title: Quantifying nucleoporin stoichiometry inside single nuclear pore complexes in vivo.
abstract: 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.
authors: 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.
authors: Lan
authors: L
authors: Mi
authors: Department of Biology, Temple University, Philadelphia, PA 19122.
authors: Alexander
authors: A
authors: Goryaynov
authors: Department of Molecular, Cell and Development Biology, University of California, Santa Cruz, CA 95064.
authors: Andre
authors: A
authors: Lindquist
authors: Department of Molecular, Cell and Development Biology, University of California, Santa Cruz, CA 95064.
authors: Michael
authors: M
authors: Rexach
authors: Department of Biology, Temple University, Philadelphia, PA 19122.
authors: Weidong
authors: W
authors: Yang
mesh_terms: D021581:Active Transport, Cell Nucleus; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D022022:Nuclear Pore / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D028861:Nuclear Pore Complex Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D009687:Nuclear Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D055503:Protein Multimerization; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: C082533:NIC96 protein, S cerevisiae; C062748:NSP1 protein, S cerevisiae; D028861:Nuclear Pore Complex Proteins; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D049452:Green Fluorescent Proteins