Lipoplex-mediated transfection of mammalian cells occurs through the cholesterol-dependent clathrin-mediated pathway of endocytosis
Lipoplex-mediated transfection of mammalian cells occurs through the cholesterol-dependent clathrin-mediated pathway of endocytosis
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DOI：
10.1074/jbc.m111257200
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发表时间：
2002-05-17
影响因子：
4.8
通讯作者：
Hoekstra, D
中科院分区：
文献类型：
--
作者：
Zuhorn, IS;Kalicharan, R;Hoekstra, D
合成的两亲物被广泛用作载体系统。然而，为了匹配病毒载体获得的转染效率，需要进一步深入了解脂质复合物的性质，决定转染效率，包括传递机制。虽然内吞作用通常被称为脂质复合物进入和转染的途径，但其确切性质一直不清楚。在这里，我们证明，脂质体介导的转染被抑制超过80%，当质膜胆固醇耗尽与甲基-β-环糊精。胆固醇补充恢复转染能力。胆固醇耗竭后的脂复合物的细胞分布的调查显示，独家抑制内化，而细胞协会保持不受影响。这些数据有力地支持了这样的观点，即复杂的内化，而不是质粒穿过质膜的直接易位，是实现有效的lipoplex介导的转染的先决条件。我们表明，内化lipoplex与转铁蛋白共定位在早期内吞隔室和lipoplex的内化抑制钾耗竭的细胞和细胞中过表达显性负Eps 15突变体。结合的概念，可以排除eaveolae介导的内化，我们得出结论，有效的lipoplex介导的转染需要复杂的内化通过胆固醇依赖性网格蛋白介导的内吞途径。
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-beta-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 eaveolae-mediated internalization can be excluded, we conclude that efficient lipoplex-mediated transfection requires complex internalization via the cholesterol-dependent clathrin-mediated pathway of endocytosis.