The Nuclear Pore Complex consists of two independent scaffolds

Regmi, Saroj G.; Lee, Hangnoh; Kaufhold, Ross; Fichtman, Boris; Chen, Shane; Aksenova, Vasilisa; Turcotte, Elizabeth; Harel, Amnon; Arnaoutov, Alexei; Dasso, Mary

doi:10.1101/2020.11.13.381947

Cited by 12 publications

(12 citation statements)

References 42 publications

(68 reference statements)

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“…Thus, the order of NPC disassembly in the C. elegans embryo appears to be different from that of human cells, with some nucleoporin subcomplexes leaving the NPC earlier without affecting the permeability barrier. This echoes one study in human cells in which Auxin-mediated depletion of the entire Y-complex had little impact on the stability of the inner ring complex and the central channel nucleoporins ( 60 ). The functional relevance of the earlier departure of the nuclear basket and the Y complex subunits from the NPC in C. elegans as compared to human cells is presently unclear.…”

Section: Discussionsupporting

confidence: 68%

Mechanisms of Nuclear Pore Complex disassembly by the mitotic Polo-Like Kinase 1 (PLK-1) inC. elegansembryos

Nkoula

Velez-Aguilera

Ossareh-Nazari

et al. 2023

Preprint

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The nuclear envelope, which protects and organizes the interphase genome, is dismantled during mitosis. In the C. elegans zygote, nuclear envelope breakdown (NEBD) of the parental pronuclei is spatially and temporally regulated during mitosis to promote the unification of the parental genomes. During NEBD, Nuclear Pore Complex (NPC) disassembly is critical for rupturing the nuclear permeability barrier and removing the NPCs from the membranes near the centrosomes and between the juxtaposed pronuclei. By combining live imaging, biochemistry, and phosphoproteomics, we characterized NPC disassembly and unveiled the exact role of the mitotic kinase PLK-1 in this process. We show that PLK-1 disassembles the NPC by targeting multiple NPC sub-complexes, including the cytoplasmic filaments, the central channel, and the inner ring. Notably, PLK-1 is recruited to and phosphorylates intrinsically disordered regions of several multivalent linker nucleoporins, a mechanism that appears to be an evolutionarily conserved driver of NPC disassembly during mitosis.

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Section: Discussionsupporting

confidence: 68%

Mechanisms of Nuclear Pore Complex disassembly by the mitotic Polo-Like Kinase 1 (PLK-1) inC. elegansembryos

Nkoula

Velez-Aguilera

Ossareh-Nazari

et al. 2023

Preprint

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“…The unexpected discovery of the presence and unique role of NUP93 in crosslinking the outer ring spokes of the human NPC, along with its organizing role in the inner ring as both scaffold and linker, exemplifies the reuse of linker-scaffold functional units at completely different locations of the NPC. Its ubiquity rationalizes the observation that rapid degron-induced depletion of NUP93 leads to the concomitant loss of both inner and outer rings from the nuclear pore, legitimizing NUP93 as a 'lynchpin' of the NPC (76). These findings further inform the mechanistic bases for pathologies like steroid-resistant 30 (which was not certified by peer review) is the author/funder.…”

Section: Discussionmentioning

confidence: 69%

Architecture of the linker-scaffold in the nuclear pore

Petrovic

Samanta

Perriches

et al. 2022

Science

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INTRODUCTION In eukaryotic cells, the selective bidirectional transport of macromolecules between the nucleus and cytoplasm occurs through the nuclear pore complex (NPC). Embedded in nuclear envelope pores, the ~110-MDa human NPC is an ~1200-Å-wide and ~750-Å-tall assembly of ~1000 proteins, collectively termed nucleoporins. Because of the NPC’s eightfold rotational symmetry along the nucleocytoplasmic axis, each of the ~34 different nucleoporins occurs in multiples of eight. Architecturally, the NPC’s symmetric core is composed of an inner ring encircling the central transport channel and two outer rings anchored on both sides of the nuclear envelope. Because of its central role in the flow of genetic information from DNA to RNA to protein, the NPC is commonly targeted in viral infections and its nucleoporin constituents are associated with a plethora of diseases. RATIONALE Although the arrangement of most scaffold nucleoporins in the NPC’s symmetric core was determined by quantitative docking of crystal structures into cryo–electron tomographic (cryo-ET) maps of intact NPCs, the topology and molecular details of their cohesion by multivalent linker nucleoporins have remained elusive. Recently, in situ cryo-ET reconstructions of NPCs from various species have indicated that the NPC’s inner ring is capable of reversible constriction and dilation in response to variations in nuclear envelope membrane tension, thereby modulating the diameter of the central transport channel by ~200 Å. We combined biochemical reconstitution, high-resolution crystal and single-particle cryo–electron microscopy (cryo-EM) structure determination, docking into cryo-ET maps, and physiological validation to elucidate the molecular architecture of the linker-scaffold interaction network that not only is essential for the NPC’s integrity but also confers the plasticity and robustness necessary to allow and withstand such large-scale conformational changes. RESULTS By biochemically mapping scaffold-binding regions of all fungal and human linker nucleoporins and determining crystal and single-particle cryo-EM structures of linker-scaffold complexes, we completed the characterization of the biochemically tractable linker-scaffold network and established its evolutionary conservation, despite considerable sequence divergence. We determined a series of crystal and single-particle cryo-EM structures of the intact Nup188 and Nup192 scaffold hubs bound to their Nic96, Nup145N, and Nup53 linker nucleoporin binding regions, revealing that both proteins form distinct question mark–shaped keystones of two evolutionarily conserved hetero‑octameric inner ring complexes. Linkers bind to scaffold surface pockets through short defined motifs, with flanking regions commonly forming additional disperse interactions that reinforce the binding. Using a structure‑guided functional analysis in Saccharomyces cerevisiae , we confirmed the robustness of linker‑scaffold interactions and established the physiological relevance of our biochemical and structural findings. The near-atomic composite structures resulting from quantitative docking of experimental structures into human and S. cerevisiae cryo-ET maps of constricted and dilated NPCs structurally disambiguated the positioning of the Nup188 and Nup192 hubs in the intact fungal and human NPC and revealed the topology of the linker-scaffold network. The linker-scaffold gives rise to eight relatively rigid inner ring spokes that are flexibly interconnected to allow for the formation of lateral channels. Unexpectedly, we uncovered that linker‑scaffold interactions play an opposing role in the outer rings by forming tight cross-link staples between the eight nuclear and cytoplasmic outer ring spokes, thereby limiting the dilatory movements to the inner ring. CONCLUSION We have substantially advanced the structural and biochemical characterization of the symmetric core of the S. cerevisiae and human NPCs and determined near-atomic composite structures. The composite structures uncover the molecular mechanism by which the evolutionarily conserved linker‑scaffold establishes the NPC’s integrity while simultaneously allowing for the observed plasticity of the central transport channel. The composite structures are roadmaps for the mechanistic dissection of NPC assembly and disassembly, the etiology of NPC‑associated diseases, the role of NPC dilation in nucleocytoplasmic transport of soluble and integral membrane protein cargos, and the anchoring of asymmetric nucleoporins. Linker-scaffold architecture in the human NPC’s symmetric core. Near‑atomic composite structure of the NPC’s symmetric core obtained by quantitative docking of high-resolution crystal and single-particle cryo-EM structures into a cryo-ET reconstruction of the intact human NPC. Schematic representations of the intricate linker-scaffold topology of the cytoplasmic outer ring, inner ring, and nuclear outer ring (clockwise from top) are depicted for the boxed regions. C, C terminus; N, N terminus.

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“…Cell culture and Nup96 depletion NUP96::Neon-AID DLD-1 cells 23 were grown in standard tissue culture conditions (37 °C with 5% CO 2 ) using DMEM medium supplemented with 4.5 g l −1 glucose and 4.5 g l −1 sodium pyruvate (Corning), 10% FBS (Gemini Bio) and 2 mM GlutaMAX (Gibco). Next, cells were cultured for 18-24 h on freshly glow-discharged carbon-coated EM gold grids (200 mesh, R1.2/1.3 or R2/1; Quantifoil Micro Tools) before plunge-freezing.…”

Section: Methodsmentioning

confidence: 99%

“…We used cryo-FIB-milled lamellae of human DLD-1 cells containing an auxin-inducible degron (AID) tag at the NUP96 (HGNC symbol: NUP98) loci (homozygous NUP96::Neon-AID) 23 for the targeted depletion of Nup96. Such Nup96 depletion leads to a loss of both the CR and NR elements, whereas the IR is unaffected.…”

Section: The Architecture Of the Npc In Dld-1 Cellsmentioning

confidence: 99%

The cellular environment shapes the nuclear pore complex architecture

Schuller

Wojtynek

Mankus

et al. 2021

Nature

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172

161

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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.

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The Nuclear Pore Complex consists of two independent scaffolds

Cited by 12 publications

References 42 publications

Mechanisms of Nuclear Pore Complex disassembly by the mitotic Polo-Like Kinase 1 (PLK-1) inC. elegansembryos

Mechanisms of Nuclear Pore Complex disassembly by the mitotic Polo-Like Kinase 1 (PLK-1) inC. elegansembryos

Architecture of the linker-scaffold in the nuclear pore

The cellular environment shapes the nuclear pore complex architecture

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