Postmitotic Nuclear Pore Complex Assembly Research Articles

Biallelic NDC1 variants that interfere with ALADIN binding are associated with neuropathy and triple A-like syndrome

Nuclear pore complexes (NPC) regulate nucleocytoplasmic transport and are anchored in the nuclear envelope by the transmembrane nucleoporin NDC1. NDC1 is essential for post-mitotic NPC assembly and the recruitment of ALADIN to the nuclear envelope. While no human disorder has been associated to one of the three transmembrane nucleoporins, biallelic variants in AAAS, encoding ALADIN, cause Triple-A syndrome (Allgrove syndrome). Triple A syndrome, characterized by alacrima, achalasia and adrenal insufficiency, often includes progressive demyelinating polyneuropathy and other neurological complaints. In this report, diagnostic exome and/or RNA-sequencing was performed in 7 individuals from 4 unrelated consanguineous families with AAAS negative triple A syndrome. Molecular and clinical studies followed to elucidate the pathogenic mechanism. The affected individuals presented with intellectual disability, motor impairment, severe demyelinating with secondary axonal polyneuropathy, alacrima and achalasia.. None of the affected individuals has adrenal insufficiency. All individuals presented with biallelic NDC1 in-frame deletions or missense variants, that affect amino acids and protein domains required for ALADIN binding. No other significant variants associated with the phenotypic features were reported. Skin fibroblasts derived from affected individuals show decreased recruitment of ALADIN to the NE and decreased post-mitotic NPC insertion, confirming pathogenicity of the variants. Taken together, our results implicate biallelic NDC1 variants in the pathogenesis of polyneuropathy and a Triple-A-like disorder without adrenal insufficiency, by interfering with physiological NDC1 functions, including the recruitment of ALADIN to the NPC.

A checkpoint function for Nup98 in nuclear pore formation suggested by novel inhibitory nanobodies

Nuclear pore complex (NPC) biogenesis is a still enigmatic example of protein self-assembly. We now introduce several cross-reacting anti-Nup nanobodies for imaging intact nuclear pore complexes from frog to human. We also report a simplified assay that directly tracks postmitotic NPC assembly with added fluorophore-labeled anti-Nup nanobodies. During interphase, NPCs are inserted into a pre-existing nuclear envelope. Monitoring this process is challenging because newly assembled NPCs are indistinguishable from pre-existing ones. We overcame this problem by inserting Xenopus-derived NPCs into human nuclear envelopes and using frog-specific anti-Nup nanobodies for detection. We further asked whether anti-Nup nanobodies could serve as NPC assembly inhibitors. Using a selection strategy against conserved epitopes, we obtained anti-Nup93, Nup98, and Nup155 nanobodies that block Nup–Nup interfaces and arrest NPC assembly. We solved structures of nanobody-target complexes and identified roles for the Nup93 α-solenoid domain in recruiting Nup358 and the Nup214·88·62 complex, as well as for Nup155 and the Nup98 autoproteolytic domain in NPC scaffold assembly. The latter suggests a checkpoint linking pore formation to the assembly of the Nup98-dominated permeability barrier.

Phosphorylation of ELYS promotes its interaction with VAPB at decondensing chromosomes during mitosis.

ELYS is a nucleoporin that localizes to the nuclear side of the nuclear pore complex (NPC) in interphase cells. In mitosis, it serves as an assembly platform that interacts with chromatin and then with nucleoporin subcomplexes to initiate post-mitotic NPC assembly. Here we identify ELYS as a major binding partner of the membrane protein VAPB during mitosis. In mitosis, ELYS becomes phosphorylated at many sites, including a predicted FFAT (two phenylalanines in an acidic tract) motif, which mediates interaction with the MSP (major sperm protein)-domain of VAPB. Binding assays using recombinant proteins or cell lysates and co-immunoprecipitation experiments show that VAPB binds the FFAT motif of ELYS in a phosphorylation-dependent manner. In anaphase, the two proteins co-localize to the non-core region of the newly forming nuclear envelope. Depletion of VAPB results in prolonged mitosis, slow progression from meta- to anaphase and in chromosome segregation defects. Together, our results suggest a role of VAPB in mitosis upon recruitment to or release from ELYS at the non-core region of the chromatin in a phosphorylation-dependent manner.

A quantitative map of nuclear pore assembly reveals two distinct mechanisms

Understanding how the nuclear pore complex (NPC) is assembled is of fundamental importance to grasp the mechanisms behind its essential function and understand its role during the evolution of eukaryotes1–4. There are at least two NPC assembly pathways—one during the exit from mitosis and one during nuclear growth in interphase—but we currently lack a quantitative map of these events. Here we use fluorescence correlation spectroscopy calibrated live imaging of endogenously fluorescently tagged nucleoporins to map the changes in the composition and stoichiometry of seven major modules of the human NPC during its assembly in single dividing cells. This systematic quantitative map reveals that the two assembly pathways have distinct molecular mechanisms, in which the order of addition of two large structural components, the central ring complex and nuclear filaments are inverted. The dynamic stoichiometry data was integrated to create a spatiotemporal model of the NPC assembly pathway and predict the structures of postmitotic NPC assembly intermediates.

Inherited nuclear pore substructures template post-mitotic pore assembly.

Nuclear envelope assembly during late mitosis includes rapid formation of several thousand complete nuclear pore complexes (NPCs). This efficient use of NPC components (nucleoporins or "NUPs") is essential for ensuring immediate nucleocytoplasmic communication in each daughter cell. We show that octameric subassemblies of outer and inner nuclear pore rings remain intact in the mitotic endoplasmic reticulum (ER) after NPC disassembly during prophase. These "inherited" subassemblies then incorporate into NPCs during post-mitotic pore formation. We further show that the stable subassemblies persist through multiple rounds of cell division and the accompanying rounds of NPC mitotic disassembly and post-mitotic assembly. De novo formation of NPCs from newly synthesized NUPs during interphase will then have a distinct initiation mechanism. We postulate that a yet-to-be-identified modification marks and "immortalizes" one or more components of the specific octameric outer and inner ring subcomplexes that then template post-mitotic NPC assembly during subsequent cell cycles.

Nuclear Pore Complexes Cluster in Dysmorphic Nuclei of Normal and Progeria Cells during Replicative Senescence.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare premature aging disease caused by a mutation in LMNA. A G608G mutation in exon 11 of LMNA is responsible for most HGPS cases, generating a truncated protein called “progerin”. Progerin is permanently farnesylated and accumulates in HGPS cells, causing multiple cellular defects such as nuclear dysmorphism, a thickened lamina, loss of heterochromatin, premature senescence, and clustering of Nuclear Pore Complexes (NPC). To identify the mechanism of NPC clustering in HGPS cells, we evaluated post-mitotic NPC assembly in control and HGPS cells and found no defects. Next, we examined the occurrence of NPC clustering in control and HGPS cells during replicative senescence. We reported that NPC clustering occurs solely in the dysmorphic nuclei of control and HGPS cells. Hence, NPC clustering occurred at a higher frequency in HGPS cells compared to control cells at early passages; however, in late cultures with similar senescence index, NPCs clustering occurred at a similar rate in both control and HGPS. Our results show that progerin does not disrupt post-mitotic reassembly of NPCs. However, NPCs frequently cluster in dysmorphic nuclei with a high progerin content. Additionally, nuclear envelope defects that arise during replicative senescence cause NPC clustering in senescent cells with dysmorphic nuclei.

The nucleoporin ELYS regulates nuclear size by controlling NPC number and nuclear import capacity.

How intracellular organelles acquire their characteristic sizes is a fundamental question in cell biology. Given stereotypical changes in nuclear size in cancer, it is important to understand the mechanisms that control nuclear size in human cells. Using a high-throughput imaging RNAi screen, we identify and mechanistically characterize ELYS, a nucleoporin required for post-mitotic nuclear pore complex (NPC) assembly, as a determinant of nuclear size in mammalian cells. ELYS knockdown results in small nuclei, reduced nuclear lamin B2 localization, lower NPC density, and decreased nuclear import. Increasing nuclear import by importin α overexpression rescues nuclear size and lamin B2 import, while inhibiting importin α/β-mediated nuclear import decreases nuclear size. Conversely, ELYS overexpression increases nuclear size, enriches nuclear lamin B2 at the nuclear periphery, and elevates NPC density and nuclear import. Consistent with these observations, knockdown or inhibition of exportin 1 increases nuclear size. Thus, we identify ELYS as a novel positive effector of mammalian nuclear size and propose that nuclear size is sensitive to NPC density and nuclear import capacity.

Chapter 10 - Imaging the Assembly, Structure, and Function of the Nuclear Pore Inside Cells

The nuclear pore complex (NPC) mediates selective transport across the nuclear envelope (NE) and plays crucial roles in several additional cellular functions. In higher eukaryotes, the NPC and the NE disassemble and reassemble during cell division and live-cell imaging has been a powerful tool to analyze these dynamic processes. Here, we present a method for the kinetic analysis of postmitotic NPC assembly and reestablishment of transport competence in intact cells by multicolor 4D imaging and photoswitching. By applying the methods we have established previously using normal rat kidney to HeLa cells, we demonstrate the conservation of NPC assembly in different mammalian cells. We recently showed that the molecular organization of the NPC can be studied by combining stochastic super-resolution microscopy with single-particle averaging and present this method here in detail.

Structural and Functional Studies of the 252 kDa Nucleoporin ELYS Reveal Distinct Roles for Its Three Tethered Domains

In metazoa, the nuclear envelope (NE), together with the embedded nuclear pore complexes (NPCs), breaks down and reassembles during cell division. It is suggested that ELYS, a nucleoporin, binds to chromatin in an initial step of postmitotic NPC assembly and subsequently recruits the essential Y-subcomplex, the major scaffolding unit of the NPC. Here, we show that ELYS contains three domains: an N-terminal β-propeller domain, a central α-helical domain, and a C-terminal disordered region. While the disordered region is responsible for the interactions with chromatin, the two preceding domains synergistically mediate tethering to the NPC. We present the crystal structure of the seven-bladed β-propeller domain at 1.9Å resolution. Analysis of the β-propeller surface reveals the regions that are required for NPC anchorage. We discuss the possible roles of ELYS in the context of the NPC scaffold architecture.

Nuclear pore formation but not nuclear growth is governed by cyclin-dependent kinases (Cdks) during interphase

Nuclear volume and the number of nuclear pore complexes (NPCs) on the nucleus almost double during interphase in dividing cells. How these events are coordinated with the cell cycle is poorly understood, particularly in mammalian cells. We report here, based on newly developed techniques for visualizing NPC formation, that cyclin-dependent kinases (Cdks), especially Cdk1 and Cdk2, promote interphase NPC formation in human dividing cells. Cdks seem to drive an early step of NPC formation because Cdk inhibition suppressed generation of 'nascent pores', which we argue are immature NPCs under the formation process. Consistent with this, Cdk inhibition disturbed proper expression and localization of some nucleoporins, including Elys/Mel-28, which triggers postmitotic NPC assembly. Strikingly, Cdk suppression did not notably affect nuclear growth, suggesting that interphase NPC formation and nuclear growth have distinct regulation mechanisms.

Importin β Regulates the Seeding of Chromatin with Initiation Sites for Nuclear Pore Assembly

The nuclear envelope of higher eukaryotic cells reforms at the exit from mitosis, in concert with the assembly of nuclear pore complexes (NPCs). The first step in postmitotic NPC assembly involves the "seeding" of chromatin with ELYS and the Nup107-160 complex. Subsequent steps in the assembly process are poorly understood and different mechanistic models have been proposed to explain the formation of the full supramolecular structure. Here, we show that the initial step of chromatin seeding is negatively regulated by importin beta. Direct imaging of the chromatin attachment sites reveals single sites situated predominantly on the highest substructures of chromatin surface and lacking any sign of annular structures or oligomerized pre-NPCs. Surprisingly, the inhibition by importin beta is only partially reversed by RanGTP. Importin beta forms a high-molecular-weight complex with both ELYS and the Nup107-160 complex in cytosol. We suggest that initiation sites for NPC assembly contain single copies of chromatin-bound ELYS/Nup107-160 and that the lateral oligomerization of these subunits depends on the recruitment of membrane components. We predict that additional regulators, besides importin beta and Ran, may be involved in coordinating the initial seeding of chromatin with subsequent steps in the NPC assembly pathway.

MEL‐28/ELYS is required for the recruitment of nucleoporins to chromatin and postmitotic nuclear pore complex assembly

The metazoan nuclear envelope (NE) breaks down and re-forms during each cell cycle. Nuclear pore complexes (NPCs), which allow nucleocytoplasmic transport during interphase, assemble into the re-forming NE at the end of mitosis. Using in vitro NE assembly, we show that the vertebrate homologue of MEL-28 (maternal effect lethal), a recently discovered NE component in Caenorhabditis elegans, functions in postmitotic NPC assembly. MEL-28 interacts with the Nup107-160 complex (Nup for nucleoporin), an important building block of the NPC, and is essential for the recruitment of the Nup107-160 complex to chromatin. We suggest that MEL-28 acts as a seeding point for NPC assembly.

The Conserved Nup107-160 Complex Is Critical for Nuclear Pore Complex Assembly

Nuclear pore complexes (NPCs) are large multiprotein assemblies that allow traffic between the cytoplasm and the nucleus. During mitosis in higher eukaryotes, the Nuclear Envelope (NE) breaks down and NPCs disassemble. How NPCs reassemble and incorporate into the NE upon mitotic exit is poorly understood. We demonstrate a function for the conserved Nup107-160 complex in this process. Partial in vivo depletion of Nup133 or Nup107 via RNAi in HeLa cells resulted in reduced levels of multiple nucleoporins and decreased NPC density in the NE. Immunodepletion of the entire Nup107-160 complex from in vitro nuclear assembly reactions produced nuclei with a continuous NE but no NPCs. This phenotype was reversible only if Nup107-160 complex was readded before closed NE formation. Depletion also prevented association of FG-repeat nucleoporins with chromatin. We propose a stepwise model in which postmitotic NPC assembly initiates on chromatin via early recruitment of the Nup107-160 complex.