title: Nuclear pore assembly proceeds by an inside-out extrusion of the nuclear envelope.
abstract: The nuclear pore complex (NPC) mediates nucleocytoplasmic transport through the nuclear envelope. How the NPC assembles into this double membrane boundary has remained enigmatic. Here, we captured temporally staged assembly intermediates by correlating live cell imaging with high-resolution electron tomography and super-resolution microscopy. Intermediates were dome-shaped evaginations of the inner nuclear membrane (INM), that grew in diameter and depth until they fused with the flat outer nuclear membrane. Live and super-resolved fluorescence microscopy revealed the molecular maturation of the intermediates, which initially contained the nuclear and cytoplasmic ring component Nup107, and only later the cytoplasmic filament component Nup358. EM particle averaging showed that the evagination base was surrounded by an 8-fold rotationally symmetric ring structure from the beginning and that a growing mushroom-shaped density was continuously associated with the deforming membrane. Quantitative structural analysis revealed that interphase NPC assembly proceeds by an asymmetric inside-out extrusion of the INM.
authors.affiliation: Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg, Germany.
authors.forename: Shotaro
authors.identifier: 0000-0003-3976-0843
authors.initials: S
authors.lastname: Otsuka
authors.affiliation: Structural and Computational Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany.
authors.forename: Khanh Huy
authors.initials: KH
authors.lastname: Bui
authors.affiliation: Structural and Computational Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany.|Electron Microscopy Core Facility, European Molecular Biology Laboratory, Heidelberg, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Schorb
authors.affiliation: Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg, Germany.
authors.forename: M Julius
authors.identifier: 0000-0003-3303-5755
authors.initials: MJ
authors.lastname: Hossain
authors.affiliation: Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg, Germany.
authors.forename: Antonio Z
authors.initials: AZ
authors.lastname: Politi
authors.affiliation: Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg, Germany.
authors.forename: Birgit
authors.initials: B
authors.lastname: Koch
authors.affiliation: Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg, Germany.
authors.forename: Mikhail
authors.initials: M
authors.lastname: Eltsov
authors.affiliation: Structural and Computational Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Beck
authors.affiliation: Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg, Germany.
authors.forename: Jan
authors.identifier: 0000-0001-5909-701X
authors.initials: J
authors.lastname: Ellenberg
mesh_terms: D021581:Active Transport, Cell Nucleus / Q000235:genetics*; D000818:Animals; D002467:Cell Nucleus / Q000235:genetics / Q000648:ultrastructure; D006367:HeLa Cells; D006801:Humans; D008856:Microscopy, Fluorescence; D009685:Nuclear Envelope / Q000737:chemistry / Q000235:genetics* / Q000648:ultrastructure; D022022:Nuclear Pore / Q000737:chemistry / Q000235:genetics* / Q000648:ultrastructure; D028861:Nuclear Pore Complex Proteins / Q000737:chemistry* / Q000235:genetics / Q000648:ultrastructure
chemical_list: D028861:Nuclear Pore Complex Proteins
keywords: correlative light-electron microscopy; electron tomography; human; live cell imaging; nuclear envelope; nuclear pore complex; super-resolution microscopy