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J. Biol. Chem., Vol. 277, Issue 20, 18021-18028, May 17, 2002
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From the Synthetic amphiphiles are widely used as a
carrier system. However, to match transfection efficiencies as obtained
for viral vectors, further insight is required into the properties of
lipoplexes that dictate transfection efficiency, including the
mechanism of delivery. Although endocytosis is often referred to as the pathway of lipoplex entry and transfection, its precise nature has been
poorly defined. Here, we demonstrate that lipoplex-mediated transfection is inhibited by more than 80%, when plasma membrane cholesterol is depleted with methyl-
Lipoplex-mediated Transfection of Mammalian Cells Occurs
through the Cholesterol-dependent Clathrin-mediated
Pathway of Endocytosis*
,¶
Department of Membrane Cell Biology,
University of Groningen, A. Deusinglaan 1, Groningen 9713 AV, and
the § Laboratory for Cell Biology and Electron Microscopy,
University of Groningen, Oostersingel 69, Groningen 9713 EZ, The
Netherlands
-cyclodextrin. Cholesterol replenishment restores the transfection capacity. Investigation of the
cellular distribution of lipoplexes after cholesterol depletion revealed an exclusive inhibition of internalization, whereas
cell-association remained unaffected. These data strongly support the
notion that complex internalization, rather than the direct
translocation of plasmid across the plasma membrane, is a prerequisite
for accomplishing effective lipoplex-mediated transfection. We
demonstrate that internalized lipoplexes colocalize with transferrin in
early endocytic compartments and that lipoplex internalization is
inhibited in potassium-depleted cells and in cells overexpressing
dominant negative Eps15 mutants. In conjunction with the notion that
caveolae-mediated internalization can be excluded, we conclude that
efficient lipoplex-mediated transfection requires complex
internalization via the cholesterol-dependent clathrin-mediated pathway of endocytosis.
*
This work was supported by the Netherlands Foundation for
Chemical Research (to C. W.)/Netherlands Technology Foundation (to S. T. W.) (349-40001).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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