Mi Lan, Goryaynov Alexander, Lindquist Andre, Rexach Michael, Yang Weidong
1] Department of Biology, Temple University, Philadelphia, PA 19122 [2] Department of Optical Science and Engineering, Shanghai Engineering Research Center of Ultra-Precision Optical Manufacturing, Fudan University, Shanghai 200433, China.
Department of Biology, Temple University, Philadelphia, PA 19122.
Sci Rep. 2015 Mar 23;5:9372. doi: 10.1038/srep09372.
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.
核孔复合体（NPC）是真核细胞中最大的超分子结构之一。其八角形环状支架贯穿核膜，具有调节核质运输的独特分子机制。NPC由约30种不同的核孔蛋白（Nup）组成，每个NPC中平均有8、16或32个拷贝。由于在单个超分子复合物中计数蛋白质存在固有困难，这一估计尚未在活细胞中的单个NPC上得到证实。在这里，我们使用单分子SPEED显微镜直接计数活酵母单个NPC内24种不同Nup的拷贝数，发现与先前的估计既有一致之处，也有显著偏差。正如预期的那样，我们计数到四个外周Nup各有8个拷贝，14个支架Nup各有16个拷贝。出乎意料的是，我们计数到Nsp1和Nic96最多有16个拷贝，而不是先前估计的32个；并且发现其他六种Nup只有10 - 15个拷贝，而不是预期的8个或16个拷贝。这种原位分子计数技术可以测试NPC和细胞中其他超分子结构的结构 - 功能模型。
