bims-nucpor 2023-04-30 papers

Issue of 2023‒04‒30
seven papers selected by
Sara Mingu
Johannes Gutenberg University

Nature. 2023 Apr 26.

Visualizing the disordered nuclear transport machinery in situ.

Miao Yu, Maziar Heidari, Sofya Mikhaleva, Piau Siong Tan, Sara Mingu, Hao Ruan, Christopher D Reinkemeier, Agnieszka Obarska-Kosinska, Marc Siggel, Martin Beck, Gerhard Hummer, Edward A Lemke.

The approximately 120 MDa mammalian nuclear pore complex (NPC) acts as a gatekeeper for the transport between the nucleus and cytosol1. The central channel of the NPC is filled with hundreds of intrinsically disordered proteins (IDPs) called FG-nucleoporins (FG-NUPs)2,3. Although the structure of the NPC scaffold has been resolved in remarkable detail, the actual transport machinery built up by FG-NUPs-about 50 MDa-is depicted as an approximately 60-nm hole in even highly resolved tomograms and/or structures computed with artificial intelligence4-11. Here we directly probed conformations of the vital FG-NUP98 inside NPCs in live cells and in permeabilized cells with an intact transport machinery by using a synthetic biology-enabled site-specific small-molecule labelling approach paired with highly time-resolved fluorescence microscopy. Single permeabilized cell measurements of the distance distribution of FG-NUP98 segments combined with coarse-grained molecular simulations of the NPC allowed us to map the uncharted molecular environment inside the nanosized transport channel. We determined that the channel provides-in the terminology of the Flory polymer theory12-a 'good solvent' environment. This enables the FG domain to adopt expanded conformations and thus control transport between the nucleus and cytoplasm. With more than 30% of the proteome being formed from IDPs, our study opens a window into resolving disorder-function relationships of IDPs in situ, which are important in various processes, such as cellular signalling, phase separation, ageing and viral entry.

DOI: https://doi.org/10.1038/s41586-023-05990-0

Biochem Soc Trans. 2023 04 26. 51(2): 871-886

Improving the hole picture: towards a consensus on the mechanism of nuclear transport.

David Cowburn, Michael Rout.

Nuclear pore complexes (NPCs) mediate the exchange of materials between the nucleoplasm and cytoplasm, playing a key role in the separation of nucleic acids and proteins into their required compartments. The static structure of the NPC is relatively well defined by recent cryo-EM and other studies. The functional roles of dynamic components in the pore of the NPC, phenylalanyl-glycyl (FG) repeat rich nucleoporins, is less clear because of our limited understanding of highly dynamic protein systems. These proteins form a 'restrained concentrate' which interacts with and concentrates nuclear transport factors (NTRs) to provide facilitated nucleocytoplasmic transport of cargoes. Very rapid on- and off-rates among FG repeats and NTRs supports extremely fast facilitated transport, close to the rate of macromolecular diffusion in cytoplasm, while complexes without specific interactions are entropically excluded, though details on several aspects of the transport mechanism and FG repeat behaviors remain to be resolved. However, as discussed here, new technical approaches combined with more advanced modeling methods will likely provide an improved dynamic description of NPC transport, potentially at the atomic level in the near future. Such advances are likely to be of major benefit in comprehending the roles the malfunctioning NPC plays in cancer, ageing, viral diseases, and neurodegeneration.

Keywords: cell nucleus; nuclear pores; nuclear protein transportport proteins; nucleic acids

DOI: https://doi.org/10.1042/BST20220494

Dev Cell. 2023 Apr 24. pii: S1534-5807(23)00156-9. [Epub ahead of print]

A tubule-sheet continuum model for the mechanism of nuclear envelope assembly.

Gengjing Zhao, Shiwei Liu, Sanjana Arun, Fioranna Renda, Alexey Khodjakov, David Pellman.

Nuclear envelope (NE) assembly defects cause chromosome fragmentation, cancer, and aging. However, major questions about the mechanism of NE assembly and its relationship to nuclear pathology are unresolved. In particular, how cells efficiently assemble the NE starting from vastly different, cell type-specific endoplasmic reticulum (ER) morphologies is unclear. Here, we identify a NE assembly mechanism, "membrane infiltration," that defines one end of a continuum with another NE assembly mechanism, "lateral sheet expansion," in human cells. Membrane infiltration involves the recruitment of ER tubules or small sheets to the chromatin surface by mitotic actin filaments. Lateral sheet expansion involves actin-independent envelopment of peripheral chromatin by large ER sheets that then extend over chromatin within the spindle. We propose a "tubule-sheet continuum" model that explains the efficient NE assembly from any starting ER morphology, the cell type-specific patterns of nuclear pore complex (NPC) assembly, and the obligatory NPC assembly defect of micronuclei.

Keywords: endoplasmic reticulum; micronuclei; mitosis; mitotic actin; myosin V; nuclear envelope; nuclear pore complexes

DOI: https://doi.org/10.1016/j.devcel.2023.04.003

Plant Sci. 2023 Apr 26. pii: S0168-9452(23)00136-X. [Epub ahead of print] 111719

Characterisation of the Trans-membrane Nucleoporins GP210 and NDC1 in Arabidopsis thaliana.

Patrick P Collins, Ronan C Broad, Krithika Yogeeswaran, Arvind Varsani, Anthony M Poole, David A Collings.

The nuclear pore is structurally conserved across eukaryotes as are many of the pore's constituent proteins. The transmembrane nuclear pore proteins GP210 and NDC1 span the nuclear envelope holding the nuclear pore in place. Orthologues of GP210 and NDC1 in Arabidopsis were investigated through characterisation of T-DNA insertional mutants. While the T-DNA insert into GP210 reduced expression of the gene, the insert in the NDC1 gene resulted in increased expression in both the ndc1 mutant as well as the ndc1/gp210 double mutant. The ndc1 and gp210 individual mutants showed little phenotypic difference from wild-type plants, but the ndc1/gp210 mutant showed a range of phenotypic effects. As with many plant nuclear pore protein mutants, these effects included non-nuclear phenotypes such as reduced pollen viability, reduced growth and glabrous leaves in mature plants. Importantly, however, ndc1/gp210 exhibited nuclear-specific effects including modifications to nuclear shape in different cell types. We also observed functional changes to nuclear transport in ndc1/gp210 plants, with low levels of cytoplasmic fluorescence observed in cells expressing nuclear-targeted GFP. The lack of phenotypes in individual insertional lines, and the relatively mild phenotype suggests that additional transmembrane nucleoporins, such as the recently-discovered CPR5, likely compensate for their loss.

Keywords: Arabidopsis thaliana; GP210; NDC1; nuclear pore; nucleoporin

DOI: https://doi.org/10.1016/j.plantsci.2023.111719

Epigenetics Chromatin. 2023 Apr 28. 16(1): 14

An optimized approach for multiplexing single-nuclear ATAC-seq using oligonucleotide-conjugated antibodies.

Betelehem Solomon Bera, Taylor V Thompson, Eric Sosa, Hiroko Nomaru, David Reynolds, Robert A Dubin, Shahina B Maqbool, Deyou Zheng, Bernice E Morrow, John M Greally, Masako Suzuki.

BACKGROUND: Single-cell technologies to analyze transcription and chromatin structure have been widely used in many research areas to reveal the functions and molecular properties of cells at single-cell resolution. Sample multiplexing techniques are valuable when performing single-cell analysis, reducing technical variation and permitting cost efficiencies. Several commercially available methods have been used in many scRNA-seq studies. On the other hand, while several methods have been published, multiplexing techniques for single nuclear assay for transposase-accessible chromatin (snATAC)-seq assays remain under development. We developed a simple nucleus hashing method using oligonucleotide-conjugated antibodies recognizing nuclear pore complex proteins, NuHash, to perform snATAC-seq library preparations by multiplexing.RESULTS: We performed multiplexing snATAC-seq analyses on a mixture of human and mouse cell samples (two samples, 2-plex, and four samples, 4-plex) using NuHash. The analyses on nuclei with at least 10,000 read counts showed that the demultiplexing accuracy of NuHash was high, and only ten out of 9144 nuclei (2-plex) and 150 of 12,208 nuclei (4-plex) had discordant classifications between NuHash demultiplexing and discrimination using reference genome alignments. The differential open chromatin region (OCR) analysis between female and male samples revealed that male-specific OCRs were enriched in chromosome Y (four out of nine). We also found that five female-specific OCRs (20 OCRs) were on chromosome X. A comparative analysis between snATAC-seq and deeply sequenced bulk ATAC-seq on the same samples revealed that the bulk ATAC-seq signal intensity was positively correlated with the number of cell clusters detected in snATAC-seq. Moreover, when we categorized snATAC-seq peaks based on the number of cell clusters in which the peak was present, we observed different distributions over different genomic features between the groups. This result suggests that the peak intensities of bulk ATAC-seq can be used to identify different types of functional loci.
CONCLUSIONS: Our multiplexing method using oligo-conjugated anti-nuclear pore complex proteins, NuHash, permits high-accuracy demultiplexing of samples. The NuHash protocol is straightforward, works on frozen samples, and requires no modifications for snATAC-seq library preparation.

Keywords: Assay for transposase-accessible chromatin (ATAC); Multiplexing; Open-chromatin regions; Single-cell

DOI: https://doi.org/10.1186/s13072-023-00486-7

Genes (Basel). 2023 Mar 23. pii: 775. [Epub ahead of print]14(4):

Close Ties between the Nuclear Envelope and Mammalian Telomeres: Give Me Shelter.

Gaëlle Pennarun, Julien Picotto, Pascale Bertrand.

The nuclear envelope (NE) in eukaryotic cells is essential to provide a protective compartment for the genome. Beside its role in connecting the nucleus with the cytoplasm, the NE has numerous important functions including chromatin organization, DNA replication and repair. NE alterations have been linked to different human diseases, such as laminopathies, and are a hallmark of cancer cells. Telomeres, the ends of eukaryotic chromosomes, are crucial for preserving genome stability. Their maintenance involves specific telomeric proteins, repair proteins and several additional factors, including NE proteins. Links between telomere maintenance and the NE have been well established in yeast, in which telomere tethering to the NE is critical for their preservation and beyond. For a long time, in mammalian cells, except during meiosis, telomeres were thought to be randomly localized throughout the nucleus, but recent advances have uncovered close ties between mammalian telomeres and the NE that play important roles for maintaining genome integrity. In this review, we will summarize these connections, with a special focus on telomere dynamics and the nuclear lamina, one of the main NE components, and discuss the evolutionary conservation of these mechanisms.

Keywords: DNA repair; alternative lengthening of telomeres; genome stability; lamins; nuclear envelope; nuclear pore; shelterin; telomere maintenance

DOI: https://doi.org/10.3390/genes14040775

Proc Natl Acad Sci U S A. 2023 May 02. 120(18): e2220545120

Structural insights into HIV-1 polyanion-dependent capsid lattice formation revealed by single particle cryo-EM.

Carolyn M Highland, Aaron Tan, Clifton L Ricaña, John A G Briggs, Robert A Dick.

The HIV-1 capsid houses the viral genome and interacts extensively with host cell proteins throughout the viral life cycle. It is composed of capsid protein (CA), which assembles into a conical fullerene lattice composed of roughly 200 CA hexamers and 12 CA pentamers. Previous structural analyses of individual CA hexamers and pentamers have provided valuable insight into capsid structure and function, but detailed structural information about these assemblies in the broader context of the capsid lattice is lacking. In this study, we combined cryoelectron tomography and single particle analysis (SPA) cryoelectron microscopy to determine structures of continuous regions of the capsid lattice containing both hexamers and pentamers. We also developed a method of liposome scaffold-based in vitro lattice assembly ("lattice templating") that enabled us to directly study the lattice under a wider range of conditions than has previously been possible. Using this approach, we identified a critical role for inositol hexakisphosphate in pentamer formation and determined the structure of the CA lattice bound to the capsid-targeting antiretroviral drug GS-6207 (lenacapavir). Our work reveals key structural details of the mature HIV-1 CA lattice and establishes the combination of lattice templating and SPA as a robust strategy for studying retroviral capsid structure and capsid interactions with host proteins and antiviral compounds.

Keywords: HIV-1; capsid; cryoelectron microscopy; cryoelectron tomography; retrovirus structure

DOI: https://doi.org/10.1073/pnas.2220545120