Dynein recruitment to nuclear pores activates apical nuclear migration and mitotic entry in brain progenitor cells.

Radial glial progenitors (RGPs) are elongated epithelial cells that give rise to neurons, glia, and adult stem cells during brain development. RGP nuclei migrate basally during G1, apically using cytoplasmic dynein during G2, and undergo mitosis at the ventricular surface. By live imaging of in utero electroporated rat brain, we find that two distinct G2-specific mechanisms for dynein nuclear pore recruitment are essential for apical nuclear migration. The "RanBP2-BicD2" and "Nup133-CENP-F" pathways act sequentially, with Nup133 or CENP-F RNAi arresting nuclei close to the ventricular surface in a premitotic state. Forced targeting of dynein to the nuclear envelope rescues nuclear migration and cell-cycle progression, demonstrating that apical nuclear migration is not simply correlated with cell-cycle progression from G2 to mitosis, but rather, is a required event. These results reveal that cell-cycle control of apical nuclear migration occurs by motor protein recruitment and identify a role for nucleus- and centrosome-associated forces in mitotic entry. PAPERCLIP:

Y-complex nucleoporins independently contribute to nuclear pore assembly and gene regulation in neuronal progenitors.

Besides assembling nuclear pore complexes, the conduits of nuclear transport, many nucleoporins also contribute to chromatin organization and gene expression, with critical roles in development and pathologies. We previously reported that Nup133 and Seh1, two components of the Y-complex subassembly of the nuclear pore scaffold, are dispensable for mouse embryonic stem cell viability but required for their survival during neuroectodermal differentiation. Here, a transcriptomic analysis revealed that Nup133 regulates a subset of genes at early stages of neuroectodermal differentiation, including Lhx1 and Nup210l, which encodes a newly validated nucleoporin. These genes are also misregulated in Nup133ΔMid neuronal progenitors, in which nuclear pore basket assembly is impaired. However, a four-fold reduction of Nup133 levels, despite also affecting basket assembly, is not sufficient to alter Nup210l and Lhx1 expression. Finally, these two genes are also misregulated in Seh1-deficient neural progenitors, which only show a mild reduction in nuclear pore density. Together these data reveal a shared function of Y-complex nucleoporins in gene regulation during neuroectodermal differentiation, apparently independent of nuclear pore basket integrity.

Integrity of the short arm of the nuclear pore Y-complex is required for mouse embryonic stem cell growth and differentiation.

Many cellular processes, ranging from cell division to differentiation, are controlled by nuclear pore complexes (NPCs). However, studying the contributions of individual NPC subunits to these processes in vertebrates has long been impeded by their complexity and the lack of efficient genetic tools. Here, we use genome editing in mouse embryonic stem cells (mESCs) to characterize the role of NPC structural components, focusing on the short arm of the Y-complex that comprises Nup85, Seh1 and Nup43. We show that Seh1 and Nup43, although dispensable in pluripotent mESCs, are required for their normal cell growth rates, their viability upon differentiation and for the maintenance of proper NPC density. mESCs with an N-terminally truncated Nup85 mutation (in which interaction with Seh1 is greatly impaired) feature a similar reduction of NPC density. However, their proliferation and differentiation are unaltered, indicating that it is the integrity of the Y-complex, rather than the number of NPCs, that is critical to ensure these processes.

Disentangling the molecular determinants for Cenp-F localization to nuclear pores and kinetochores.

Cenp-F is a multifaceted protein implicated in cancer and developmental pathologies. The Cenp-F C-terminal region contains overlapping binding sites for numerous proteins that contribute to its functions throughout the cell cycle. Here, we focus on the nuclear pore protein Nup133 that interacts with Cenp-F both at nuclear pores in prophase and at kinetochores in mitosis, and on the kinase Bub1, known to contribute to Cenp-F targeting to kinetochores. By combining in silico structural modeling and yeast two-hybrid assays, we generate an interaction model between a conserved helix within the Nup133 ß-propeller and a short leucine zipper-containing dimeric segment of Cenp-F. We thereby create mutants affecting the Nup133/Cenp-F interface and show that they prevent Cenp-F localization to the nuclear envelope, but not to kinetochores. Conversely, a point mutation within an adjacent leucine zipper affecting the kinetochore targeting of Cenp-F KT-core domain impairs its interaction with Bub1, but not with Nup133, identifying Bub1 as the direct KT-core binding partner of Cenp-F. Finally, we show that Cenp-E redundantly contributes together with Bub1 to the recruitment of Cenp-F to kinetochores.

Formation of Nup98-containing nuclear bodies in HeLa sublines is linked to genomic rearrangements affecting chromosome 11.

Nup98 is an important component of the nuclear pore complex (NPC) and also a rare but recurrent target for chromosomal translocation in leukaemogenesis. Nup98 contains multiple cohesive Gly-Leu-Phe-Gly (GLFG) repeats that are critical notably for the formation of intranuclear GLFG bodies. Previous studies have reported the existence of GLFG bodies in cells overexpressing exogenous Nup98 or in a HeLa subline (HeLa-C) expressing an unusual elevated amount of endogenous Nup98. Here, we have analysed the presence of Nup98-containing bodies in several human cell lines. We found that HEp-2, another HeLa subline, contains GLFG bodies that are distinct from those identified in HeLa-C. Rapid amplification of cDNA ends (RACE) revealed that HEp-2 cells express additional truncated forms of Nup98 fused to a non-coding region of chromosome 11q22.1. Cytogenetic analyses using FISH and array-CGH further revealed chromosomal rearrangements that were distinct from those observed in leukaemic cells. Indeed, HEp-2 cells feature a massive amplification of juxtaposed NUP98 and 11q22.1 loci on a chromosome marker derived from chromosome 3. Unexpectedly, minor co-amplifications of NUP98 and 11q22.1 loci were also observed in other HeLa sublines, but on rearranged chromosomes 11. Altogether, this study reveals that distinct genomic rearrangements affecting NUP98 are associated with the formation of GLFG bodies in specific HeLa sublines.

Nuclear pore targeting of the yeast Pom33 nucleoporin depends on karyopherin and lipid binding.

Pom33 is an integral membrane protein of the yeast nuclear pore complex (NPC), and it is required for proper NPC distribution and assembly. To characterize the Pom33 NPC-targeting determinants, we performed immunoprecipitation experiments followed by mass spectrometry analyses. This identified a new Pom33 partner, the nuclear import factor Kap123. In vitro experiments revealed a direct interaction between the Pom33 C-terminal domain (CTD) and Kap123. In silico analysis predicted the presence of two amphipathic α-helices within Pom33-CTD. Circular dichroism and liposome co-flotation assays showed that this domain is able to fold into α-helices in the presence of liposomes and preferentially binds to highly curved lipid membranes. When expressed in yeast, under conditions abolishing Pom33-CTD membrane association, this domain behaves as a Kap123-dependent nuclear localization signal (NLS). Although deletion of Pom33 C-terminal domain (Pom33(ΔCTD)-GFP) impaired Pom33 stability and NPC targeting, mutants affecting either Kap123 binding or the amphipathic properties of the α-helices did not display any detectable defect. However, combined impairment of lipid and Kap123 binding affects targeting of Pom33 to NPCs. These data highlight the requirement of multiple determinants and mechanisms for proper NPC localization of Pom33.

Probing nuclear pore complex architecture with proximity-dependent biotinylation.

Proximity-dependent biotin identification (BioID) is a method for identifying protein associations that occur in vivo. By fusing a promiscuous biotin ligase to a protein of interest expressed in living cells, BioID permits the labeling of proximate proteins during a defined labeling period. In this study we used BioID to study the human nuclear pore complex (NPC), one of the largest macromolecular assemblies in eukaryotes. Anchored within the nuclear envelope, NPCs mediate the nucleocytoplasmic trafficking of numerous cellular components. We applied BioID to constituents of the Nup107-160 complex and the Nup93 complex, two conserved NPC subcomplexes. A strikingly different set of NPC constituents was detected depending on the position of these BioID-fusion proteins within the NPC. By applying BioID to several constituents located throughout the extremely stable Nup107-160 subcomplex, we refined our understanding of this highly conserved subcomplex, in part by demonstrating a direct interaction of Nup43 with Nup85. Furthermore, by using the extremely stable Nup107-160 structure as a molecular ruler, we defined the practical labeling radius of BioID. These studies further our understanding of human NPC organization and demonstrate that BioID is a valuable tool for exploring the constituency and organization of large protein assemblies in living cells.

Sumoylation of the THO complex regulates the biogenesis of a subset of mRNPs.

Assembly of messenger ribonucleoparticles (mRNPs) is a pivotal step in gene expression, but only a few molecular mechanisms contributing to its regulation have been described. Here, through a comprehensive proteomic survey of mRNP assembly, we demonstrate that the SUMO pathway specifically controls the association of the THO complex with mRNPs. We further show that the THO complex, a key player in the interplay between gene expression, mRNA export and genetic stability, is sumoylated on its Hpr1 subunit and that this modification regulates its association with mRNPs. Altered recruitment of the THO complex onto mRNPs in sumoylation-defective mutants does not affect bulk mRNA export or genetic stability, but impairs the expression of acidic stress-induced genes and, consistently, compromises viability in acidic stress conditions. Importantly, inactivation of the nuclear exosome suppresses the phenotypes of the hpr1 non-sumoylatable mutant, showing that SUMO-dependent mRNP assembly is critical to allow a specific subset of mRNPs to escape degradation. This article thus provides the first example of a SUMO-dependent mRNP-assembly event allowing a refined tuning of gene expression, in particular under specific stress conditions.

Probing a continuum of macro-molecular assembly models with graph templates of complexes.

Reconstruction by data integration is an emerging trend to reconstruct large protein assemblies, but uncertainties on the input data yield average models whose quantitative interpretation is challenging. This article presents methods to probe fuzzy models of large assemblies against atomic resolution models of subsystems. Consider a toleranced model (TOM) of a macromolecular assembly, namely a continuum of nested shapes representing the assembly at multiple scales. Also consider a template namely an atomic resolution 3D model of a subsystem (a complex) of this assembly. We present graph-based algorithms performing a multi-scale assessment of the complexes of the TOM, by comparing the pairwise contacts which appear in the TOM against those of the template. We apply this machinery on TOM derived from an average model of the nuclear pore complex, to explore the connections among members of its well-characterized Y-complex.

Metazoan nuclear pore complexes in gene regulation and genome stability.

The nuclear pore complexes (NPCs), one of the hallmarks of eukaryotic nuclei, allow selective transport of macromolecules between the cytoplasm and the nucleus. Besides this canonical function, an increasing number of additional roles have been attributed to the NPCs and their constituents, the nucleoporins. Here we review recent insights into the mechanisms by which NPCs and nucleoporins affect transcription and DNA repair in metazoans. In the first part, we discuss how gene expression can be affected by the localization of genome-nucleoporin interactions at pores or "off-pores", by the role of nucleoporins in chromatin organization at different scales, or by the physical properties of nucleoporins. In the second part, we review the contribution of NPCs to genome stability, including transport-dependent and -independent functions and the role of positioning at NPCs in the repair of heterochromatic breaks and the regulation of replication stress.

Assessing the reconstruction of macromolecular assemblies with toleranced models.

We introduce toleranced models (TOMs), a generic and versatile framework meant to handle models of macromolecular assemblies featuring uncertainties on the shapes and the positions of proteins. A TOM being a continuum of nested shapes, the inner (resp. outer) ones representing high (low) confidence regions, we present topological and geometric statistics assessing features of this continuum at multiple scales. While the topological statistics qualify contacts between instances of protein types and complexes involving prescribed protein types, the geometric statistics scale the geometric accuracy of these complexes. We validate the TOM framework on recent average models of the entire nuclear pore complex (NPC) obtained from reconstruction by data integration, and confront our quantitative analysis against experimental findings related to complexes of the NPC, namely the Y-complex, the T-complex, and the Nsp1-Nup82-Nup159 complex. In the three cases, our analysis bridges the gap between global qualitative models of the entire NPC, and atomic resolution models or putative models of the aforementioned complexes. In a broader perspective, the quantitative assessments provided by the TOM framework should prove instrumental to implement a virtuous loop "model reconstruction-model selection", in the context of reconstruction by data integration.

Mapping the dynamic organization of the nuclear pore complex inside single living cells.

Most cellular activities are executed by multi-protein complexes that form the basic functional modules of their molecular machinery. Proteomic approaches can provide an evermore detailed picture of their composition, but do not reveal how these machines are organized dynamically to accomplish their biological function. Here, we present a method to determine the dissociation rates of protein subunits from complexes that have a traceable localization inside single living cells. As a case study, we systematically analysed the dynamic organization of vertebrate nuclear pore complexes (NPCs), large supramolecular complexes of about 30 different polypeptides. NPC components exhibited a wide range of residence times covering five orders of magnitude from seconds to days. We found the central parts of the NPC to be very stable, consistent with a function as a structural scaffold, whereas more peripheral components exhibited more dynamic behaviour, suggesting adaptor as well as regulatory functions. The presented strategy can be applied to many multi-protein complexes and will help to characterize the dynamic behaviour of complex networks of proteins in live cells.

Nuclear transport and the mitotic apparatus: an evolving relationship.

The trafficking of macromolecules between the cytoplasm and the nucleus is controlled by the nuclear pore complexes (NPCs) and various transport factors that facilitate the movement of cargos through the NPCs and their accumulation in the target compartment. While their functions in transport are well established, an ever-growing number of observations have also linked components of the nuclear transport machinery to processes that control chromosome segregation during mitosis, including spindle assembly, kinetochore function, and the spindle assembly checkpoint. In this review, we will discuss this evolving area of study and emerging hypotheses that propose key roles for components of the nuclear transport apparatus in mitotic progression.

Nucleoporins prevent DNA damage accumulation by modulating Ulp1-dependent sumoylation processes.

Increasing evidences suggest that nuclear pore complexes (NPCs) control different aspects of nuclear metabolism, including transcription, nuclear organization, and DNA repair. We previously established that the Nup84 complex, a major NPC building block, is part of a genetic network involved in DNA repair. Here, we show that double-strand break (DSB) appearance is linked to a shared function of the Nup84 and the Nup60/Mlp1-2 complexes. Mutants within these complexes exhibit similar genetic interactions and alteration in DNA repair processes as mutants of the SUMO-protease Ulp1. Consistently, these nucleoporins are required for maintenance of proper Ulp1 levels at NPCs and for the establishment of the appropriate sumoylation of several cellular proteins, including the DNA repair factor Yku70. Moreover, restoration of nuclear envelope-associated Ulp1 in nucleoporin mutants reestablishes proper sumoylation patterns and suppresses DSB accumulation and genetic interactions with DNA repair genes. Our results thus provide a molecular mechanism that underlies the connection between NPC and genome stability.

Publisher Correction: Moderate Nucleoporin 133 deficiency leads to glomerular damage in zebrafish.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Author Correction: Moderate Nucleoporin 133 deficiency leads to glomerular damage in zebrafish.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Molecular rearrangements within the nuclear pore complexes: a new way to regulate nucleocytoplasmic transport.

Until now, regulation of nucleocytoplasmic transport of macromolecules has been thought to occur mainly through modifications of the cargo molecules. However, in the December 26 issue of Cell, Makhnevych et al. describe a novel mechanism controlling cell cycle progression in yeast that involves subtle molecular rearrangements within the nuclear pore complexes.

Moderate Nucleoporin 133 deficiency leads to glomerular damage in zebrafish.

Although structural nuclear pore proteins (nucleoporins) are seemingly required in every cell type to assemble a functional nuclear transport machinery, mutations or deregulation of a subset of them have been associated with specific human hereditary diseases. In particular, previous genetic studies of patients with nephrotic syndrome identified mutations in Nup107 that impaired the expression or the localization of its direct partner at nuclear pores, Nup133. In the present study, we characterized the zebrafish nup133 orthologous gene and its expression pattern during larval development. Using a morpholino-mediated gene knockdown, we show that partial depletion of Nup133 in zebrafish larvae leads to the formation of kidney cysts, a phenotype that can be rescued by co-injection of wild type mRNA. Analysis of different markers for tubular and glomerular development shows that the overall kidney development is not affected by nup133 knockdown. Likewise, no gross defect in nuclear pore complex assembly was observed in these nup133 morphants. On the other hand, nup133 downregulation results in proteinuria and moderate foot process effacement, mimicking some of the abnormalities typically featured by patients with nephrotic syndrome. These data indicate that nup133 is a new gene required for proper glomerular structure and function in zebrafish.

Pml39, a novel protein of the nuclear periphery required for nuclear retention of improper messenger ribonucleoparticles.

Using a genetic screen, we have identified a previously uncharacterized Saccharomyces cerevisiae open reading frame (renamed PML39) that displays a specific interaction with nucleoporins of the Nup84 complex. Localization of a Pml39-green fluorescent protein (GFP) fusion and two-hybrid studies revealed that Pml39 is mainly docked to a subset of nuclear pore complexes opposite to the nucleolus through interactions with Mlp1 and Mlp2. The absence of Pml39 leads to a specific leakage of unspliced mRNAs that is not enhanced upon MLP1 deletion. In addition, overexpression of PML39-GFP induces a specific trapping of mRNAs transcribed from an intron-containing reporter and of the heterogenous nuclear ribonucleoprotein Nab2 within discrete nuclear domains. In a nup60delta mutant, Pml39 is mislocalized together with Mlp1 and Mlp2 in intranuclear foci that also recruit Nab2. Moreover, pml39delta partially rescues the thermosensitive phenotypes of messenger ribonucleoparticles (mRNPs) assembly mutants, indicating that PML39 deletion also bypasses the requirement for normally assembled mRNPs. Together, these data indicate that Pml39 is an upstream effector of the Mlps, involved in the retention of improper mRNPs in the nucleus before their export.

Regulation of Cenp-F localization to nuclear pores and kinetochores.

In metazoans, the assembly of kinetochores on centrometric chromatin and the dismantling of nuclear pore complexes are processes that have to be tightly coordinated to ensure the proper assembly of the mitotic spindle and a successful mitosis. It is therefore noteworthy that these two macromolecular assemblies share a subset of constituents. One of these multifaceted components is Cenp-F, a protein implicated in cancer and developmental pathologies. During the cell cycle, Cenp-F localizes in multiple cellular structures including the nuclear envelope in late G2/early prophase and kinetochores throughout mitosis. We recently characterized the molecular determinants of Cenp-F interaction with Nup133, a structural nuclear pore constituent. In parallel with two other independent studies, we further elucidated the mechanisms governing Cenp-F kinetochore recruitment that mainly relies on its interaction with Bub1, with redundant contribution of Cenp-E upon acute microtubule depolymerisation. Here we synthesize the current literature regarding the dual location of Cenp-F at nuclear pores and kinetochores and extend our discussion to the regulation of these NPC and kinetochore localizations by mitotic kinase and spindle microtubules.