ProtectNPC · In cellulo cryo electron tomography study on the structural regulation of nuclear pore complexes by the Nup210 luminal ring

The nuclear envelope (NE) is a double-membrane structure that encloses and protects the genetic material in eukaryotes. It is intermitted by nuclear pore complexes (NPCs), which form channels across the NE and mediate nucleocytoplasmic exchange. High tension in the NE is known to invoke NPC dilation, involving structural changes of the NPC scaffold. It is unknown to date, which NPC constituents limit maximum dilation to prevent NPC disintegration under high NE tension, and if disintegration of NPCs can result in NE rupture and nucleocytoplasmic mixing. A putative candidate for protecting NPC structure under mechanical strain is the nucleoporin 210 (Nup210). It is upregulated in several cancers, emerging as a biomarker for breast cancer metastasis and important for cellular adhesion and migration. Nup210 mainly resides in the lumen of the NPC-associated NE und forms a luminal ring (LR), which surrounds the entire residual NPC. In the proposed project, I will assess the role of the Nup210 LR in maintaining NPC structure to prevent disintegration and potential NE rupture under mechanical strain. For this, I will apply focused ion beam milling, cryo electron tomography and advanced image processing techniques to control and Nup210 depleted human cells under differing conditions of NE tension. The comparison of NPC structures will reveal how the Nup210 LR contributes to NPC structural integrity on a molecular level. I will further resolve the conformational landscape of the Nup210 LR to determine whether it elastically accommodates NPC dilation and re-constriction. These structural studies will be complemented and functionally validated by analysing nucleocytoplasmic exchange and NE rupture in the presence and absence of Nup210 in cells. The insights into NPC scaffold integrity and the contribution of the Nup210 LR, and the workflows developed in this project will be of great interest to the structural biology, NPC, NE and mechanobiology research communities.