Parihar, P. S. | Search bioRxiv

bioRxiv2026

Concanavalin A as a pan-eukaryotic nuclear envelope marker for expansion microscopy

Across eukaryotes, the nuclear envelope exhibits distinct remodelling strategies during mitosis: complete breakdown (open mitosis), partial disruption (intermediate), or full retention (closed). However, both light and electron microscopy of the nuclear envelope, at sufficient resolution to determine integrity and mode of remodelling, have proven technically challenging. Here, Concanavalin A (ConA), a glycan-binding plant lectin, provides discernible nuclear envelope/endoplasmic reticulum (NE/ER) labelling in Ultrastructure Expansion Microscopy (U-ExM). Applied post-expansion, ConA circumvents antibody optimisation and labels the NE across mammalian cells and diverse microbial eukaryotes. By imaging four opisthokont and amoebozoan species with established mitotic strategies, C. perkinsii, D. discoideum, S. pombe, and S. arctica, we distinguished the full spectrum of NE remodelling. Applied to species with poorly characterised mitotic strategies, ConA revealed intermediate mitosis with polar fenestrae in the multinucleate stramenopile A. limacinum and a life cycle-coupled switch between open and closed mitosis in the amoebozoan P. polycephalum. These observations support the hypothesis that multinucleated life cycles favour closed or intermediate mitosis, protecting chromosomes from capture by neighbouring spindles during synchronous divisions. Together, these results establish ConA in combination with expansion microscopy as a broadly applicable tool for uncovering the diversity and evolution of NE remodelling across eukaryotes.

cell biology↗

bioRxiv2023

Extended regulation interface coupled to the allosteric network and disease mutations in the PP2A-B56 delta holoenzyme

An increasing number of mutations associated with devastating human diseases are diagnosed by whole-genome/exon sequencing. Recurrent de novo missense mutations have been discovered in B56{delta} (encoded by PPP2R5D), a regulatory subunit of protein phosphatase 2A (PP2A), that cause intellectual disabilities (ID), macrocephaly, Parkinsonism, and a broad range of neurological symptoms. Single-particle cryo-EM structures show that the PP2A-B56{delta} holoenzyme possesses closed latent and open active forms. In the closed form, the long, disordered arms of B56{delta} termini fold against each other and the holoenzyme core, establishing dual autoinhibition of the phosphatase active site and the substrate-binding protein groove. The resulting interface spans over 190 [A] and harbors unfavorable contacts, activation phosphorylation sites, and nearly all residues with ID-associated mutations. Our studies suggest that this dynamic interface is close to an allosteric network responsive to activation phosphorylation and altered globally by mutations. Furthermore, we found that ID mutations perturb the activation phosphorylation rates, and the severe variants significantly increase the mitotic duration and error rates compared to the wild variant.

biochemistry↗