bims-nucpor 2021-08-01 papers

bims-nucpor

Biomed News

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

Issue of 2021–08–01
eight papers selected by
Sara Mingu, Johannes Gutenberg University

Structure, Maintenance, and Regulation of Nuclear Pore Complexes: The Gatekeepers of the Eukaryotic Genome.
Torsin and NEP1R1-CTDNEP1 phosphatase affect interphase nuclear pore complex insertion by lipid-dependent and lipid-independent mechanisms.
Nuclear accumulation of CHMP7 initiates nuclear pore complex injury and subsequent TDP-43 dysfunction in sporadic and familial ALS.
New Family Members of FG Repeat Proteins and Their Unexplored Roles During Phase Separation.
Nuclear Transport Assays in Permeabilized Mouse Cortical Neurons.
DNA-Origami NanoTrap for Studying the Selective Barriers Formed by Phenylalanine-Glycine-Rich Nucleoporins.
Overexpression of the nucleoporin Nup88 stimulates migration and invasion of HeLa cells.
Nuclear exportin 1 facilitates turnip mosaic virus infection by exporting the sumoylated viral replicase and by repressing plant immunity.

Cold Spring Harb Perspect Biol. 2021 Jul 26. pii: a040691. [Epub ahead of print]

Structure, Maintenance, and Regulation of Nuclear Pore Complexes: The Gatekeepers of the Eukaryotic Genome.

Marcela Raices, Maximiliano A D'Angelo.

In eukaryotic cells, the genetic material is segregated inside the nucleus. This compartmentalization of the genome requires a transport system that allows cells to move molecules across the nuclear envelope, the membrane-based barrier that surrounds the chromosomes. Nuclear pore complexes (NPCs) are the central component of the nuclear transport machinery. These large protein channels penetrate the nuclear envelope, creating a passage between the nucleus and the cytoplasm through which nucleocytoplasmic molecule exchange occurs. NPCs are one of the largest protein assemblies of eukaryotic cells and, in addition to their critical function in nuclear transport, these structures also play key roles in many cellular processes in a transport-independent manner. Here we will review the current knowledge of the NPC structure, the cellular mechanisms that regulate their formation and maintenance, and we will provide a brief description of a variety of processes that NPCs regulate.

DOI: https://doi.org/10.1101/cshperspect.a040691
EMBO J. 2021 Jul 27. e106914

Torsin and NEP1R1-CTDNEP1 phosphatase affect interphase nuclear pore complex insertion by lipid-dependent and lipid-independent mechanisms.

Julie Jacquemyn, Joyce Foroozandeh, Katlijn Vints, Jef Swerts, Patrik Verstreken, Natalia V Gounko, Sandra F Gallego, Rose Goodchild.

  The interphase nuclear envelope (NE) is extensively remodeled during nuclear pore complex (NPC) insertion. How this remodeling occurs and why it requires Torsin ATPases, which also regulate lipid metabolism, remains poorly understood. Here, we show that Drosophila Torsin (dTorsin) affects lipid metabolism via the NEP1R1-CTDNEP1 phosphatase and the Lipin phosphatidic acid (PA) phosphatase. This includes that Torsins remove NEP1R1-CTDNEP1 from the NE in fly and mouse cells, leading to subsequent Lipin exclusion from the nucleus. NEP1R1-CTDNEP1 downregulation also restores nuclear pore membrane fusion in post-mitotic dTorsinKO fat body cells. However, dTorsin-associated nuclear pore defects do not correlate with lipidomic abnormalities and are not resolved by silencing of Lipin. Further testing confirmed that membrane fusion continues in cells with hyperactivated Lipin. It also led to the surprising finding that excessive PA metabolism inhibits recruitment of the inner ring complex Nup35 subunit, resulting in elongated channel-like structures in place of mature nuclear pores. We conclude that the NEP1R1-CTDNEP1 phosphatase affects interphase NPC biogenesis by lipid-dependent and lipid-independent mechanisms, explaining some of the pleiotropic effects of Torsins.

Keywords:  Drosophila fat body; Lipin; Torsin; nuclear membrane; nuclear pore

DOI:  https://doi.org/10.15252/embj.2020106914
Sci Transl Med. 2021 Jul 28. pii: eabe1923. [Epub ahead of print]13(604):

Nuclear accumulation of CHMP7 initiates nuclear pore complex injury and subsequent TDP-43 dysfunction in sporadic and familial ALS.

Alyssa N Coyne, Victoria Baskerville, Benjamin L Zaepfel, Dennis W Dickson, Frank Rigo, Frank Bennett, C Patrick Lusk, Jeffrey D Rothstein.

Alterations in the components [nucleoporins (Nups)] and function of the nuclear pore complex (NPC) have been implicated as contributors to the pathogenesis of genetic forms of neurodegeneration including C9orf72 amyotrophic lateral sclerosis/frontotemporal dementia (ALS/FTD). We hypothesized that Nup alterations and the consequential loss of NPC function may lie upstream of TDP-43 dysfunction and mislocalization widely observed in ALS, FTD, and related neurodegenerative diseases. Here, we provide evidence that CHMP7, a critical mediator of NPC quality control, is increased in nuclei of C9orf72 and sporadic ALS induced pluripotent stem cell (iPSC)-derived spinal neurons (iPSNs) and postmortem human motor cortex before the emergence of Nup alterations. Inhibiting the nuclear export of CHMP7 triggered Nup reduction and TDP-43 dysfunction and pathology in human neurons. Knockdown of CHMP7 alleviated disease-associated Nup alterations, deficits in Ran GTPase localization, defects in TDP-43-associated mRNA expression, and downstream glutamate-induced neuronal death. Thus, our data support a role for altered CHMP7-mediated Nup homeostasis as a prominent initiating pathological mechanism for familial and sporadic ALS and highlight the potential for CHMP7 as therapeutic target.

DOI: https://doi.org/10.1126/scitranslmed.abe1923
Front Cell Dev Biol. 2021 ;9 708702

New Family Members of FG Repeat Proteins and Their Unexplored Roles During Phase Separation.

Yoichi Shinkai, Masahiro Kuramochi, Takamitsu Miyafusa.

  The condensation and compartmentalization of biomacromolecules in the cell are driven by the process of phase separation. The main effectors of phase separation are intrinsically disordered proteins, which include proteins with a phenylalanine-glycine (FG) repeat domain. Our understanding of the biological function of FG repeat proteins during phase separation has been mainly derived from recent research on a member of the nuclear pore complex proteins, nucleoporins containing FG repeat domain (FG-NUPs). FG-NUPs form meshwork structures by inter- and intra-molecular FG domain interactions, which confine the nucleo-cytoplasmic exchange. Whereas FG-NUPs localize in the nuclear membrane, other FG repeat proteins reside in the cytoplasm and the nucleoplasm, and the biological function of the FG repeat domain of these proteins is not well described. In the present review, we list the FG repeat proteins that are known to phase separate in the cell, and review their biological functions. We extract the unraveled features of FG repeat proteins as an activator of barrier formation and homotypic cell-cell interactions. Understanding the regulatory mechanisms of FG repeat proteins will provide a potential delivery tool for therapeutic reagents.

Keywords:  FG repeat; P granule; intrinsically disordered protein; keratohyalin granule; nuclear pore; phase separation

DOI:  https://doi.org/10.3389/fcell.2021.708702
J Vis Exp. 2021 Jul 09.

Nuclear Transport Assays in Permeabilized Mouse Cortical Neurons.

Lindsey R Hayes, Lauren Duan, Svetlana Vidensky, Petr Kalab.

Disruption of nucleocytoplasmic transport is increasingly implicated in the pathogenesis of neurodegenerative diseases. Moreover, there is a growing recognition of cell-specific differences in nuclear pore complex structure, prompting a need to adapt nuclear transport methods for use in neurons. Permeabilized cell assays, in which the plasma membrane is selectively perforated by digitonin, are widely used to study passive and active nuclear transport in immortalized cell lines but have not been applied to neuronal cultures. In our initial attempts, we observed the rapid loss of nuclear membrane integrity in primary mouse cortical neurons exposed to even low concentrations of digitonin. We hypothesized that neuronal nuclear membranes may be uniquely vulnerable to the loss of cytoplasmic support. After testing multiple approaches to improve nuclear stability, we observed optimal nuclear integrity following hypotonic lysis in the presence of a concentrated bovine serum albumin cushion. Neuronal nuclei prepared by this approach reliably import recombinant fluorescent cargo in an energy-dependent manner, facilitating analysis of nuclear import by high content microscopy with automated analysis. We anticipate that this method will be broadly applicable to studies of passive and active nuclear transport in primary neurons.

DOI: https://doi.org/10.3791/62710
J Am Chem Soc. 2021 Jul 29.

DNA-Origami NanoTrap for Studying the Selective Barriers Formed by Phenylalanine-Glycine-Rich Nucleoporins.

Qi Shen, Taoran Tian, Qiancheng Xiong, Patrick D Ellis Fisher, Yong Xiong, Thomas J Melia, C Patrick Lusk, Chenxiang Lin.

DNA nanotechnology provides a versatile and powerful tool to dissect the structure-function relationship of biomolecular machines like the nuclear pore complex (NPC), an enormous protein assembly that controls molecular traffic between the nucleus and cytoplasm. To understand how the intrinsically disordered, Phe-Gly-rich nucleoporins (FG-nups) within the NPC establish a selective barrier to macromolecules, we built a DNA-origami NanoTrap. The NanoTrap comprises precisely arranged FG-nups in an NPC-like channel, which sits on a baseplate that captures macromolecules that pass through the FG network. Using this biomimetic construct, we determined that the FG-motif type, grafting density, and spatial arrangement are critical determinants of an effective diffusion barrier. Further, we observed that diffusion barriers formed with cohesive FG interactions dominate in mixed-FG-nup scenarios. Finally, we demonstrated that the nuclear transport receptor, Ntf2, can selectively transport model cargo through NanoTraps composed of FxFG but not GLFG Nups. Our NanoTrap thus recapitulates the NPC's fundamental biological activities, providing a valuable tool for studying nuclear transport.

DOI: https://doi.org/10.1021/jacs.1c05550
Histochem Cell Biol. 2021 Jul 31.

Overexpression of the nucleoporin Nup88 stimulates migration and invasion of HeLa cells.

Masaki Makise, Ryota Uchimura, Kumiko Higashi, Yasumi Mashiki, Rikako Shiraishi, Yuumi Shutoku, Akihiko Kuniyasu.

  Elevated expression of the nucleoporin Nup88, a constituent of the nuclear pore complex, is seen in various types of malignant tumors, but whether this overexpression contributes to the malignant phenotype has yet to be determined. Here, we investigated the effect of the overexpression of Nup88 on the migration and invasion of cervical cancer HeLa cells. The overexpression of Nup88 promoted a slight but significant increase in both migration and invasion, whereas knockdown of Nup88 by RNA interference suppressed these phenotypes. The observed phenotypes in Nup88-overexpressing HeLa cells were not due to the progression of the epithelial-to-mesenchymal transition or activation of NF-κB, which are known to be important for cell migration and invasion. Instead, we identified an upregulation of matrix metalloproteinase-12 (MMP-12) at both the gene and protein levels in Nup88-overexpressing HeLa cells. Upregulation of MMP-12 protein by the overexpression of Nup88 was also observed in one other cervical cancer cell line and two prostate cancer cell lines but not 293 cells. Treatment with a selective inhibitor against MMP-12 enzymatic activity significantly suppressed the invasive ability of HeLa cells induced by Nup88 overexpression. Taken together, our results suggest that overexpression of Nup88 can stimulate malignant phenotypes including invasive ability, which is promoted by MMP-12 expression.

Keywords:  HeLa cells; Invasion; MMP-12; Migration; NF-κB; Nup88

DOI:  https://doi.org/10.1007/s00418-021-02020-w
New Phytol. 2021 Jul 30.

Nuclear exportin 1 facilitates turnip mosaic virus infection by exporting the sumoylated viral replicase and by repressing plant immunity.

Mingzhen Zhang, Pan Gong, Linhao Ge, Zhaoyang Chang, Xiaofei Cheng, Xueping Zhou, Aiming Wang, Fangfang Li.

  Exportin 1/XPO1 is an important nuclear export receptor that binds directly to cargo proteins and translocates the cargo proteins to the cytoplasm. To understand XPO1 protein functions during potyvirus infections, we investigated the nuclear export of the NIb protein encoding the RNA-dependent RNA polymerase (RdRp) of turnip mosaic virus (TuMV). Previously, we found that NIb is transported to the nucleus after translation and sumoylated by the sumoylation (small ubiquitin-like modifier) pathway to support viral infection. Here, we report that XPO1 interacts with NIb to facilitate translocation from the nucleus to the viral replication complexes (VRCs) that accumulate in the perinuclear regions of TuMV infected cells. XPO1 contains two NIb-binding domains that recognize and interact with NIb in the nucleus and in the perinuclear regions, respectively, which facilitates TuMV replication. Moreover, XPO1 is involved in nuclear export of the sumoylated NIb and host factors tagged with SUMO3 that is essential for suppression of plant immunity in the nucleus. Deficiencies of XPO1 in Arabidopsis and Nicotiana benthamiana plants inhibit TuMV replication and infection. These data demonstrate that XPO1 functions as a host factor in TuMV infection by regulating NIb nucleo-cytoplasmic transport and plant immunity.

Keywords:  RNA dependent RNA polymerase (RdRp); SUMO3; XPO1; nuclear export; plant immunity; turnip mosaic virus (TuMV)

DOI:  https://doi.org/10.1111/nph.17657