bims-nucpor 2022-12-18 papers

J Cell Biol. 2023 Feb 06. pii: e202204039. [Epub ahead of print]222(2):

Meiotic nuclear pore complex remodeling provides key insights into nuclear basket organization.

Grant A King, Rahel Wettstein, Joseph M Varberg, Keerthana Chetlapalli, Madison E Walsh, Ludovic C J Gillet, Claudia Hernández-Armenta, Pedro Beltrao, Ruedi Aebersold, Sue L Jaspersen, Joao Matos, Elçin Ünal.

Nuclear pore complexes (NPCs) are large proteinaceous assemblies that mediate nuclear compartmentalization. NPCs undergo large-scale structural rearrangements during mitosis in metazoans and some fungi. However, our understanding of NPC remodeling beyond mitosis remains limited. Using time-lapse fluorescence microscopy, we discovered that NPCs undergo two mechanistically separable remodeling events during budding yeast meiosis in which parts or all of the nuclear basket transiently dissociate from the NPC core during meiosis I and II, respectively. Meiosis I detachment, observed for Nup60 and Nup2, is driven by Polo kinase-mediated phosphorylation of Nup60 at its interface with the Y-complex. Subsequent reattachment of Nup60-Nup2 to the NPC core is facilitated by a lipid-binding amphipathic helix in Nup60. Preventing Nup60-Nup2 reattachment causes misorganization of the entire nuclear basket in gametes. Strikingly, meiotic nuclear basket remodeling also occurs in the distantly related fission yeast, Schizosaccharomyces pombe. Our study reveals a conserved and developmentally programmed aspect of NPC plasticity, providing key mechanistic insights into the nuclear basket organization.

DOI: https://doi.org/10.1083/jcb.202204039

Nanoscale Adv. 2022 Nov 22. 4(23): 4925-4937

Pore performance: artificial nanoscale constructs that mimic the biomolecular transport of the nuclear pore complex.

John Andersson, Justas Svirelis, Jesper Medin, Julia Järlebark, Rebekah Hailes, Andreas Dahlin.

The nuclear pore complex is a nanoscale assembly that achieves shuttle-cargo transport of biomolecules: a certain cargo molecule can only pass the barrier if it is attached to a shuttle molecule. In this review we summarize the most important efforts aiming to reproduce this feature in artificial settings. This can be achieved by solid state nanopores that have been functionalized with the most important proteins found in the biological system. Alternatively, the nanopores are chemically modified with synthetic polymers. However, only a few studies have demonstrated a shuttle-cargo transport mechanism and due to cargo leakage, the selectivity is not comparable to that of the biological system. Other recent approaches are based on DNA origami, though biomolecule transport has not yet been studied with these. The highest selectivity has been achieved with macroscopic gels, but they are yet to be scaled down to nano-dimensions. It is concluded that although several interesting studies exist, we are still far from achieving selective and efficient artificial shuttle-cargo transport of biomolecules. Besides being of fundamental interest, such a system could be potentially useful in bioanalytical devices.

DOI: https://doi.org/10.1039/d2na00389a

Int J Mol Sci. 2022 Dec 05. pii: 15333. [Epub ahead of print]23(23):

Deciphering the Binding of the Nuclear Localization Sequence of Myc Protein to the Nuclear Carrier Importin α3.

Bruno Rizzuti, Juan L Iovanna, José L Neira.

The oncoprotein Myc is a transcription factor regulating global gene expression and modulating cell proliferation, apoptosis, and metabolism. Myc has a nuclear localization sequence (NLS) comprising residues Pro320 to Asp328, to allow for nuclear translocation. We designed a peptide comprising such region and the flanking residues (Ala310-Asn339), NLS-Myc, to study, in vitro and in silico, the ability to bind importin α3 (Impα3) and its truncated species (ΔImpα3) depleted of the importin binding domain (IBB), by using fluorescence, circular dichroism (CD), biolayer interferometry (BLI), nuclear magnetic resonance (NMR), and molecular simulations. NLS-Myc interacted with both importin species, with affinity constants of ~0.5 µM (for Impα3) and ~60 nM (for ΔImpα3), as measured by BLI. The molecular simulations predicted that the anchoring of NLS-Myc took place in the major binding site of Impα3 for the NLS of cargo proteins. Besides clarifying the conformational behavior of the isolated NLS of Myc in solution, our results identified some unique properties in the binding of this localization sequence to the nuclear carrier Impα3, such as a difference in the kinetics of its release mechanism depending on the presence or absence of the IBB domain.

Keywords: binding; biolayer interferometry; calorimetry; fluorescence; molecular docking; nuclear localization signal

DOI: https://doi.org/10.3390/ijms232315333

J Cell Sci. 2022 Dec 15. pii: jcs260240. [Epub ahead of print]135(24):

Ultrastructure expansion microscopy reveals the cellular architecture of budding and fission yeast.

Kerstin Hinterndorfer, Marine H Laporte, Felix Mikus, Lucas Tafur, Clélia Bourgoint, Manoel Prouteau, Gautam Dey, Robbie Loewith, Paul Guichard, Virginie Hamel.

The budding and fission yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe have served as invaluable model organisms to study conserved fundamental cellular processes. Although super-resolution microscopy has in recent years paved the way to a better understanding of the spatial organization of molecules in cells, its wide use in yeasts has remained limited due to the specific know-how and instrumentation required, contrasted with the relative ease of endogenous tagging and live-cell fluorescence microscopy. To facilitate super-resolution microscopy in yeasts, we have extended the ultrastructure expansion microscopy (U-ExM) method to both S. cerevisiae and S. pombe, enabling a 4-fold isotropic expansion. We demonstrate that U-ExM allows imaging of the microtubule cytoskeleton and its associated spindle pole body, notably unveiling the Sfi1p-Cdc31p spatial organization on the appendage bridge structure. In S. pombe, we validate the method by monitoring the homeostatic regulation of nuclear pore complex number through the cell cycle. Combined with NHS-ester pan-labelling, which provides a global cellular context, U-ExM reveals the subcellular organization of these two yeast models and provides a powerful new method to augment the already extensive yeast toolbox. This article has an associated First Person interview with Kerstin Hinterndorfer and Felix Mikus, two of the joint first authors of the paper.

Keywords: S. cerevisiae ; S. pombe ; Microtubules; Nuclear pore complex; Spindle pole body duplication; U-ExM; Ultrastructure expansion microscopy

DOI: https://doi.org/10.1242/jcs.260240

Science. 2022 Dec 16. 378(6625): eadf3971

Protein import into peroxisomes occurs through a nuclear pore-like phase.

Yuan Gao, Michael L Skowyra, Peiqiang Feng, Tom A Rapoport.

Peroxisomes are ubiquitous organelles whose dysfunction causes fatal human diseases. Most peroxisomal proteins are imported from the cytosol in a folded state by the soluble receptor PEX5. How folded cargo crosses the membrane is unknown. Here, we show that peroxisomal import is similar to nuclear transport. The peroxisomal membrane protein PEX13 contains a conserved tyrosine (Y)- and glycine (G)-rich YG domain, which forms a selective phase resembling that formed by phenylalanine-glycine (FG) repeats within nuclear pores. PEX13 resides in the membrane in two orientations that oligomerize and suspend the YG meshwork within the lipid bilayer. Purified YG domains form hydrogels into which PEX5 selectively partitions, by using conserved aromatic amino acid motifs, bringing cargo along. The YG meshwork thus forms an aqueous conduit through which PEX5 delivers folded proteins into peroxisomes.

DOI: https://doi.org/10.1126/science.adf3971

Trends Microbiol. 2022 Dec 12. pii: S0966-842X(22)00336-5. [Epub ahead of print]

Unraveling the mystery of viroid nuclear import.

Ashish Prasad, Amita Sharma, Jayanwita Sarkar.

Viroids are closed-circular infectious RNAs that are known to infect plants. Despite their small noncoding genome, they have the ability to cause disease. The nuclear import mechanism of nucleus replicating viroids is not well understood. Ma et al. have recently highlighted the route of viroid entry into the nucleus.

Keywords: IMPa-4; VIRP1; viroids

DOI: https://doi.org/10.1016/j.tim.2022.12.002