Role of clathrin- and caveolae-mediated endocytosis in gene transfer mediated by lipo- and polyplexes.
We investigated the effects of inhibitors of clathrin-mediated endocytosis (chlorpromazine and K(+) depletion) and of caveolae-mediated uptake (filipin and genistein) on internalization of FITC-poly-l-lysine-labeled DOTAP/DNA lipoplexes and PEI/DNA polyplexes by A549 pneumocytes and HeLa cells and on the transfection efficiencies of these complexes with the luciferase gene. Uptake of the complexes was assayed by fluorescence-activated cell sorting. Lipoplex internalization was inhibited by chlorpromazine and K(+) depletion but unaffected by filipin and genistein. In contrast, polyplex internalization was inhibited by all four inhibitors. We conclude that lipoplex uptake proceeds only by clathrin-mediated endocytosis, while polyplexes are taken up by two mechanisms, one involving caveolae and the other clathrin-coated pits. Transfection by lipoplexes was entirely abolished by blocking clathrin-mediated endocytosis, whereas inhibition of the caveolae pathway had no effect. By contrast, transfection mediated by polyplexes was completely blocked by genistein and filipin but was unaffected by inhibitors of clathrin-mediated endocytosis. Fluorescence colocalization studies with a lysosomal marker, AlexaFluor-dextran, revealed that polyplexes taken up by clathrin-mediated endocytosis are targeted to the lysosomal compartment for degradation, while the polyplexes internalized via caveolae escape this compartment, permitting efficient transfection.
Rejman
Joanna
J
Institute for Experimental Treatment of Cystic Fibrosis, HSR Scientific Institute, Via Olgettina 58, 20132 Milan, Italy.
Bragonzi
Alessandra
A
Conese
Massimo
M
D021941:Caveolae / Q000378:metabolism*; D045744:Cell Line, Tumor; D002469:Cell Separation; D002966:Clathrin / Q000737:chemistry*; D004705:Endocytosis; D005229:Fatty Acids, Monounsaturated / Q000737:chemistry; D005372:Filipin / Q000494:pharmacology; D005434:Flow Cytometry; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes / Q000494:pharmacology; D018014:Gene Transfer Techniques; D015316:Genetic Therapy; D005822:Genetic Vectors; D019833:Genistein / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008247:Lysosomes / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011188:Potassium / Q000737:chemistry; D000644:Quaternary Ammonium Compounds / Q000737:chemistry; D014162:Transfection; D014343:Trypan Blue / Q000494:pharmacology
D002966:Clathrin; D005229:Fatty Acids, Monounsaturated; D005456:Fluorescent Dyes; D000644:Quaternary Ammonium Compounds; D005372:Filipin; D019833:Genistein; D016650:Fluorescein-5-isothiocyanate; D014343:Trypan Blue; C070046:1,2-dioleoyloxy-3-(trimethylammonium)propane; D011188:Potassium