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Tachikawa, M.

Publications and source records attributed to Tachikawa, M..

8 recordsLinked to original sources
bioRxiv2026

CCAN forms the spherical shell-like structure in interphase kinetochores

The kinetochore is an essential structure for the faithful chromosome segregation during mitosis. The 16-protein complex known as the constitutive centromere-associated network (CCAN) localizes the kinetochore throughout the cell cycle and forms the basis of the kinetochore. Although a structural model of an individual CCAN unit has been proposed through cryo-electron microscopy analysis, little is known about how multiple CCAN units are organized within cells. Here, using in silico and in vivo analyses, we demonstrate that multiple CCAN units form a spherical, shell-like structure in interphase DT40 kinetochores. CENP-T and CENP-N localize to the CCAN shell, and the CENP-C cupin domain clusters at the center. Molecular dynamics simulations of CCAN assembly suggest that CENP-C contributes to the orientation of CCAN units, leading to the formation of a spherical shell-like structure via CENP-C oligomerization. Super-resolution imaging reveals the spherical shell-like structure at DT40 kinetochores, and knocking out CENP-C abolishes the structure, supporting the reliability of the predicted models. These findings provide important insights into the spatial organization of functional kinetochores within cells.

molecular biology↗
bioRxiv2024

Differential clearance rate of proteins encoded on a self-amplifying mRNA vaccine in muscle and lymph nodes

ARCT-154, a recently approved self-amplifying mRNA (saRNA) vaccine, has been shown in clinical trials to induce higher levels of neutralizing antibodies and sustain them for a longer time than the conventional mRNA vaccine BNT162b2. However, the scientific evidence explaining this superiority remain elusive. Hence, we explored the temporal changes in spike protein and replicase components following a single dose of ARCT-154 vaccination in mice. The encoded spike protein reached its highest level approximately 3 days after vaccination and quickly disappeared from the rectus femoris muscle, the injection site. Although the spike protein levels also peaked at an early time point in the lymph nodes, it remained detectable 28 days after the vaccination and then disappeared by 44 days after the vaccination. Expression of nsP1, nsP2 and nsP4 was observed in the injected muscle and/or the lymph nodes for up to 15 days post-vaccination. These data suggest that prolonged expression of spike proteins in lymph nodes may, if not entirely, be responsible for the induction of higher and prolonged levels of neutralizing antibodies by the saRNA vaccine.

molecular biology↗
bioRxiv2023

Actin dynamics switches two distinct modes of endosomal fusion in yolk sac visceral endoderm cells

Membranes undergo various patterns of deformation during vesicle fusion, but how this membrane deformation is regulated and contributes to fusion remains unknown. In this study, we developed a new method of observing the fusion of individual late endosomes and lysosomes by using yolk sac visceral endoderm cells that have huge endocytic vesicles. We found that there were two distinct fusion modes that were differently regulated. In homotypic fusion, two late endosomes fused quickly, whereas in heterotypic fusion they fused to lysosomes slowly. Mathematical modeling showed that vesicle size is a critical determinant of these fusion types and that membrane fluctuation forces can overcome the vesicle size effects. We found that actin filaments were bound to late endosomes and forces derived from dynamic actin remodeling were necessary for quick fusion during homotypic fusion. Furthermore, cofilin played a role in endocytic fusion by regulating actin turnover. These data suggest that actin promotes vesicle fusion for efficient membrane trafficking in visceral endoderm cells.

cell biology↗
bioRxiv2020

Plant genome response to incoming coding sequences: stochastic transcriptional activation independent of chromatin configuration

Horizontal gene transfer can occur between phylogenetically distant organisms, such as prokaryotes and eukaryotes. In these cases, how do the translocated genes acquire transcriptional competency in the alien eukaryotic genome? According to the conventional view, specific loci of the eukaryotic genome are thought to provide transcriptional competency to the incoming coding sequences. To examine this possibility, we randomly introduced the promoterless luciferase (LUC)-coding sequences into the genome of Arabidopsis thaliana cultured cells and performed a genome-wide "transgene location vs. expression" scan. We mapped 4,504 promoterless LUC inserts on the A. thaliana chromosomes, and found that about 30% of them were transcribed. Only a small portion of them were explained by the conventional transcriptional fusions with the annotated genes, and the remainder occurred in a quite different manner; (1) they occurred all over the chromosomal regions, (2) independently of the insertion sites relative to the annotated gene loci, inherent transcribed regions, or heterochromatic regions, and (3) with one magnitude lower transcriptional level than the conventional transcriptional fusions. This type of transcriptional activation occurred at about 30% of the inserts, raising a question as to what this 30% means. We tested two hypotheses: the activation occurred at 30% of the entire chromosomal regions, or stochastically at 30% of each insertion event. Our experimental analysis indicates that the latter model could explain this transcriptional activation, a new type of plant genome response to the incoming coding sequences. We discuss the possible mechanisms and evolutionary roles of this phenomenon in the plant genome.

evolutionary biology↗
bioRxiv2020

De novo activated transcription of newborn coding sequences is inheritable in the plant genome

The manner in which inserted foreign coding sequences become transcriptionally activated and fixed in the plant genome is poorly understood. To examine such processes of gene evolution, we performed an artificial evolutionary experiment in Arabidopsis thaliana. As a model of gene-birth events, we introduced a promoterless coding sequence of the firefly luciferase (LUC) gene and established 386 T2-generation transgenic lines. Among them, we determined the individual LUC insertion loci in 76 lines and found that one-third of them were transcribed de novo even in the intergenic or inherently unexpressed regions. In the transcribed lines, transcription-related chromatin marks were detected across the newly activated transcribed regions. These results agreed with our previous findings in A. thaliana cultured cells under a similar experimental scheme. A comparison of the results of the T2-plant and cultured cell experiments revealed that the de novo-activated transcription concomitant with local chromatin remodelling was inheritable. During one-generation inheritance, it seems likely that the transcription activities of the LUC inserts trapped by the endogenous genes/transcripts became stronger, while those of de novo transcription in the intergenic/untranscribed regions became weaker. These findings may offer a clue for the elucidation of the mechanism by which inserted foreign coding sequences become transcriptionally activated and fixed in the plant genome.

evolutionary biology↗
bioRxiv2020

Evaluation of origin of driving force for loop formation in a chromatin fiber

Chromosome condensation results from the formation of consecutive chromatin loops in which excluded volume interactions lead to chromosome stiffness. Formation of chromatin loops requires energy, but the source of such energy remains controversial. Here, we quantified the energy balance during chromatin loop formation by calculating the free energies of unlooped and looped chromatins using a lattice model of polymer chains. We tested two hypothetical energy sources: thermal fluctuation and ATP hydrolysis. We evaluated the free energy difference of the chain loop model without accounting for excluded volume interactions (phantom loop model), and integrated those interactions by employing the mean-field theory (interacting loop model), where we introduced the parameter of excluded volume interaction within a single loop vex. Using our strategy, we confirmed that loop-growth efficiency calculated by the phantom loop model is too high to explain the experimental data. Comparing loop-growth efficiencies for each energy source, and using the interacting loop model, we found that excluded volume interaction is essential for chromatins resistance to looping, regardless of the energy source. We predict that the quantitative measurement of vex determines which energy source is more plausible. Author summaryBefore mitosis, the chromatin fibers of eukaryotic cells fold into consecutive loop structures and condense into rod-like chromosomes. Chromosome stiffness results from the interaction of the excluded volume between chromatin loops. The driving force of loop formation and growth is still controversial, despite the many efforts undertaken to clarify it. Two possible origins can be considered: the energy provided by thermal fluctuations or the energy gained from ATP hydrolysis. To discuss the validity of each, we constructed a theoretical model of chromatin loop formation that includes excluded volume interactions. Using this model, we calculated the free energy difference before and after chromatin loop formation, which corresponds to the energy that fuels chromatin looping. By comparing the results for each energy source, we conclude that the spatial distribution of chromatin loops should be relatively wide, given the large excluded volume interaction within a single loop, irrespective of which energy source is valid. Moreover, our results imply that intra-loop interactions are key to determine the driving force of chromatin loop formation.

biophysics↗
bioRxiv2019

Large DNA virus promoted the endosymbiotic evolution to make a photosynthetic eukaryote

Chloroplasts in photosynthetic eukaryotes originated from a cyanobacterial endosymbiosis far more than 1 billion years ago1-3. Due to this ancientness, it remains unclear how this evolutionary process proceeded. To unveil this mystery, we analysed the whole genome sequence of a photosynthetic rhizarian amoeba4, Paulinella micropora5,6, which has a chloroplast-like organelle that originated from another cyanobacterial endosymbiosis7-10 about 0.1 billion years ago11. Here we show that the predacious amoeba that engulfed cyanobacteria evolved into a photosynthetic organism very quickly in the evolutionary time scale, probably aided by the drastic genome reorganization activated by large DNA virus. In the endosymbiotic evolution of eukaryotic cells, gene transfer from the endosymbiont genome to the host nucleus is essential for the evolving host cell to control the endosymbiont-derived organelle12. In P. micropora, we found that the gene transfer from the free-living and endosymbiotic bacteria to the amoeba nucleus was rapidly activated but both simultaneously ceased within the initiation period of the endosymbiotic evolution, suggesting that the genome reorganization drastically proceeded and completed. During this period, large DNA virus appeared to have infected the amoeba, followed by the rapid amplification and diversification of virus-related genes. These findings led us to re-examine the conventional endosymbiotic evolutionary scenario that exclusively deals with the host and the symbiont, and to extend it by incorporating a third critical player, large DNA virus, which activates the drastic gene transfer and genome reorganization between them. This Paulinella version of the evolutionary hypothesis deserves further testing of its generality in evolutionary systems and could shed light on the unknown roles of large DNA viruses13 in the evolution of terrestrial life.

evolutionary biology↗