title: Super-resolution mapping of scaffold nucleoporins in the nuclear pore complex.
abstract: The nuclear pore complex (NPC), composed of ∼30 different nucleoporins (Nups), 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. However, the precise copy number and the spatial location of each Nup in the native NPC remain obscure due to the inherent difficulty of counting and localizing proteins inside of the sub-micrometer supramolecular complex. Here, we combined super-resolution single-point edge-excitation subdiffraction (SPEED) microscopy and nanobody-specific labeling to reveal the spatial distribution of scaffold Nups within three separate layers in the native NPC with a precision of ∼3 nm. Our data reveal both the radial and axial spatial distributions for Pom121, Nup37 and Nup35 and provide evidence for their copy numbers of 8, 32 and 16, respectively, per NPC. This approach can help pave the path for mapping the entirety of Nups in native NPCs and also other structural components of macromolecular complexes.
authors.affiliation: Department of Biology, Temple University, 1900 N. 12th Street, Philadelphia, PA, 19122, USA.|Department of Optical Science and Engineering, Fudan University, 220 Handan Road, Shanghai, 200433, PR China.
authors.forename: Jiong
authors.initials: J
authors.lastname: Ma
authors.affiliation: Department of Biology, Temple University, 1900 N. 12th Street, Philadelphia, PA, 19122, USA.
authors.forename: Joseph M
authors.initials: JM
authors.lastname: Kelich
authors.affiliation: Department of Biology, Temple University, 1900 N. 12th Street, Philadelphia, PA, 19122, USA.
authors.forename: Samuel L
authors.initials: SL
authors.lastname: Junod
authors.affiliation: Department of Biology, Temple University, 1900 N. 12th Street, Philadelphia, PA, 19122, USA weidong.yang@temple.edu.
authors.forename: Weidong
authors.identifier: 0000-0002-3554-3035
authors.initials: W
authors.lastname: Yang
mesh_terms: D018628:Gene Dosage; D006367:HeLa Cells; D006801:Humans; D008853:Microscopy / Q000379:methods*; D022022:Nuclear Pore / Q000378:metabolism*; D028861:Nuclear Pore Complex Proteins / Q000378:metabolism*
chemical_list: D028861:Nuclear Pore Complex Proteins
keywords: Nanobody; Nuclear pore complex; Super resolution