bims-nucpor 2023-12-24 papers

bims-nucpor

Biomed News

on Nuclear pore complex and nucleoporins in stress, aging and disease

Issue of 2023–12–24
nine papers selected by
Sara Mingu, Johannes Gutenberg University

Protocol for live-cell super-resolution imaging of transport of pre-ribosomal subunits through the nuclear pore complex.
Pitstop-2 Upsets The Integrity of Nuclear Pore Complexes (NPCs) by Interaction with β-Propeller Folds of Npc Scaffold Proteins.
The scaffold nucleoporins SAR1 and SAR3 are essential for proper meiotic progression in Arabidopsis thaliana.
A functional and structural comparative analysis of Large Tumor Antigens reveals evolution of different importin α-dependent nuclear localization signals.
Human RTEL1 Interacts with KPNB1 (Importin β) and NUP153 and Connects Nuclear Import to Nuclear Envelope Stability in S-Phase.
NUP85 as a Neurodevelopmental Gene: From Podocyte to Neuron.
Special Nuclear Structures in the Germinal Vesicle of the Common Frog with Emphasis on the So-Called Karyosphere Capsule.
Glucose stress causes mRNA retention in nuclear Nab2 condensates.
Spermatozoa in mice lacking the nucleoporin NUP210L show defects in head shape and motility but not in nuclear compaction or histone replacement.

STAR Protoc. 2023 Dec 18. pii: S2666-1667(23)00757-8. [Epub ahead of print]5(1): 102790

Protocol for live-cell super-resolution imaging of transport of pre-ribosomal subunits through the nuclear pore complex.

Mark Tingey, Samuel L Junod, Coby Rush, Weidong Yang.

  Here, we present a protocol for single-molecule super-resolution imaging of the nuclear export of pre-ribosomal subunits pre-40S and pre-60S through nuclear pore complexes. We describe steps for plating cells and co-transfecting cells. We then detail steps for using single-point edge-excitation sub-diffraction microscopy, allowing visualization of real-time dynamics of the pre-ribosomal subunits. For complete details on the use and execution of this protocol, please refer to Junod et al. (2023).1.

Keywords:  Biophysics; Cell Biology; Cell Membrane; Microscopy; Single-molecule Assays

DOI:  https://doi.org/10.1016/j.xpro.2023.102790
Adv Biol (Weinh). 2023 Dec 21. e2300360

Pitstop-2 Upsets The Integrity of Nuclear Pore Complexes (NPCs) by Interaction with β-Propeller Folds of Npc Scaffold Proteins.

Sílvio Terra Stefanello, Caren Rigon Mizdal, Victor Shahin.

  The small compound Pitstop-2 is a recent potent inhibitor of clathrin-mediated endocytosis (CME), widely used in biomedical research areas. In recent years, however, it is observed that it exhibits CME-independent inhibitory effects on nuclear pore complexes (NPCs), the nucleocytoplasmic gatekeepers. NPCs are elaborate proteinaceous transport nano-machineries of crucial physiological importance rendering them novel targets for various medical applications. They mediate all nucleocytoplasmic transport forming a physiologically essential selective nucleocytoplasmic barrier. The direct Pitstop-2 disruptive effects on NPCs manifested themselves at both the structural and functional integrity levels. Moreover, they are massive, acute, and detectable at concentrations equal to CME-inhibitory concentrations. Pitstop-2 inhibits CME by binding to the terminal β-propeller domain of the heavy chain of clathrin. Several NPC scaffold proteins, critical for the structural and functional integrity of the NPC, possess β-propeller folds. Herein, utilizing computational docking analysis, it is demonstrated that Pitstop-2 exhibits particularly high binding affinities to β-propeller folds of NPC scaffold proteins, similar to its binding affinity to the terminal β-propeller domain of clathrin. The authors, therefore, conclude that Pitstop-2 is a potent disruptor of NPCs, an activity which, separately or in synergy with CME inhibition, may be exploited for a myriad of pharmacological applications.

Keywords:  NPC-associated diseases; Nuclear pore complexes (NPCs); clathrin; nanomedicine; nucleocytoplasmic transport

DOI:  https://doi.org/10.1002/adbi.202300360
Front Cell Dev Biol. 2023 ;11 1285695

The scaffold nucleoporins SAR1 and SAR3 are essential for proper meiotic progression in Arabidopsis thaliana.

Nadia Fernández-Jiménez, Marina Martinez-Garcia, Javier Varas, Félix Gil-Dones, Juan Luis Santos, Mónica Pradillo.

  Nuclear Pore Complexes (NPCs) are embedded in the nuclear envelope (NE), regulating macromolecule transport and physically interacting with chromatin. The NE undergoes dramatic breakdown and reformation during plant cell division. In addition, this structure has a specific meiotic function, anchoring and positioning telomeres to facilitate the pairing of homologous chromosomes. To elucidate a possible function of the structural components of the NPCs in meiosis, we have characterized several Arabidopsis lines with mutations in genes encoding nucleoporins belonging to the outer ring complex. Plants defective for either SUPPRESSOR OF AUXIN RESISTANCE1 (SAR1, also called NUP160) or SAR3 (NUP96) present condensation abnormalities and SPO11-dependent chromosome fragmentation in a fraction of meiocytes, which is increased in the double mutant sar1 sar3. We also observed these meiotic defects in mutants deficient in the outer ring complex protein HOS1, but not in mutants affected in other components of this complex. Furthermore, our findings may suggest defects in the structure of NPCs in sar1 and a potential link between the meiotic role of this nucleoporin and a component of the RUBylation pathway. These results provide the first insights in plants into the role of nucleoporins in meiotic chromosome behavior.

Keywords:  Arabidopsis; NUP160; NUP96; SAR1; SAR3; meiosis; nuclear pore complex

DOI:  https://doi.org/10.3389/fcell.2023.1285695
Protein Sci. 2023 Dec 18.

A functional and structural comparative analysis of Large Tumor Antigens reveals evolution of different importin α-dependent nuclear localization signals.

Emily M Cross, Nasim Akbari, Hanieh Ghassabian, Mikayla Hoad, Silvia Pavan, Daryl Ariawan, Camilla M Donnelly, Enrico Lavezzo, Gayle F Petersen, Jade K Forwood, Gualtiero Alvisi.

  Nucleocytoplasmic transport regulates the passage of proteins between the nucleus and cytoplasm. In the best characterized pathway, importin (IMP) α bridges cargoes bearing basic, classical nuclear localization signals (cNLSs) to IMPβ1, which mediates transport through the nuclear pore complex. IMPα recognizes three types of cNLSs via two binding sites: the major binding site accommodates monopartite cNLSs, the minor binding site recognizes atypical cNLSs, whilst bipartite cNLSs simultaneously interact with both major and minor sites. Despite the growing knowledge regarding IMPα-cNLS interactions, our understanding of the evolution of cNLSs is limited. We combined bioinformatic, biochemical, functional, and structural approaches to study this phenomenon, using polyomaviruses (PyVs) Large Tumor Antigens (LTAs) as a model. We characterized functional cNLSs from all human (H)PyV LTAs, located between the LXCXE motif and origin binding domain. Surprisingly, the prototypical SV40 monopartite NLS is not well conserved; HPyV LTA NLSs are extremely heterogenous in terms of structural organization, IMPα isoform binding, and nuclear targeting abilities, thus influencing the nuclear accumulation properties of full-length proteins. While several LTAs possess bipartite cNLSs, Merkel Cell (MC) PyV contains a hybrid bipartite cNLS whose upstream stretch of basic amino acids can function as an atypical cNLS, specifically binding to the IMPα minor site upon deletion of the downstream amino acids after viral integration in the host genome. Therefore, duplication of a monopartite cNLS and subsequent accumulation of point mutations, optimizing interaction with distinct IMPα binding sites, led to the evolution of bipartite and atypical NLSs binding at the minor site. This article is protected by copyright. All rights reserved.

Keywords:  Merkel cell polyomavirus; NLS; NLS evolution; cNLS; importin alpha structure; importins; large T antigens; nuclear transport; oncogenes

DOI:  https://doi.org/10.1002/pro.4876
Cells. 2023 Dec 08. pii: 2798. [Epub ahead of print]12(24):

Human RTEL1 Interacts with KPNB1 (Importin β) and NUP153 and Connects Nuclear Import to Nuclear Envelope Stability in S-Phase.

Michael Schertzer, Laurent Jullien, André L Pinto, Rodrigo T Calado, Patrick Revy, Arturo Londoño-Vallejo.

  Regulator of TElomere Length Helicase 1 (RTEL1) is a helicase required for telomere maintenance and genome replication and repair. RTEL1 has been previously shown to participate in the nuclear export of small nuclear RNAs. Here we show that RTEL1 deficiency leads to a nuclear envelope destabilization exclusively in cells entering S-phase and in direct connection to origin firing. We discovered that inhibiting protein import also leads to similar, albeit non-cell cycle-related, nuclear envelope disruptions. Remarkably, overexpression of wild-type RTEL1, or of its C-terminal part lacking the helicase domain, protects cells against nuclear envelope anomalies mediated by protein import inhibition. We identified distinct domains in the C-terminus of RTEL1 essential for the interaction with KPNB1 (importin β) and NUP153, respectively, and we demonstrated that, on its own, the latter domain can promote the dynamic nuclear internalization of peptides that freely diffuse through the nuclear pore. Consistent with putative functions exerted in protein import, RTEL1 can be visualized on both sides of the nuclear pore using high-resolution microscopy. In all, our work points to an unanticipated, helicase-independent, role of RTEL1 in connecting both nucleocytoplasmic trafficking and nuclear envelope integrity to genome replication initiation in S-phase.

Keywords:  KPNB1 (importin β); NUP153; Regulator of TElomere Length Helicase 1; S-phase; nuclear envelope; nuclear pore; protein import

DOI:  https://doi.org/10.3390/cells12242798
Genes (Basel). 2023 Nov 27. pii: 2143. [Epub ahead of print]14(12):

NUP85 as a Neurodevelopmental Gene: From Podocyte to Neuron.

Antonella Gambadauro, Giuseppe Donato Mangano, Karol Galletta, Francesca Granata, Antonella Riva, Laura Massella, Isabella Guzzo, Giovanni Farello, Giovanna Scorrano, Ludovica Di Francesco, Giulio Di Donato, Carolina Ianni, Armando Di Ludovico, Saverio La Bella, Pasquale Striano, Stephanie Efthymiou, Henry Houlden, Rosaria Nardello, Roberto Chimenz.

  Pathogenic gene variants encoding nuclear pore complex (NPC) proteins were previously implicated in the pathogenesis of steroid-resistant nephrotic syndrome (SRNS). The NUP85 gene, encoding nucleoporin, is related to a very rare form of SRNS with limited genotype-phenotype information. We identified an Italian boy affected with an SRNS associated with severe neurodevelopmental impairment characterized by microcephaly, axial hypotonia, lack of achievement of motor milestones, and refractory seizures with an associated hypsarrhythmic pattern on electroencephalography. Brain magnetic resonance imaging (MRI) showed hypoplasia of the corpus callosum and a simplified gyration of the cerebral cortex. Since the age of 3 years, the boy was followed up at our Pediatric Nephrology Department for an SRNS, with a focal segmental glomerulosclerosis at renal biopsy. The boy died 32 months after SRNS onset, and a Whole-Exome Sequencing analysis revealed a novel compound heterozygous variant in NUP85 (NM_024844.5): 611T>A (p.Val204Glu), c.1904T>G (p.Leu635Arg), inherited from the father and mother, respectively. We delineated the clinical phenotypes of NUP85-related disorders, reviewed the affected individuals so far reported in the literature, and overall expanded both the phenotypic and the molecular spectrum associated with this ultra-rare genetic condition. Our study suggests a potential occurrence of severe neurological phenotypes as part of the NUP85-related clinical spectrum and highlights an important involvement of nucleoporin in brain developmental processes and neurological function.

Keywords:  NUP85; developmental delay; epileptic spasm; microcephaly; nephrotic syndrome type 17; steroid-resistant nephrotic syndrome

DOI:  https://doi.org/10.3390/genes14122143
J Dev Biol. 2023 Dec 12. pii: 44. [Epub ahead of print]11(4):

Special Nuclear Structures in the Germinal Vesicle of the Common Frog with Emphasis on the So-Called Karyosphere Capsule.

Dmitry S Bogolyubov, Sergey V Shabelnikov, Alexandra O Travina, Maksim I Sulatsky, Irina O Bogolyubova.

  The karyosphere (karyosome) is a structure that forms in the oocyte nucleus-germinal vesicle (GV)-at the diplotene stage of meiotic prophase due to the assembly of all chromosomes in a limited portion of the GV. In some organisms, the karyosphere has an extrachromosomal external capsule, the marker protein of which is nuclear F-actin. Despite many years of theories about the formation of the karyosphere capsule (KC) in the GV of the common frog Rana temporaria, we present data that cast doubt on its existence, at least in this species. Specific extrachromosomal strands, which had been considered the main elements of the frog's KC, do not form a continuous layer around the karyosphere and, according to immunogold labeling, do not contain structural proteins, such as actin and lamin B. At the same time, F-actin is indeed noticeably concentrated around the karyosphere, creating the illusion of a capsule at the light microscopy/fluorescence level. The barrier-to-autointegration factor (BAF) and one of its functional partners-LEMD2, an inner nuclear membrane protein-are not localized in the strands, suggesting that the strands are not functional counterparts of the nuclear envelope. The presence of characteristic strands in the GV of R. temporaria late oocytes may reflect an excess of SMC1 involved in the structural maintenance of diplotene oocyte chromosomes at the karyosphere stage, since SMC1 has been shown to be the most abundant protein in the strands. Other characteristic microstructures-the so-called annuli, very similar in ultrastructure to the nuclear pore complexes-do not contain nucleoporins Nup35 and Nup93, and, therefore, they cannot be considered autonomous pore complexes, as previously thought. Taken together, our data indicate that traditional ideas about the existence of the R. temporaria KC as a special structural compartment of the GV are to be revisited.

Keywords:  Rana temporaria; electron microscopy; germinal vesicle; karyosome; karyosphere; karyosphere capsule; nuclear actin; nuclear compartments

DOI:  https://doi.org/10.3390/jdb11040044
Cell Rep. 2023 Dec 18. pii: S2211-1247(23)01605-4. [Epub ahead of print]43(1): 113593

Glucose stress causes mRNA retention in nuclear Nab2 condensates.

Stephanie Heinrich, Maria Hondele, Désirée Marchand, Carina Patrizia Derrer, Mostafa Zedan, Alexandra Oswald, Liliana Malinovska, Federico Uliana, Sarah Khawaja, Roberta Mancini, David Grunwald, Karsten Weis.

  Nuclear mRNA export via nuclear pore complexes is an essential step in eukaryotic gene expression. Although factors involved in mRNA transport have been characterized, a comprehensive mechanistic understanding of this process and its regulation is lacking. Here, we use single-RNA imaging in yeast to show that cells use mRNA retention to control mRNA export during stress. We demonstrate that, upon glucose withdrawal, the essential RNA-binding factor Nab2 forms RNA-dependent condensate-like structures in the nucleus. This coincides with a reduced abundance of the DEAD-box ATPase Dbp5 at the nuclear pore. Depleting Dbp5, and consequently blocking mRNA export, is necessary and sufficient to trigger Nab2 condensation. The state of Nab2 condensation influences the extent of nuclear mRNA accumulation and can be recapitulated in vitro, where Nab2 forms RNA-dependent liquid droplets. We hypothesize that cells use condensation to regulate mRNA export and control gene expression during stress.

Keywords:  CP: Cell biology; DEAD-box ATPase Dbp5; Nab2; RNA; condensation; glucose stress; mRNA export

DOI:  https://doi.org/10.1016/j.celrep.2023.113593
Clin Genet. 2023 Dec 21.

Spermatozoa in mice lacking the nucleoporin NUP210L show defects in head shape and motility but not in nuclear compaction or histone replacement.

Maha Al Dala Ali, Guy Longepied, Aurore Nicolet, Catherine Metzler-Guillemain, Michael J Mitchell.

  Biallelic loss-of-function mutation of NUP210L, encoding a testis-specific nucleoporin, has been reported in an infertile man whose spermatozoa show uncondensed heads and histone retention. Mice with a homozygous transgene intronic insertion in Nup210l were infertile but spermatozoa had condensed heads. Expression from this insertion allele is undefined, however, and residual NUP210L production could underlie the milder phenotype. To resolve this issue, we have created Nup210lem1Mjmm , a null allele of Nup210l, in the mouse. Nup210lem1Mjmm homozygotes show uniform mild anomalies of sperm head morphology and decreased motility, but nuclear compaction and histone removal appear unaffected. Thus, our mouse model does not support that NUP210L loss alone blocks spermatid nuclear compaction. Re-analyzing the patient's exome data, we identified a rare, potentially pathogenic, heterozygous variant in nucleoporin gene NUP153 (p.Pro485Leu), and showed that, in mouse and human, NUP210L and NUP153 colocalize at the caudal nuclear pole in elongating spermatids and spermatozoa. Unexpectedly, in round spermatids, NUP210L and NUP153 localisation differs between mouse (nucleoplasm) and human (nuclear periphery). Our data suggest two explanations for the increased phenotypic severity associated with NUP210L loss in human compared to mouse: a genetic variant in human NUP153 (p.Pro485Leu), and inter-species divergence in nuclear pore function in round spermatids.

Keywords:  chromatin; humans; infertility; mice; nuclear pore complex proteins; sperm head; spermatid; spermatogenesis; transgenes

DOI:  https://doi.org/10.1111/cge.14468