdomains and the efficacy of transfection monitored by measuring the expression level of GFP in different cell lines transfected by lipofection using two different transfection agents. These findings imply that GM1-enriched membrane microdomains interfere with the process of lipofection. The blocked step must be endocytosis since the accumulation of fluorescently labeled plasmids was lower in cells with high content of GM1-enriched membrane microdomains. Such a correlation was not observed in cells transfected by electroporation. By comparing the efficiency of lipofection in several cell lines we found that those with a high density of GM1-enriched membrane microdomains were the most resistant to transfection. We conclude that the inhibition of lipofection by GM1-enriched membrane microdomains is a general rule, and that endocytosis of lipoplexes can be rate limiting in cells with high density of GM1-enriched membrane rafts.
Kovács
Tamás
T
Department of Biophysics and Cell Biology, University of Debrecen, Debrecen 4012, Hungary.
Kárász
Andrea
A
Szöllosi
János
J
Nagy
Peter
P
D000818:Animals; D002412:Cations; D045744:Cell Line, Tumor; D002772:Cholera Toxin / Q000378:metabolism; D002966:Clathrin / Q000378:metabolism; D004705:Endocytosis; D005456:Fluorescent Dyes; D005677:G(M1) Ganglioside / Q000378:metabolism*; D006801:Humans; D008081:Liposomes / Q000378:metabolism*; D021962:Membrane Microdomains / Q000378:metabolism*; D051379:Mice; D010957:Plasmids / Q000378:metabolism; D013194:Staining and Labeling; D014162:Transfection; D014168:Transferrin / Q000378:metabolism
D002412:Cations; D002966:Clathrin; D005456:Fluorescent Dyes; D008081:Liposomes; D014168:Transferrin; D005677:G(M1) Ganglioside; D002772:Cholera Toxin