Cell entry of arginine-rich peptides is independent of endocytosis.
Cell entry of arginine-rich peptides is independent of endocytosis.
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DOI：
10.1074/jbc.m805550200
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发表时间：
2009-02-06
期刊：
影响因子：
--
通讯作者：
Cardoso MC
中科院分区：
文献类型：
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作者：
Ter-Avetisyan G;Tünnemann G;Nowak D;Nitschke M;Herrmann A;Drab M;Cardoso MC
富含精氨酸的肽是细胞穿透肽的一个亚类，其被活细胞摄取，并且可以在细胞质和核质内自由扩散。这种现象已被归因于内吞模式的摄取和随后从囊泡释放或直接膜渗透（转导）。为了区分这两种可能性，我们阻断了内吞途径，这些途径被认为参与了细胞穿透肽的摄取。然后，我们通过共聚焦显微镜监测细胞穿透转录反式激活因子（达特）肽随时间推移进入活的哺乳动物细胞的摄取和分布。为了防止化学抑制剂的副作用，我们使用了基因工程细胞以及不同的温度。我们发现，敲除网格蛋白介导的内吞作用和敲除小窝蛋白介导的内吞作用并不影响达特进入细胞的能力。此外，达特肽在整个细胞质和细胞核中显示出与对照细胞相同的细胞内分布。即使在4 °C下孵育细胞也不会消除达特摄取，也不会改变其细胞内分布。因此，我们得出结论，这种分布的结果从达特肽直接穿透（转导）质膜。不太可能形成非选择性孔，因为同时加入的荧光团不会与达特肽一起吸收。总之，尽管达特转导的频率和动力学在细胞类型之间变化，但它不依赖于内吞作用。
Arginine-rich peptides are a subclass of cell-penetrating peptides that are taken up by living cells and can be detected freely diffusing inside the cytoplasm and nucleoplasm. This phenomenon has been attributed to either an endocytic mode of uptake and a subsequent release from vesicles or to direct membrane penetration (transduction). To distinguish between both possibilities, we have blocked endocytic pathways suggested to be involved in uptake of cell-penetrating peptides. We have then monitored by confocal microscopy the uptake and distribution of the cell-penetrating transactivator of transcription (TAT) peptide into living mammalian cells over time. To prevent side effects of chemical inhibitors, we used genetically engineered cells as well as different temperature. We found that a knockdown of clathrin-mediated endocytosis and a knock-out of caveolin-mediated endocytosis did not affect the ability of TAT to enter cells. In addition, the TAT peptide showed the same intracellular distribution throughout the cytoplasm and nucleus as in control cells. Even incubation of cells at 4 °C did not abrogate TAT uptake nor change its intracellular distribution. We therefore conclude that this distribution results from TAT peptide that directly penetrated (transduced) the plasma membrane. The formation of nonselective pores is unlikely, because simultaneously added fluorophores were not taken up together with the TAT peptide. In summary, although the frequency and kinetics of TAT transduction varied between cell types, it was independent of endocytosis.