Phosphorylation of Nup98 by multiple kinases is crucial for NPC disassembly during mitotic entry.

Disassembly of nuclear pore complexes (NPCs) is a decisive event during mitotic entry in cells undergoing open mitosis, yet the molecular mechanisms underlying NPC disassembly are unknown. Using chemical inhibition and depletion experiments we show that NPC disassembly is a phosphorylation-driven process, dependent on CDK1 activity and supported by members of the NIMA-related kinase (Nek) family. We identify phosphorylation of the GLFG-repeat nucleoporin Nup98 as an important step in mitotic NPC disassembly. Mitotic hyperphosphorylation of Nup98 is accomplished by multiple kinases, including CDK1 and Neks. Nuclei carrying a phosphodeficient mutant of Nup98 undergo nuclear envelope breakdown slowly, such that both the dissociation of Nup98 from NPCs and the permeabilization of the nuclear envelope are delayed. Together, our data provide evidence for a phosphorylation-dependent mechanism underlying disintegration of NPCs during prophase. Moreover, we identify mitotic phosphorylation of Nup98 as a rate-limiting step in mitotic NPC disassembly.

The nucleoporin Nup50 activates the Ran guanine nucleotide exchange factor RCC1 to promote NPC assembly at the end of mitosis.

During mitotic exit, thousands of nuclear pore complexes (NPCs) assemble concomitant with the nuclear envelope to build a transport-competent nucleus. Here, we show that Nup50 plays a crucial role in NPC assembly independent of its well-established function in nuclear transport. RNAi-mediated downregulation in cells or immunodepletion of Nup50 protein in Xenopus egg extracts interferes with NPC assembly. We define a conserved central region of 46 residues in Nup50 that is crucial for Nup153 and MEL28/ELYS binding, and for NPC interaction. Surprisingly, neither NPC interaction nor binding of Nup50 to importin α/ß, the GTPase Ran, or chromatin is crucial for its function in the assembly process. Instead, an N-terminal fragment of Nup50 can stimulate the Ran GTPase guanine nucleotide exchange factor RCC1 and NPC assembly, indicating that Nup50 acts via the Ran system in NPC reformation at the end of mitosis. In support of this conclusion, Nup50 mutants defective in RCC1 binding and stimulation cannot replace the wild-type protein in in vitro NPC assembly assays, whereas excess RCC1 can compensate the loss of Nup50.

The second half of mitosis and its implications in cancer biology.

The nucleus undergoes dramatic structural and functional changes during cell division. With the entry into mitosis, in human cells the nuclear envelope breaks down, chromosomes rearrange into rod-like structures which are collected and segregated by the spindle apparatus. While these processes in the first half of mitosis have been intensively studied, much less is known about the second half of mitosis, when a functional nucleus reforms in each of the emerging cells. Here we review our current understanding of mitotic exit and nuclear reformation with spotlights on the links to cancer biology.

FXR proteins bring new perspectives to nucleoporins' homeostasis.

FXR proteins are part of ribonucleoprotein granules controlling stability, translation, and cellular localization of target mRNAs. In the current issue, Agote-Aran et al extend the repertoire of FXR protein action and show that they are also involved in the transport of nuclear pore complex components to the nuclear envelope and thereby prevent cytoplasmic aggregation of these proteins.

Looking outside the box: a comparative cross-kingdom view on the cell biology of the three major lineages of eukaryotic multicellular life.

Many cell biological facts that can be found in dedicated scientific textbooks are based on findings originally made in humans and/or other mammals, including respective tissue culture systems. They are often presented as if they were universally valid, neglecting that many aspects differ-in part considerably-between the three major kingdoms of multicellular eukaryotic life, comprising animals, plants and fungi. Here, we provide a comparative cross-kingdom view on the basic cell biology across these lineages, highlighting in particular essential differences in cellular structures and processes between phyla. We focus on key dissimilarities in cellular organization, e.g. regarding cell size and shape, the composition of the extracellular matrix, the types of cell-cell junctions, the presence of specific membrane-bound organelles and the organization of the cytoskeleton. We further highlight essential disparities in important cellular processes such as signal transduction, intracellular transport, cell cycle regulation, apoptosis and cytokinesis. Our comprehensive cross-kingdom comparison emphasizes overlaps but also marked differences between the major lineages of the three kingdoms and, thus, adds to a more holistic view of multicellular eukaryotic cell biology.

Biallelic variants in CRIPT cause a Rothmund-Thomson-like syndrome with increased cellular senescence.

PURPOSE: Rothmund-Thomson syndrome (RTS) is characterized by poikiloderma, sparse hair, small stature, skeletal defects, cancer, and cataracts, resembling features of premature aging. RECQL4 and ANAPC1 are the 2 known disease genes associated with RTS in >70% of cases. We describe RTS-like features in 5 individuals with biallelic variants in CRIPT (OMIM 615789). METHODS: Two newly identified and 4 published individuals with CRIPT variants were systematically compared with those with RTS using clinical data, computational analysis of photographs, histologic analysis of skin, and cellular studies on fibroblasts. RESULTS: All CRIPT individuals fulfilled the diagnostic criteria for RTS and additionally had neurodevelopmental delay and seizures. Using computational gestalt analysis, CRIPT individuals showed greatest facial similarity with individuals with RTS. Skin biopsies revealed a high expression of senescence markers (p53/p16/p21) and the senescence-associated ß-galactosidase activity was elevated in CRIPT-deficient fibroblasts. RECQL4- and CRIPT-deficient fibroblasts showed an unremarkable mitotic progression and unremarkable number of mitotic errors and no or only mild sensitivity to genotoxic stress by ionizing radiation, mitomycin C, hydroxyurea, etoposide, and potassium bromate. CONCLUSION: CRIPT causes an RTS-like syndrome associated with neurodevelopmental delay and epilepsy. At the cellular level, RECQL4- and CRIPT-deficient cells display increased senescence, suggesting shared molecular mechanisms leading to the clinical phenotypes.

In situ structural analysis of the human nuclear pore complex.

Nuclear pore complexes are fundamental components of all eukaryotic cells that mediate nucleocytoplasmic exchange. Determining their 110-megadalton structure imposes a formidable challenge and requires in situ structural biology approaches. Of approximately 30 nucleoporins (Nups), 15 are structured and form the Y and inner-ring complexes. These two major scaffolding modules assemble in multiple copies into an eight-fold rotationally symmetric structure that fuses the inner and outer nuclear membranes to form a central channel of ~60 nm in diameter. The scaffold is decorated with transport-channel Nups that often contain phenylalanine-repeat sequences and mediate the interaction with cargo complexes. Although the architectural arrangement of parts of the Y complex has been elucidated, it is unclear how exactly it oligomerizes in situ. Here we combine cryo-electron tomography with mass spectrometry, biochemical analysis, perturbation experiments and structural modelling to generate, to our knowledge, the most comprehensive architectural model of the human nuclear pore complex to date. Our data suggest previously unknown protein interfaces across Y complexes and to inner-ring complex members. We show that the transport-channel Nup358 (also known as Ranbp2) has a previously unanticipated role in Y-complex oligomerization. Our findings blur the established boundaries between scaffold and transport-channel Nups. We conclude that, similar to coated vesicles, several copies of the same structural building block--although compositionally identical--engage in different local sets of interactions and conformations.

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 ß-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.

Correction to: Biallelic Variants in the Nuclear Pore Complex Protein NUP93 Are Associated with Non-progressive Congenital Ataxia.

The original version of this article unfortunately contained mistake in Fig. 3 image.

Biallelic Variants in the Nuclear Pore Complex Protein NUP93 Are Associated with Non-progressive Congenital Ataxia.

Nuclear pore complexes (NPCs) are the gateways of the nuclear envelope mediating transport between cytoplasm and nucleus. They form huge complexes of 125 MDa in vertebrates and consist of about 30 different nucleoporins present in multiple copies in each complex. Here, we describe pathogenic variants in the nucleoporin 93 (NUP93) associated with an autosomal recessive form of congenital ataxia. Two rare compound heterozygous variants of NUP93 were identified by whole exome sequencing in two brothers with isolated cerebellar atrophy: one missense variant (p.R537W) results in a protein which does not localize to NPCs and cannot functionally replace the wild type protein, whereas the variant (p.F699L) apparently supports NPC assembly. In addition to its recently described pathological role in steroid-resistant nephrotic syndrome, our work identifies NUP93 as a candidate gene for non-progressive congenital ataxia.

Perforating the nuclear boundary - how nuclear pore complexes assemble.

The nucleus is enclosed by the nuclear envelope, a double membrane which creates a selective barrier between the cytoplasm and the nuclear interior. Its barrier and transport characteristics are determined by nuclear pore complexes (NPCs) that are embedded within the nuclear envelope, and control molecular exchange between the cytoplasm and nucleoplasm. In this Commentary, we discuss the biogenesis of these huge protein assemblies from approximately one thousand individual proteins. We will summarize current knowledge about distinct assembly modes in animal cells that are characteristic for different cell cycle phases and their regulation.

The lysine demethylase LSD1 is required for nuclear envelope formation at the end of mitosis.

The metazoan nucleus breaks down and reassembles during each cell division. Upon mitotic exit, the successful reestablishment of an interphase nucleus requires the coordinated reorganization of chromatin and formation of a functional nuclear envelope. Here, we report that the histone demethylase LSD1 (also known as KDM1A) plays a crucial role in nuclear assembly at the end of mitosis. Downregulation of LSD1 in cells extends telophase and impairs nuclear pore complex assembly. In vitro, LSD1 demethylase activity is required for the recruitment of MEL28 (also known as ELYS and AHCTF1) and nuclear envelope precursor vesicles to chromatin, crucial steps in nuclear reassembly. Accordingly, the formation of a closed nuclear envelope and nuclear pore complex assembly are impaired upon depletion of LSD1 or inhibition of its activity. Our results identify histone demethylation by LSD1 as a new regulatory mechanism linking the chromatin state and nuclear envelope formation at the end of mitosis.

A single herpesvirus protein can mediate vesicle formation in the nuclear envelope.

Herpesviruses assemble capsids in the nucleus and egress by unconventional vesicle-mediated trafficking through the nuclear envelope. Capsids bud at the inner nuclear membrane into the nuclear envelope lumen. The resulting intralumenal vesicles fuse with the outer nuclear membrane, delivering the capsids to the cytoplasm. Two viral proteins are required for vesicle formation, the tail-anchored pUL34 and its soluble interactor, pUL31. Whether cellular proteins are involved is unclear. Using giant unilamellar vesicles, we show that pUL31 and pUL34 are sufficient for membrane budding and scission. pUL34 function can be bypassed by membrane tethering of pUL31, demonstrating that pUL34 is required for pUL31 membrane recruitment but not for membrane remodeling. pUL31 can inwardly deform membranes by oligomerizing on their inner surface to form buds that constrict to vesicles. Therefore, a single viral protein can mediate all events necessary for membrane budding and abscission.

Dimerization and direct membrane interaction of Nup53 contribute to nuclear pore complex assembly.

Nuclear pore complexes (NPCs) fuse the two membranes of the nuclear envelope (NE) to a pore, connecting cytoplasm and nucleoplasm and allowing exchange of macromolecules between these compartments. Most NPC proteins do not contain integral membrane domains and thus it is largely unclear how NPCs are embedded and anchored in the NE. Here, we show that the evolutionary conserved nuclear pore protein Nup53 binds independently of other proteins to membranes, a property that is crucial for NPC assembly and conserved between yeast and vertebrates. The vertebrate protein comprises two membrane binding sites, of which the C-terminal domain has membrane deforming capabilities, and is specifically required for de novo NPC assembly and insertion into the intact NE during interphase. Dimerization of Nup53 contributes to its membrane interaction and is crucial for its function in NPC assembly.

Interaction of Nup53 with Ndc1 and Nup155 is 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.

The diverse roles of the Nup93/Nic96 complex proteins - structural scaffolds of the nuclear pore complex with additional cellular functions.

Nuclear pore complexes mediate the transport between the cell nucleoplasm and cytoplasm. These 125 MDa structures are among the largest assemblies found in eukaryotes, built from proteins organized in distinct subcomplexes that act as building blocks during nuclear pore complex biogenesis. In this review, we focus on one of these subcomplexes, the Nup93 complex in metazoa and its yeast counterpart, the Nic96 complex. We discuss its essential function in nuclear pore complex assembly as a linker between the nuclear membrane and the central part of the pore and its various roles in nuclear transport processes and beyond.

Building a nuclear envelope at the end of mitosis: coordinating membrane reorganization, nuclear pore complex assembly, and chromatin de-condensation.

The metazoan nucleus is disassembled and re-built at every mitotic cell division. The nuclear envelope, including nuclear pore complexes, breaks down at the beginning of mitosis to accommodate the capture of massively condensed chromosomes by the spindle apparatus. At the end of mitosis, a nuclear envelope is newly formed around each set of segregating and de-condensing chromatin. We review the current understanding of the membrane restructuring events involved in the formation of the nuclear membrane sheets of the envelope, the mechanisms governing nuclear pore complex assembly and integration in the nascent nuclear membranes, and the regulated coordination of these events with chromatin de-condensation.

Distinct domains in Ndc1 mediate its interaction with the Nup84 complex and the nuclear membrane.

Nuclear pore complexes (NPCs) are embedded in the nuclear envelope and built from ∼30 different nucleoporins (Nups) in multiple copies, few are integral membrane proteins. One of these transmembrane nucleoporins, Ndc1, is thought to function in NPC assembly at the fused inner and outer nuclear membranes. Here, we show a direct interaction of Ndc1's transmembrane domain with Nup120 and Nup133, members of the pore membrane coating Y-complex. We identify an amphipathic helix in Ndc1's C-terminal domain binding highly curved liposomes. Upon overexpression, this amphipathic motif is toxic and dramatically alters the intracellular membrane organization in yeast. Ndc1's amphipathic motif functionally interacts with related motifs in the C-terminus of the nucleoporins Nup53 and Nup59, important for pore membrane binding and interconnecting NPC modules. The essential function of Ndc1 can be suppressed by deleting the amphipathic helix from Nup53. Our data indicate that nuclear membrane and presumably NPC biogenesis depends on a balanced ratio between amphipathic motifs in diverse nucleoporins.