Nuclear transport of single molecules: dwell times at the nuclear pore complex.
Nuclear transport of single molecules: dwell times at the nuclear pore complex.
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DOI：
10.1083/jcb.200411005
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发表时间：
2005-01-17
期刊：
影响因子：
--
通讯作者：
Peters R
中科院分区：
文献类型：
--
作者：
Kubitscheck U;Grünwald D;Hoekstra A;Rohleder D;Kues T;Siebrasse JP;Peters R
大分子通过核孔复合物（NPC）选择性移位的机制仍然基本上没有解决。单分子方法可以提供独特的信息的地形性质和动力学过程的异步超分子组装具有良好的空间和时间分辨率。在这里，单分子远场荧光显微镜应用于透化细胞的NPC。核孔蛋白Nup 358可以定位在距离POM 121-GFP沿着NPC轴70 nm处。RanGDP的转运受体NTF 2的结合位点在NPC的细胞质丝和中央框架中观察到，但不在核质丝中观察到。NTF 2和转运蛋白1在其NPC结合位点的停留时间分别为5.8 ± 0.2和7.1 ± 0.2 ms。值得注意的是，这些受体的停留时间减少后，结合到一个特定的运输底物，表明易位加速加载受体分子。连同已知的转运率，我们的数据表明，核质转运发生在单个NPC内通过多个平行的途径。
The mechanism by which macromolecules are selectively translocated through the nuclear pore complex (NPC) is still essentially unresolved. Single molecule methods can provide unique information on topographic properties and kinetic processes of asynchronous supramolecular assemblies with excellent spatial and time resolution. Here, single-molecule far-field fluorescence microscopy was applied to the NPC of permeabilized cells. The nucleoporin Nup358 could be localized at a distance of 70 nm from POM121-GFP along the NPC axis. Binding sites of NTF2, the transport receptor of RanGDP, were observed in cytoplasmic filaments and central framework, but not nucleoplasmic filaments of the NPC. The dwell times of NTF2 and transportin 1 at their NPC binding sites were 5.8 ± 0.2 and 7.1 ± 0.2 ms, respectively. Notably, the dwell times of these receptors were reduced upon binding to a specific transport substrate, suggesting that translocation is accelerated for loaded receptor molecules. Together with the known transport rates, our data suggest that nucleocytoplasmic transport occurs via multiple parallel pathways within single NPCs.
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