Biology subjects

Arimura, Y.

Publications and source records attributed to Arimura, Y..

6 recordsLinked to original sources

bioRxiv2026

Native yeast kinetochore structures identify an essential inner kinetochore interaction

Kinetochores must accurately assemble on centromeres for faithful chromosome segregation. Although a conserved centromeric nucleosome is essential for kinetochore assembly, budding yeast centromeric DNA is a poor template for nucleosome formation in vitro, perhaps due to its intrinsic rigidity. To better understand yeast inner kinetochore assembly, we developed a one-step protocol to purify native inner kinetochore subcomplexes for structural studies. We performed cryo-electron microscopy on the purifications and generated density maps of four separate inner kinetochore complexes, two of which had not previously been visualized and may represent intermediate assembly states. We identified an Ndc10 trimerization domain that engages centromeric DNA and a pair of CBF3 complexes and is associated with substantial bending of centromeric DNA. Ndc10 trimerization is essential for kinetochore assembly and chromosome segregation. We propose that Ndc10 trimerization facilitates centromeric DNA bending to stabilize the centromeric nucleosome and inner kinetochore.

biochemistry↗

bioRxiv2025

IMPACTS OF DNA METHYLATION ON H2A.Z DEPOSITION AND NUCLEOSOME STABILITY

The histone variant H2A.Z and DNA methylation are enriched at mutually exclusive genomic segments, though its mechanistic bases remain unclear. Here, we examine impacts of DNA methylation on the intrinsic stability of the H2A.Z nucleosome and chaperone-mediated H2A.Z deposition. Cryo-EM and endonuclease analyses suggest that DNA methylation subtly increases the openness and accessibility of the H2A.Z nucleosome on satellite II-derived DNA sequences. In transcriptionally silent Xenopus egg extracts, H2A.Z preferentially associates with unmethylated DNA though a substantial proportion of H2A.Z is recruited to methylated DNA. Preferential H2A.Z deposition to unmethylated DNA depends on the SRCAP complex, whose DNA binding is suppressed by methylation, while a SRCAP-independent and DNA methylation-insensitive mechanism for H2A.Z deposition also exists. Altogether, we propose that SRCAP drives the biased association of H2A.Z to unmethylated DNA, while additional mechanisms, potentially taking advantage of the subtle DNA methylation-induced physical effects, further assist the exclusion of H2A.Z from methylated DNA.

molecular biology↗

bioRxiv2024

Architecture and flexibility of native kinetochores revealed by structural studies utilizing a thermophilic yeast

Eukaryotic chromosome segregation requires kinetochores, multi-megadalton protein machines that assemble on the centromeres of chromosomes and mediate attachments to dynamic spindle microtubules. Kinetochores are built from numerous complexes, and understanding how they are arranged is key to understanding how kinetochores perform their multiple functions. However, an integrated understanding of kinetochore architecture has not yet been established. To address this, we purified functional, native kinetochores from Kluyveromyces marxianus and examined them by electron microscopy, cryo-electron tomography and atomic force microscopy. The kinetochores are extremely large, flexible assemblies that exhibit features consistent with prior models. We assigned kinetochore polarity by visualizing their interactions with microtubules and locating the microtubule binder Ndc80c. This work shows that isolated kinetochores are more dynamic and complex than what might be anticipated based on the known structures of recombinant subassemblies, and provides the foundation to study the global architecture and functions of kinetochores at a structural level.

biophysics↗

bioRxiv2024

Cryo-EM analysis 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. Here, we devise Magnetic Isolation and Concentration (MagIC)-cryo-EM, a technique enabling direct structural analysis of targets captured on magnetic beads, thereby reducing the targets concentration requirement to < 0.0005 mg/ml. Adapting MagIC-cryo-EM to a Chromatin Immunoprecipitation protocol, we characterized structural variations of the linker histone H1.8-associated nucleosomes that were isolated from interphase and metaphase chromosomes in Xenopus egg extract. Combining Duplicated Selection To Exclude Rubbish particles (DuSTER), a particle curation method that excludes low signal-to-noise ratio particles, we also resolved the 3D cryo-EM structures of nucleoplasmin NPM2 co-isolated with the linker histone H1.8 and revealed distinct open and closed structural variants. Our study demonstrates the utility of MagIC-cryo-EM for structural analysis of scarce macromolecules in heterogeneous samples and provides structural insights into the cell cycle-regulation of H1.8 association to nucleosomes.

molecular biology↗

bioRxiv2021

Structural Mechanics of the Alpha-2-Macroglobulin Transformation

Alpha-2-Macroglobulin (A2M) is the critical pan-protease inhibitor of the innate immune system. When proteases cleave the A2M bait region, global structural transformation of the A2M tetramer is triggered to entrap the protease. The structural basis behind the cleavage-induced transformation and the protease entrapment remains unclear. Here, we report cryo-EM structures of native- and intermediate-forms of the Xenopus laevis egg A2M homolog (A2Moo or ovomacroglobulin) tetramer at 3.7-4.1 [A] and 6.4 [A] resolution, respectively. In the native A2Moo tetramer, two pairs of dimers arrange into a cross-like configuration with four 60 [A]-wide bait-exposing grooves. Each bait in the native form threads into an aperture formed by three macroglobulin domains (MG2, MG3, MG6). The bait is released from the narrowed aperture in the induced protomer of the intermediate form. We propose that the intact bait region works as a "latch-lock" to block futile A2M transformation until its protease-mediated cleavage.

immunology↗

bioRxiv2020

Near-atomic resolution nucleosome structures and their variations in interphase and metaphase chromosomes

Structural heterogeneity of nucleosomes in functional chromosomes is unknown. Here we report cryo-EM structures of nucleosomes isolated from interphase and metaphase chromosomes at up to 3.4 [A] resolution. Averaged chromosomal nucleosome structures are highly similar to canonical left-handed recombinant nucleosome crystal structures, with DNA being selectively stabilized at two defined locations. Compared to free mono-nucleosomes, which exhibit diverse linker DNA angles and large structural variations in H3 and H4, chromosomal nucleosome structures are much more uniform, characterized by a closed linker DNA angle with interactions between the H2A C-terminal tail and DNA. Exclusively for metaphase nucleosomes, structures of the linker histone H1.8 at the on-dyad position of nucleosomes can be reconstituted at 4.4 [A] resolution. We also report diverse minor nucleosome structural variants with rearranged core histone configurations, which are more prevalent in metaphase than in interphase chromosomes. This study presents structural characteristics of nucleosomes in interphase and mitotic chromosomes. HighlightsO_LI3.4~ [A] resolution nucleosome structures from interphase and metaphase chromosomes C_LIO_LINucleosome structures in chromosomes are more uniform than in free mono-nucleosomes C_LIO_LIHistone H1.8 binds to the nucleosome dyad axis in metaphase chromosomes C_LIO_LINucleosome structural variants are more prevalent in metaphase than in interphase C_LI NOTES TO READERSWe would like to emphasize the importance of supplemental movies S1-S3, which should greatly help readers to understand characteristics of the nucleosome structural variants that we report in this study.

cell biology↗