bims-nucpor 2022-11-13 papers

Results Probl Cell Differ. 2022 ;70 315-337

The LINC Complex Assists the Nuclear Import of Mechanosensitive Transcriptional Regulators.

Mechanical forces play pivotal roles in directing cell functions and fate. To elicit gene expression, either intrinsic or extrinsic mechanical information are transmitted into the nucleus beyond the nuclear envelope via at least two distinct pathways, possibly more. The first and well-known pathway utilizes the canonical nuclear transport of mechanoresponsive transcriptional regulators through the nuclear pore complex, which is an exclusive route for macromolecular trafficking between the cytoplasm and nucleoplasm. The second pathway depends on the linker of the nucleoskeleton and cytoskeleton (LINC) complex, which is a molecular bridge traversing the nuclear envelope between the cytoskeleton and nucleoskeleton. This protein complex is a central component in mechanotransduction at the nuclear envelope that transmits mechanical information from the cytoskeleton into the nucleus to influence the nuclear structure, nuclear stiffness, chromatin organization, and gene expression. Besides the mechanical force transducing function, recent increasing evidence shows that the LINC complex plays a role in controlling nucleocytoplasmic transport of mechanoresponsive transcriptional regulators. Here we discuss recent findings regarding the contribution of the LINC complex to the regulation of intracellular localization of the most-notable mechanosensitive transcriptional regulators, β-catenin, YAP, and TAZ.

Keywords: Armadillo repeat; LINC complex; Mechanotransduction; NPC; Nesprin; Nuclear envelope; Nuclear pore complex; Nuclear trafficking; Nuclear transport; SUN; YAP; β-catenin

DOI: https://doi.org/10.1007/978-3-031-06573-6_11

G3 (Bethesda). 2021 Jul 14. pii: jkab106. [Epub ahead of print]11(7):

Mutation of the nuclear pore complex component, aladin1, disrupts asymmetric cell division in Zea mays (maize).

Norman B Best, Charles Addo-Quaye, Bong-Suk Kim, Clifford F Weil, Burkhard Schulz, Guri Johal, Brian P Dilkes.

The nuclear pore complex (NPC) regulates the movement of macromolecules between the nucleus and cytoplasm. Dysfunction of many components of the NPC results in human genetic diseases, including triple A syndrome (AAAS) as a result of mutations in ALADIN. Here, we report a nonsense mutation in the maize ortholog, aladin1 (ali1-1), at the orthologous amino acid residue of an AAAS allele from humans, alters plant stature, tassel architecture, and asymmetric divisions of subsidiary mother cells (SMCs). Crosses with the stronger nonsense allele ali1-2 identified complex allele interactions for plant height and aberrant SMC division. RNA-seq analysis of the ali1-1 mutant identified compensatory transcript accumulation for other NPC components as well as gene expression consequences consistent with conservation of ALADIN1 functions between humans and maize. These findings demonstrate that ALADIN1 is necessary for normal plant development, shoot architecture, and asymmetric cell division in maize.

Keywords: asymmetric cell division; maize; nuclear pore complex; stomata; subsidiary cells

DOI: https://doi.org/10.1093/g3journal/jkab106

Nat Biotechnol. 2022 Nov 07.

MINSTED nanoscopy enters the Ångström localization range.

Michael Weber, Henrik von der Emde, Marcel Leutenegger, Philip Gunkel, Sivakumar Sambandan, Taukeer A Khan, Jan Keller-Findeisen, Volker C Cordes, Stefan W Hell.

Super-resolution techniques have achieved localization precisions in the nanometer regime. Here we report all-optical, room temperature localization of fluorophores with precision in the Ångström range. We built on the concept of MINSTED nanoscopy where precision is increased by encircling the fluorophore with the low-intensity central region of a stimulated emission depletion (STED) donut beam while constantly increasing the absolute donut power. By blue-shifting the STED beam and separating fluorophores by on/off switching, individual fluorophores bound to a DNA strand are localized with σ = 4.7 Å, corresponding to a fraction of the fluorophore size, with only 2,000 detected photons. MINSTED fluorescence nanoscopy with single-digit nanometer resolution is exemplified by imaging nuclear pore complexes and the distribution of nuclear lamin in mammalian cells labeled by transient DNA hybridization. Because our experiments yield a localization precision σ = 2.3 Å, estimated for 10,000 detected photons, we anticipate that MINSTED will open up new areas of application in the study of macromolecular complexes in cells.

DOI: https://doi.org/10.1038/s41587-022-01519-4

Cell Rep. 2022 Nov 08. pii: S2211-1247(22)01455-3. [Epub ahead of print]41(6): 111590

Nucleoporins facilitate ORC loading onto chromatin.

Logan Richards, Christopher L Lord, Mary Lauren Benton, John A Capra, Jared T Nordman.

The origin recognition complex (ORC) binds throughout the genome to initiate DNA replication. In metazoans, it is still unclear how ORC is targeted to specific loci to facilitate helicase loading and replication initiation. Here, we perform immunoprecipitations coupled with mass spectrometry for ORC2 in Drosophila embryos. Surprisingly, we find that ORC2 associates with multiple subunits of the Nup107-160 subcomplex of the nuclear pore. Bioinformatic analysis reveals that, relative to all modENCODE factors, nucleoporins are among the most enriched factors at ORC2 binding sites. Critically, depletion of the nucleoporin Elys, a member of the Nup107-160 complex, decreases ORC2 loading onto chromatin. Depleting Elys also sensitizes cells to replication fork stalling, which could reflect a defect in establishing dormant replication origins. Our work reveals a connection between ORC, replication initiation, and nucleoporins, suggesting a function for nucleoporins in metazoan replication initiation.

Keywords: CP: Molecular biology; DNA replication; drosophila; genome stability; nuclear pore

DOI: https://doi.org/10.1016/j.celrep.2022.111590

Traffic. 2022 Nov 10.

Multiplexed cellular profiling identifies an organoselenium compound as an inhibitor of CRM1-mediated nuclear export.

Lucia Jimenez, Victor Mayoral-Varo, Carlos Amenábar, Judit Ortega, João G N Sequeira, Miguel Machuqueiro, Cristiana Mourato, Romano Silvestri, Andrea Angeli, Fabrizio Carta, Claudiu T Supuran, Diego Megías, Bibiana I Ferreira, Wolfgang Link.

Chromosomal region maintenance 1 (CRM1 also known as Xpo1 and exportin-1) is the receptor for the nuclear export controlling the intracellular localization and function of many cellular and viral proteins that play a crucial role in viral infections and cancer. The inhibition of CRM1 has emerged as a promising therapeutic approach to interfere with the lifecycle of many viruses, for the treatment of cancer and to overcome therapy resistance. Recently, selinexor has been approved as the first CRM1 inhibitor for the treatment of multiple myeloma providing the proof of concept for this therapeutic option with a new mode of action. However, selinexor is associated with dose-limiting toxicity and hence, the discovery of alternative small molecule leads that could be developed as less toxic anti-cancer and anti-viral therapeutics will have a significant impact in the clinic. Here, we report a CRM1 inhibitor discovery platform. The development of this platform includes reporter cell lines that monitor CRM1 activity by using RFP or GFP-labeled HIV-1 Rev protein with a strong heterologous nuclear export signal (NES). Simultaneously, the intracellular localization of other proteins, to be interrogated for their capacity to undergo CRM1-mediated export, can be followed by co-culturing stable cell lines expressing fluorescent fusion proteins. We used this platform to interrogate the mode of nuclear export of several proteins including PDK1, p110α, STAT5A, FOXO1, 3, 4, and TRIB2, and to screen a compound collection. We show that while p110α partially relies on CRM1-dependent nuclear export, TRIB2 is exported from the nucleus in a CRM1-independent manner. Compound screening revealed a striking activity of an organoselenium compound on the CRM1 nuclear export receptor.

Keywords: CRM1; Nuclear export; anticancer drugs; antiviral drugs; biosensors; organoselenium compounds; selinexor

DOI: https://doi.org/10.1111/tra.12872

Viruses. 2022 Nov 08. pii: 2468. [Epub ahead of print]14(11):

Ivermectin Inhibits HBV Entry into the Nucleus by Suppressing KPNA2.

Anna Nakanishi, Hiroki Okumura, Tadahiro Hashita, Aya Yamashita, Yuka Nishimura, Chihiro Watanabe, Sakina Kamimura, Sanae Hayashi, Shuko Murakami, Kyoko Ito, Takahiro Iwao, Akari Ikeda, Tomoyasu Hirose, Toshiaki Sunazuka, Yasuhito Tanaka, Tamihide Matsunaga.

Hepatitis B virus (HBV) specifically infects human hepatocytes and increases the risks of cirrhosis and liver cancer. Currently, nucleic acid analogs are the main therapeutics for chronic hepatitis caused by HBV infection. Although nucleic acid analogs can eliminate HBV DNA by inhibiting HBV reverse transcriptase, they cannot lead to negative conversion of covalently closed circular DNA (cccDNA) and hepatitis B surface antigen (HBsAg). In this study, we revealed that the antifilarial drug ivermectin suppresses HBV production by a different mechanism from the nucleic acid analog entecavir or Na+ taurocholate co-transporting polypeptide-mediated entry inhibitor cyclosporin A. Ivermectin reduced the levels of several HBV markers, including HBsAg, in HBV-infected human hepatocellular carcinoma cells (HepG2-hNTCP-C4 cells) and humanized mouse hepatocytes (PXB hepatocytes). In addition, ivermectin significantly decreased the expression of HBV core protein and the nuclear transporter karyopherin α2 (KPNA2) in the nuclei of HepG2-hNTCP-C4 cells. Furthermore, depletion of KPNA1-6 suppressed the production of cccDNA. These results suggest that KPNA1-6 is involved in the nuclear import of HBV and that ivermectin suppresses the nuclear import of HBV by inhibiting KPNA2. This study demonstrates the potential of ivermectin as a novel treatment for hepatitis B.

Keywords: hepatitis B core protein; hepatitis B surface antigen; hepatitis B virus; importin α/β; ivermectin; karyopherin α; nuclear localization signal; nuclear transfer

DOI: https://doi.org/10.3390/v14112468