Nuclear Pore Assembly Proceeds by an Inside-Out Extrusion of the Nuclear Envelope

Elife. 2016 Sep 15;5:e19071. doi: 10.7554/eLife.19071.

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.

Keywords: correlative light-electron microscopy; electron tomography; human; live cell imaging; nuclear envelope; nuclear pore complex; super-resolution microscopy.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Active Transport, Cell Nucleus / genetics*
  • Animals
  • Cell Nucleus / genetics
  • Cell Nucleus / ultrastructure
  • HeLa Cells
  • Humans
  • Microscopy, Fluorescence
  • Nuclear Envelope / chemistry
  • Nuclear Envelope / genetics*
  • Nuclear Envelope / ultrastructure
  • Nuclear Pore / chemistry
  • Nuclear Pore / genetics*
  • Nuclear Pore / ultrastructure
  • Nuclear Pore Complex Proteins / chemistry*
  • Nuclear Pore Complex Proteins / genetics
  • Nuclear Pore Complex Proteins / ultrastructure

Substances

  • Nuclear Pore Complex Proteins