The cellular environment shapes the nuclear pore complex architecture

Abstract

Nuclear pore complexes (NPCs) create large conduits for cargo transport between the nucleus and cytoplasm across the nuclear envelope (NE)1–3. These multi-megadalton structures are composed of about thirty different nucleoporins that are distributed in three main substructures (the inner, cytoplasmic and nucleoplasmic rings) around the central transport channel4–6. Here we use cryo-electron tomography on DLD-1 cells that were prepared using cryo-focused-ion-beam milling to generate a structural model for the human NPC in its native environment. We show that—compared with previous human NPC models obtained from purified NEs—the inner ring in our model is substantially wider; the volume of the central channel is increased by 75% and the nucleoplasmic and cytoplasmic rings are reorganized. Moreover, the NPC membrane exhibits asymmetry around the inner-ring complex. Using targeted degradation of Nup96, a scaffold nucleoporin of the cytoplasmic and nucleoplasmic rings, we observe the interdependence of each ring in modulating the central channel and maintaining membrane asymmetry. Our findings highlight the inherent flexibility of the NPC and suggest that the cellular environment has a considerable influence on NPC dimensions and architecture.

Nuclear pores dilate and constrict in cellulo

“…However, it remains controversial whether NPC dilation and constriction play a role during active nuclear transport (15) and whether the dilation is required to open up peripheral channels for the import of inner nuclear membrane (INM) proteins (16)(17)(18). It has been argued that the constricted state may be a result of purification (4,8). It is difficult to conceive that such large-scale conformational changes can occur on similar time scales as individual transport events (19,20), which would be the essence of a physical gate.…”

“…7 Department of Theoretical Biophysics, Max Planck Institute of Biophysics, 60438 Frankfurt am Main, Germany. 8 Institute of Biophysics, Goethe University Frankfurt, 60438 Frankfurt am Main, Germany.…”

Dunking into the Lipid Bilayer: How Direct Membrane Binding of Nucleoporins Can Contribute to Nuclear Pore Complex Structure and Assembly

“…It also remains open as to how many membrane interaction sites are minimally needed to generate and stabilize the pore membrane structure. The recent cryo-EM structure of NPCs lacking one or both outer rings and thus presumably the multiple Y-complex-membrane interaction sites indicates that a pore structure is stable with drastically reduced numbers of nucleoporin-membrane interactions [ 40 ]. Predictably, further studies of NPCs lacking specific nucleoporins and NPC substructures will reveal which membrane interactions are indeed crucial for NPC stability and/or assembly.…”

“…COPI, COPII and clathrin coats interact with their target membrane via transmembrane proteins [ 36 ] and, for the vertebrate Y-complex biochemical data, show an interaction to POM121 [ 37 ], a single spanning transmembrane nucleoporin facing with its largest parts towards the NPC [ 38 ]. Yet, the current cryo-EM reconstructions of the NPCs fail to localize POM121 [ 39 , 40 , 41 ].…”

“…This clustering might also explain the robustness of the NPC structure with regard of loss of certain nucleoporins, e.g., Nup133 [ 51 , 52 , 53 , 54 ] and Nup53/59 in budding and fission yeast [ 69 , 111 ]. Recent cryo-EM structures of NPCs lacking the cytoplasmic ring or both the cytoplasmic and nuclear rings, induced by degron-mediated degradation of the Y-complex component Nup96, show the inner ring embedded in the nuclear envelope with a smaller distance between outer and inner nuclear membrane and thus a higher positive curvature along their connection [ 40 ]. Thus, the Nup160 and Nup133 mediated membrane interactions are in this situation not required to maintain the membrane pore structure.…”