Role of clathrin- and caveolae-mediated endocytosis in gene transfer mediated by lipo- and polyplexes.
By J. Rejman, A. Bragonzi, M. Conese. Molecular Therapy, 2005
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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.
Subfields: Cell Biology, Molecular Biology
View Paper: https://www.semanticscholar.org/paper/463b3d5f9854ae86933a337e6e14f87fecee7fd0
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Citations in the corpus
- The use of inhibitors to study endocytic pathways of gene carriers: optimization and pitfalls. (2010, background): “In the literature only a few examples exist whereby the non specific effects of these agents were evaluated [22,39,43,44].”
- Gene delivery by lipoplexes and polyplexes. (2010, background): “Rejman et al. (2005) reported that in A549 and HeLa cells, PEI polyplexes are internalized by both pathways, but poly-”
- Uptake Pathways and Subsequent Intracellular Trafficking in Nonviral Gene Delivery (2006, background): “Evidence supporting the existence of a role of caveolae in the uptake of cationic polymer-DNA complexes and the class of protein transduction domains (PTDs), such as the TAT peptide , have appeared (Ferrari et al., 2003; Fittipaldi et al., 2003; Rejman et al., 2005).”