Abstract
While the static structure of the nuclear pore complex (NPC) continues to be refined with cryo-EM and x-ray crystallography, in vivo conformational changes of the NPC remain under-explored. We developed sensors that report on the orientation of NPC components by rigidly conjugating mEGFP to different NPC proteins. Our studies show conformational changes to select domains of nucleoporins (Nups) within the inner ring (Nup54, Nup58, Nup62) when transport through the NPC is perturbed and no conformational changes to Nups elsewhere in the NPC. Our results suggest that select components of the NPC are flexible and undergo conformational changes upon engaging with cargo.
Highlights
Recent advances in structural biology have allowed the characterization of the static structures of large macromolecular complexes
We establish a technique to visualize the orientations of domains of proteins in vivo and we apply this technique to study the conformational changes in the nuclear pore complex (NPC)
The orientation of Nup-mEGFP fusion proteins can be monitored with polarized-total internal reflection fluorescence microscopy
Summary
Recent advances in structural biology have allowed the characterization of the static structures of large macromolecular complexes. We establish a technique to visualize the orientations of domains of proteins in vivo and we apply this technique to study the conformational changes in the nuclear pore complex (NPC). From the scaffold of the NPC, relatively unstructured domains of Nups protrude into the lumen of the cylinder. In the adherent cell lines used in this study, the nuclei tend to be flattened ovoids and the NPCs on the basal surface share a common orientation with their central axis perpendicular to the coverslip. Cell Biology fluorescence microscopy (pol-TIRFM), we monitored the orientation of mEGFP-based sensors incorporated into different domains of individual NPCs in living cells. To monitor conformational changes in the scaffold of the NPC, we built orientational sensors by rigidly attaching mEGFP to different Nup domains. Our results show a rearrangement of the structural core of the NPC, in particular of the inner ring Nups, in response to manipulations of transport through the NPC
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