Quantifying nucleoporin stoichiometry inside single nuclear pore complexes in vivo.
Quantifying nucleoporin stoichiometry inside single nuclear pore complexes in vivo.
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DOI：
10.1038/srep09372
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发表时间：
2015-03-23
影响因子：
4.6
通讯作者：
Yang W
中科院分区：
文献类型：
--
作者：
Mi L;Goryaynov A;Lindquist A;Rexach M;Yang W
核孔复合物是真核细胞中最大的超分子结构之一。它的八角形环状支架穿透核膜，并具有独特的分子机制，调节核质运输。NPC由约30种不同的核孔蛋白（NUP）组成，平均每个NPC有8、16或32个拷贝。由于单个超分子复合物内的蛋白质计数的固有困难，该估计尚未被活细胞中的单个NPC证实。在这里，我们使用单分子SPEED显微镜直接计数活酵母的单个NPC内的24个不同NUP的拷贝数，并发现与先前估计的一致性以及显著偏差。正如预期的，我们计数了4个外周Nup的8个拷贝和14个支架Nup的16个拷贝。出乎意料的是，我们计数到Nsp 1和Nic 96的最多16个拷贝，而不是之前估计的32个;并且发现其他6个Nup的只有10-15个拷贝，而不是预期的8或16个拷贝。这种原位分子计数技术可以测试细胞中NPC和其他超分子结构的结构-功能模型。
The nuclear pore complex (NPC) is one of the largest supramolecular structures in eukaryotic cells. Its octagonal ring-scaffold perforates the nuclear envelope and features a unique molecular machinery that regulates nucleocytoplasmic transport. NPCs are composed of ~30 different nucleoporins (Nups), averaged at 8, 16 or 32 copies per NPC. This estimate has not been confirmed for individual NPCs in living cells due to the inherent difficulty of counting proteins inside single supramolecular complexes. Here we used single-molecule SPEED microscopy to directly count the copy-number of twenty-four different Nups within individual NPCs of live yeast, and found agreement as well as significant deviation from previous estimates. As expected, we counted 8 copies of four peripheral Nups and 16 copies of fourteen scaffold Nups. Unexpectedly, we counted a maximum of 16 copies of Nsp1 and Nic96, rather than 32 as previously estimated; and found only 10–15 copies of six other Nups, rather than 8 or 16 copies as expected. This in situ molecular-counting technology can test structure-function models of NPCs and other supramolecular structures in cells.