Nuclear Pore Complex Formation Research Articles

Puzzling out nuclear pore complex assembly.

Nuclear pore complexes (NPCs) are sophisticated multiprotein assemblies embedded within the nuclear envelope and controlling the exchanges of molecules between the cytoplasm and the nucleus. In this review, we summarize the mechanisms by which these elaborate complexes are built from their subunits, the nucleoporins, based on our ever-growing knowledge of NPC structural organization and on the recent identification of additional features of this process. We present the constraints faced during the production ofnucleoporins, their gathering into oligomeric complexes, and the formation of NPCs within nuclear envelopes, and review the cellular strategies at play, from co-translational assembly to the enrolment of a panel of cofactors. Remarkably, the study of NPCs can inform our perception of the biogenesis of multiprotein complexes in general - and vice versa.

Reticulon-like REEP4 at the inner nuclear membrane promotes nuclear pore complex formation.

Nuclear pore complexes (NPCs) are channels within the nuclear envelope that mediate nucleocytoplasmic transport. NPCs form within the closed nuclear envelope during interphase or assemble concomitantly with nuclear envelope reformation in late stages of mitosis. Both interphase and mitotic NPC biogenesis require coordination of protein complex assembly and membrane deformation. During early stages of mitotic NPC assembly, a seed for new NPCs is established on chromatin, yet the factors connecting the NPC seed to the membrane of the forming nuclear envelope are unknown. Here, we report that the reticulon homology domain protein REEP4 not only localizes to high-curvature membrane of the cytoplasmic endoplasmic reticulum but is also recruited to the inner nuclear membrane by the NPC biogenesis factor ELYS. This ELYS-recruited pool of REEP4 promotes NPC assembly and appears to be particularly important for NPC formation during mitosis. These findings suggest a role for REEP4 in coordinating nuclear envelope reformation with mitotic NPC biogenesis.

NSP9 of SARS-CoV-2 attenuates nuclear transport by hampering nucleoporin 62 dynamics and functions in host cells

Nuclear pore complexes (NPC) regulate molecular traffics on nuclear envelope, which plays crucial roles during cell fate specification and diseases. The viral accessory protein NSP9 of SARS-CoV-2 is reported to interact with nucleoporin 62 (NUP62), a structural component of the NPC, but its biological impact on the host cell remain obscure. Here, we established new cell line models with ectopic NSP9 expression and determined the subcellular destination and biological functions of NSP9. Confocal imaging identified NSP9 to be largely localized in close proximity to the endoplasmic reticulum. In agreement with the subcellular distribution of NSP9, association of NSP9 with NUP62 was observed in cytoplasm. Furthermore, the overexpression of NSP9 correlated with a reduction of NUP62 expression on the nuclear envelope, suggesting that attenuating NUP62 expression might have contributed to defective NPC formation. Importantly, the loss of NUP62 impaired translocation of p65, a subunit of NF-κB, upon TNF-α stimulation. Concordantly, NSP9 over-expression blocked p65 nuclear transport. Taken together, these data shed light on the molecular mechanisms underlying the modulation of host cells during SARS-CoV-2 infection.

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.

Inhibition of Nuclear Pore Complex Formation Selectively Induces Cancer Cell Death.

Nuclear pore complexes (NPC) are the central mediators of nucleocytoplasmic transport. Increasing evidence shows that many cancer cells have increased numbers of NPCs and become addicted to the nuclear transport machinery. How reducing NPC numbers affects the physiology of normal and cancer cells and whether it could be exploited for cancer therapies has not been investigated. We report that inhibition of NPC formation, a process mostly restricted to proliferating cells, causes selective cancer cell death, prevents tumor growth, and induces tumor regression. Although cancer cells die in response to NPC assembly inhibition, normal cells undergo a reversible cell-cycle arrest that allows them to survive. Mechanistically, reducing NPC numbers results in multiple alterations contributing to cancer cell death, including abnormalities in nuclear transport, catastrophic alterations in gene expression, and the selective accumulation of DNA damage. Our findings uncover the NPC formation process as a novel targetable pathway in cancer cells. SIGNIFICANCE: Reducing NPC numbers in cancer cells induces death, prevents tumor growth, and results in tumor regression. Conversely, normal cells undergo a reversible cell-cycle arrest in response to inhibition of NPC assembly. These findings expose the potential of targeting NPC formation in cancer.This article is highlighted in the In This Issue feature, p. 1.

In This Issue

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Nuclear envelope-vacuole contacts mitigate nuclear pore complex assembly stress.

The intricacy of nuclear pore complex (NPC) biogenesis imposes risks of failure that can cause defects in nuclear transport and nuclear envelope (NE) morphology; however, cellular mechanisms used to alleviate NPC assembly stress are not well defined. In the budding yeast Saccharomyces cerevisiae, we demonstrate that NVJ1- and MDM1-enriched NE-vacuole contacts increase when NPC assembly is compromised in several nup mutants, including nup116ΔGLFG cells. These interorganelle nucleus-vacuole junctions (NVJs) cooperate with lipid droplets to maintain viability and enhance NPC formation in assembly mutants. Additionally, NVJs function with ATG1 to remodel the NE and promote vacuole-dependent degradation of specific nucleoporins in nup116ΔGLFG cells. Importantly, NVJs significantly improve the physiology of NPC assembly mutants, despite having only negligible effects when NPC biogenesis is unperturbed. These results therefore define how NE-vacuole interorganelle contacts coordinate responses to mitigate deleterious cellular effects caused by disrupted NPC assembly.

Mitotic checkpoint protein Mad1 is required for early Nup153 recruitment to chromatin and nuclear envelope integrity.

Nucleoporin Nup153 is a multifunctional protein and a known binding partner of mitotic checkpoint protein Mad1 (also known as MAD1L1). The functional relevance of their interaction has remained elusive. Here, we have further dissected the interface and functional interplay of Nup153 and Mad1. Using in situ proximity ligation assays, we found that the presence of a nuclear envelope (NE) is a prerequisite for the Nup153-Mad1 association. Time-lapse microscopy revealed that depletion of Mad1 delayed recruitment of Nup153 to anaphase chromatin, which was often accompanied by a prolongation of anaphase. Furthermore, as seen by electron microscopic and three-dimensional structured illumination investigations, Nup153 and Mad1 depletion led to alterations in NE architecture, characterised by a change of membrane curvature at nuclear pore complexes (NPCs) and an expansion of the spacing between inner and outer nuclear membranes. Nup153 depletion, but not Mad1 depletion, caused defects in interphase NPC assembly, with partial displacement of cytoplasmic nucleoporins and a reduction in NPC density. Taken together, our results suggest that Nup153 has separable roles in NE and NPC formation: in post-mitotic NE re-formation in concert with Mad1 and in interphase NPC assembly, independent of Mad1.

Structural and biochemical analyses of the nuclear pore complex component ELYS identify residues responsible for nucleosome binding

The nuclear pore complex embedded within the nuclear envelope is the essential architecture for trafficking macromolecules, such as proteins and RNAs, between the cytoplasm and nucleus. The nuclear pore complex assembly occurs on chromatin in the post-mitotic phase of the cell cycle. ELYS (MEL-28/AHCTF1) binds to the nucleosome, which is the basic chromatin unit, and promotes assembly of the complex around the chromosomes in cells. Here we show that the Arg-Arg-Lys (RRK) stretch of the C-terminal ELYS region plays an essential role in the nucleosome binding. The cryo-EM structure and the crosslinking mass spectrometry reveal that the ELYS C-terminal region directly binds to the acidic patch of the nucleosome. These results provide mechanistic insight into the ELYS-nucleosome interaction, which promotes the post-mitotic nuclear pore complex formation around chromosomes in cells.

Roles of Nup133, Nup153 and membrane fenestrations in assembly of the nuclear pore complex at the end of mitosis.

Reassembly of the nuclear pore complex (NPC) at the end of mitosis is an important event for eukaryotic nuclear function. In this study, we examined the dynamic behaviors of the endoplasmic reticulum (ER) by "Live CLEM" imaging. In metaphase, numerous fenestrations on the ER membrane were observed around chromosomes. In telophase, these fenestrations became filled at the region attached to chromosomes, whereas they remained open at the region unattached to chromosomes, suggesting that NPC assembly takes place at fenestrations on the membrane. To determine the roles of nucleoporins in postmitotic NPC formation, we used artificial beads conjugated with anti-GFP antibody, which captures GFP-fused proteins on the beads when incorporated into cells. Live CLEM imaging of telophase cells containing Nup133-coated beads or Nup153-coated beads showed that Nup133 and Nup153, as the sole effector molecules, assembled the NPC-like structure on the membrane fenestrations. Indirect immunofluorescence staining of the Nup133-coated beads showed that Nup133 effectively assembled Nup107 and ELYS, whereas minimal assembly of Nup98 and Nup62 was observed; the Nup153-coated bead effectively assembled Nup98, Nup62 and Pom121, but assembled neither Nup107 nor ELYS. Our results suggest that Nup133 and Nup153 play different roles in assembling the NPC on membrane fenestrations.

A self-inhibitory interaction within Nup155 and membrane binding are required for nuclear pore complex formation.

Nuclear pore complexes (NPCs) are gateways through the nuclear envelope. How they form into a structure containing three rings and integrate into the nuclear envelope remains a challenging paradigm for coordinated assembly of macro-complexes. In vertebrates, the cytoplasmic and nucleoplasmic rings of NPCs are mostly formed by multiple copies of the Nup107-Nup160 complex, whereas the central, or inner ring is composed of Nup53, Nup93, Nup155 and the two paralogues Nup188 and Nup205. Inner ring assembly is only partially understood. Using in vitro nuclear assembly reactions, we show that direct pore membrane binding of Nup155 is crucial for NPC formation. Replacing full-length Nup155 with its N-terminal β-propeller allows assembly of the outer ring components to the NPC backbone that also contains Nup53. However, further assembly, especially recruitment of the Nup93 and Nup62 complexes, is blocked. Self-interaction between the N- and C-terminal domains of Nup155 has an auto-inhibitory function that prevents interaction between the N-terminus of Nup155 and the C-terminal region of Nup53. Nup93 can overcome this block by binding to Nup53, thereby promoting formation of the inner ring and the NPC.

Chm7 and Heh1 collaborate to link nuclear pore complex quality control with nuclear envelope sealing

The integrity of the nuclear envelope barrier relies on membrane remodeling by the ESCRTs, which seal nuclear envelope holes and contribute to the quality control of nuclear pore complexes (NPCs); whether these processes are mechanistically related remains poorly defined. Here, we show that the ESCRT-II/III chimera, Chm7, is recruited to a nuclear envelope subdomain that expands upon inhibition of NPC assembly and is required for the formation of the storage of improperly assembled NPCs (SINC) compartment. Recruitment to sites of NPC assembly is mediated by its ESCRT-II domain and the LAP2-emerin-MAN1 (LEM) family of integral inner nuclear membrane proteins, Heh1 and Heh2. We establish direct binding between Heh2 and the "open" forms of both Chm7 and the ESCRT-III, Snf7, and between Chm7 and Snf7. Interestingly, Chm7 is required for the viability of yeast strains where double membrane seals have been observed over defective NPCs; deletion of CHM7 in these strains leads to a loss of nuclear compartmentalization suggesting that the sealing of defective NPCs and nuclear envelope ruptures could proceed through similar mechanisms.

Transmembrane protein TMEM170A is a newly discovered regulator of ER and nuclear envelope morphogenesis in human cells.

ABSTRACTThe mechanism of endoplasmic reticulum (ER) morphogenesis is incompletely understood. ER tubules are shaped by the reticulons (RTNs) and DP1/Yop1p family members, but the mechanism of ER sheet formation is much less clear. Here, we characterize TMEM170A, a human transmembrane protein, which localizes in ER and nuclear envelope membranes. Silencing or overexpressing TMEM170A in HeLa K cells alters ER shape and morphology. Ultrastructural analysis reveals that downregulation of TMEM170A specifically induces tubular ER formation, whereas overexpression of TMEM170A induces ER sheet formation, indicating that TMEM170A is a newly discovered ER-sheet-promoting protein. Additionally, downregulation of TMEM170A alters nuclear shape and size, decreases the density of nuclear pore complexes (NPCs) in the nuclear envelope and causes either a reduction in inner nuclear membrane (INM) proteins or their relocalization to the ER. TMEM170A interacts with RTN4, a member of the reticulon family; simultaneous co-silencing of TMEM170A and RTN4 rescues ER, NPC and nuclear-envelope-related phenotypes, implying that the two proteins have antagonistic effects on ER membrane organization, and nuclear envelope and NPC formation.

Nup153 Recruits the Nup107-160 Complex to the Inner Nuclear Membrane for Interphasic Nuclear Pore Complex Assembly

In metazoa, nuclear pore complexes (NPCs) are assembled from constituent nucleoporins by two distinct mechanisms: in the re-forming nuclear envelope at the end of mitosis and into the intact nuclear envelope during interphase. Here, we show that the nucleoporin Nup153 is required for NPC assembly during interphase but not during mitotic exit. It functions in interphasic NPC formation by binding directly to the inner nuclear membrane via an N-terminal amphipathic helix. This binding facilitates the recruitment of the Nup107-160 complex, a crucial structural component of the NPC, to assembly sites. Our work further suggests that the nuclear transport receptor transportin and the small GTPase Ran regulate the interaction of Nup153 with the membrane and, in this way, direct pore complex assembly to the nuclear envelope during interphase.

Nuclear envelope: Curving out a nuclear pore.

The basket nucleoproteins Nup1 and Nup60 contribute to nuclear pore complex formation by inducing membrane curvature.

Surveillance of Nuclear Pore Complex Assembly by ESCRT-III/Vps4

The maintenance of nuclear compartmentalization by the nuclear envelope and nuclear pore complexes (NPCs) is essential for cell function; loss of compartmentalization is associated with cancers, laminopathies, and aging. We uncovered a pathway that surveils NPC assembly intermediates to promote the formation of functional NPCs. Surveillance is mediated by Heh2, a member of the LEM (Lap2-emerin-MAN1) family of integral inner nuclear membrane proteins, which binds to an early NPC assembly intermediate, but not to mature NPCs. Heh2 recruits the endosomal sorting complex required for transport (ESCRT)-III subunit Snf7 and the AAA-ATPase Vps4 to destabilize and clear defective NPC assembly intermediates. When surveillance or clearance is compromised, malformed NPCs accumulate in a storage of improperly assembled nuclear pore complexes compartment, or SINC. The SINC is retained in old mothers to prevent loss of daughter lifespan, highlighting a continuum of mechanisms to ensure nuclear compartmentalization.

Nucleosomal regulation of chromatin composition and nuclear assembly revealed by histone depletion.

Nucleosomes are the fundamental unit of chromatin, but analysis of transcription-independent nucleosome functions has been complicated by the gene-expression changes resulting from histone manipulation. Here we solve this dilemma by developing Xenopus laevis egg extracts deficient for nucleosome formation and by analyzing the proteomic landscape and behavior of nucleosomal chromatin and nucleosome-free DNA. We show that although nucleosome-free DNA can recruit nuclear-envelope membranes, nucleosomes are required for spindle assembly and for formation of the lamina and of nuclear pore complexes (NPCs). We show that, in addition to the Ran G-nucleotide exchange factor RCC1, ELYS, the initiator of NPC formation, fails to associate with naked DNA but directly binds histone H2A-H2B dimers and nucleosomes. Tethering ELYS and RCC1 to DNA bypasses the requirement for nucleosomes in NPC formation in a synergistic manner. Thus, the minimal essential function of nucleosomes in NPC formation is to recruit RCC1 and ELYS.

Chapter 11 - Cell-Fusion Method to Visualize Interphase Nuclear Pore Formation

In eukaryotic cells, the nucleus is a complex and sophisticated organelle that organizes genomic DNA to support essential cellular functions. The nuclear surface contains many nuclear pore complexes (NPCs), channels for macromolecular transport between the cytoplasm and nucleus. It is well known that the number of NPCs almost doubles during interphase in cycling cells. However, the mechanism of NPC formation is poorly understood, presumably because a practical system for analysis does not exist. The most difficult obstacle in the visualization of interphase NPC formation is that NPCs already exist after nuclear envelope formation, and these existing NPCs interfere with the observation of nascent NPCs. To overcome this obstacle, we developed a novel system using the cell-fusion technique (heterokaryon method), previously also used to analyze the shuttling of macromolecules between the cytoplasm and the nucleus, to visualize the newly synthesized interphase NPCs. In addition, we used a photobleaching approach that validated the cell-fusion method. We recently used these methods to demonstrate the role of cyclin-dependent protein kinases and of Pom121 in interphase NPC formation in cycling human cells. Here, we describe the details of the cell-fusion approach and compare the system with other NPC formation visualization methods.

Nup53 interaction with Ndc1 and Nup155 are required for nuclear pore complex assembly

Nuclear pore complexes (NPCs) are the gateways for nucleocytoplasmic exchange. The ordered assembly of these huge complexes from several hundred individual components into an intricate protein interaction network which deforms the two membranes of the nuclear envelope into a pore is only rudimentarily understood. Here, we show that the interaction between Nup53 and the integral pore membrane protein Ndc1 is essential for vertebrate NPC assembly. The Ndc1 binding site on Nup53 overlaps with a region that induces membrane bending and is specifically required to modulate this activity, suggesting that the membrane-deforming capability of Nup53 is adjusted during the NPC assembly process. We further demonstrate that the interaction of Nup53 and Nup155 has a crucial role in NPC formation as the main determinant of recruitment of Nup155 to the assembling pore. Overall, our results pinpoint the diversity of interaction modes accomplished by Nup53, highlighting this protein as an essential link between the pore membrane and the NPC, and as a crucial factor in the formation of the pore membrane.

Small GTPase Ran and Ran-binding proteins.

Abstract Like many other small GTPases, Ran functions in eukaryotic cells as a molecular switch that cycles between GTP- and GDP-bound forms. Through the proper modulation of the GTP/GDP cycle, Ran functions with a number of Ran-binding proteins to control a broad array of fundamental cellular functions, including nucleocytoplasmic transport, mitotic spindle assembly, and nuclear envelope and nuclear pore complex formation. Recent studies have revealed that 'Ran and Ran binding proteins' are involved in a variety of functions involving cell fate determination, including cell death, cell proliferation, cell differentiation, and malignant transformation. In this review, we discuss recent progress on the functional link between the Ran system and tumorigenesis, which give clues to the molecular understanding of cancer biology.