How condensed are mitotic chromosomes?

Chromosomes undergo dramatic compaction during mitosis, but accurately measuring their volume has been challenging. Employing serial block face scanning electron microscopy, Cisneros-Soberanis et al. (https://doi.

OpenTn5: Open-Source Resource for Robust and Scalable Tn5 Transposase Purification and Characterization.

Tagmentation combines DNA fragmentation and sequencing adapter addition by leveraging the transposition activity of the bacterial cut-and-paste Tn5 transposase, to enable efficient sequencing library preparation. Here we present an open-source protocol for the generation of multi-purpose hyperactive Tn5 transposase, including its benchmarking in CUT&Tag, bulk and single-cell ATAC-seq. The OpenTn5 protocol yields multi-milligram quantities of pG-Tn5 protein per liter of culture, sufficient for thousands of tagmentation reactions and the enzyme retains activity in storage for more than a year.

MagIC-Cryo-EM: Structural determination on magnetic beads for scarce macromolecules in heterogeneous samples.

Cryo-EM single-particle analyses typically require target macromolecule concentration at 0.05~5.0 mg/ml, which is often difficult to achieve.

Redox-Sensitive Cysteines Confer Proximal Control of the Molecular Crowding Barrier in the Nuclear Pore.

The nuclear pore complex forms a highly crowded selective barrier with intrinsically disordered regions at the nuclear membrane to coordinate nucleocytoplasmic molecular communications. Although oxidative stress is known to alter the barrier function, the molecular mechanism underlying this adaptive control of the nuclear pore complex remains unknown. Here we uncover a systematic control of the crowding barrier within the nuclear pore in response to various redox environments.

Interactions between non-structured domains of FG- and non-FG-nucleoporins coordinate the ordered assembly of the nuclear pore complex in mitosis.

In this study, we examined how channel-forming subunits of the nuclear pore complex (NPC) are assembled into a selective channel within a highly structured scaffold ring during postmitotic assembly. We focused on non-structured domains of the scaffold Nups and performed in vitro self-assembled particle assays with those derived from channel-forming FG-Nups. We found that non-structured domains of ELYS and Nup35N interacted with channel-forming FG-Nups to form a self-assembled particle.

Prolines in the α-helix confer the structural flexibility and functional integrity of importin-β.

The karyopherin family of nuclear transport receptors is composed of a long array of amphiphilic α-helices and undergoes flexible conformational changes to pass through the hydrophobic crowding barrier of the nuclear pore. Here, we focused on the characteristic enrichment of prolines in the middle of the outer α-helices of importin-β. When these prolines were substituted with alanine, nuclear transport activity was reduced drastically and , and caused a severe defect in mitotic progression.

In vivo analysis of protein crowding within the nuclear pore complex in interphase and mitosis.

The central channel of the nuclear pore complex (NPC) is occupied by non-structured polypeptides with a high content of Phe-Gly (FG) motifs. This protein-rich environment functions as an entropic barrier that prevents the passage of molecules, as well as the binding sites for karyopherins, to regulate macromolecular traffic between the nucleoplasm and the cytoplasm. In this study, we expressed individual Nups fused with a crowding-sensitive probe (GimRET) to determine the spatial distribution of protein-rich domains within the central channel in vivo, and characterize the properties of the entropic barrier.