Nup133 Is Required for Proper Nuclear Pore Basket Assembly and Dynamics in Embryonic Stem Cells

Abstract

Summary Nup133 belongs to the Y-complex, a key component of the nuclear pore complex (NPC) scaffold. Studies on a null mutation in mice previously revealed that Nup133 is essential for embryonic development but not for mouse embryonic stem cell (mESC) proliferation. Using single pore detection and average NE-fluorescence intensity, we find that Nup133 is dispensable for interphase and postmitotic NPC scaffold assembly in pluripotent mESCs. However, loss of Nup133 specifically perturbs the formation of the nuclear basket as manifested by the absence of Tpr in about half of the NPCs combined with altered dynamics of Nup153. We further demonstrate that its central domain mediates Nup133's role in assembling Tpr and Nup153 into a properly configured nuclear basket. Our findings thus revisit the role of the Y-complex in pore biogenesis and provide insights into the interplay between NPC scaffold architecture, nuclear basket assembly and the generation of heterogeneity among NPCs.

Moderate Nucleoporin 133 deficiency leads to glomerular damage in zebrafish

“…Nup133 knockdown may also alter nuclear mechanotransduction, for instance by interfering with the ‘linker of nucleoskeleton and cytoskeleton’ (LINC) complex that establish a stable and cross-linked network between the cytoskeleton and the lamina underneath the NE (reviewed in 47 ). Indeed, while Nup133 is required for the proper assembly of the NPC basket 48 , intimate links between the NPC basket, the LINC subunit Sun1 and Lamin-C have been reported 49 , 50 . As previously discussed 16 , deregulation of mechanotransduction signaling pathways may in turn affect podocytes that are subjected to mechanical stretching caused by capillary pressure.…”

“…The plasmid was generated using PCR amplification using proofreading DNA polymerases (Phusion HF, NEB) and NEBuilder HiFi DNA Assembly Cloning Kits. Briefly, the sequence encoding three copies of the influenza virus hemagglutinin ( HA ) epitope was amplified from pFA6a-3HA-kanMX6 66 , the mCherry was amplified from plasmid #1937 48 , full-length zebrafish nup133 was amplified from a Dharmacon cDNA vector (Clone ID: 2600558) and the three PCR products were inserted by recombination in a pBluescript KSM vector (Stratagene). PCR-amplified fragments and junctions were checked by sequencing.…”

G4C2 repeat RNA mediates the disassembly of the nuclear pore complex in C9orf72 ALS/FTD

“…We note that loss of POM121 alters the human neuronal nuclear repertoire of Nups and it is likely the combinatorial effect of these changes that impacts Intriguingly, POM121 overexpression or loss only affects those Nups that are disrupted in C9orf72 iPSNs or human motor cortex suggesting that this specific subset of Nups (Nup50, TPR, GP210, NDC1, POM121, Nup107, Nup133) are physically, functionally, and/or mechanistically linked in human neurons. This is supported by studies in dividing cells suggesting functional and mechanistic links between a subset of these Nups during NPC assembly (Antonin et al, 2005;Beck and Hurt, 2017;Doucet et al, 2010;Lin and Hoelz, 2019;Mitchell et al, 2010;Otsuka and Ellenberg, 2018;Souquet et al, 2018).…”

Nuclear pore complexes in development and tissue homeostasis

“…How Nup133 regulates ES differentiation is still poorly understood, but merm mutant cells and ES cells depleted of this nucleoporin do not show alterations in NPC assembly. Instead, loss of Nup133 in ES cells has been found to affect the formation of the nuclear basket and the dynamics of the basket nucleoporins Tpr and Nup153 (Souquet et al, 2018). These findings oppose studies using cancer cells, where Nup133 downregulation reduces NPC numbers and inhibit interphase NPC assembly in particular (Doucet et al, 2010).…”