Biology subjects

Chiba, S.

Publications and source records attributed to Chiba, S..

At least 19 recordsLinked to original sources

bioRxiv2026

RhoGEF Solo regulates cell density-dependent keratin remodeling and desmosome maturation

Desmosomes (DSMs) are specialized cell-cell adhesion structures that anchor keratin intermediate filaments and play a crucial role in maintaining the mechanical integrity of epithelial tissues. Keratin filaments associate with DSMs in two characteristic patterns: radial spokes extending from DSMs into the cytoplasmic keratin network and circumferential rims connecting neighboring DSMs. Solo is a Rho-guanine nucleotide exchange factor (RhoGEF) involved in cellular responses to mechanical stress and keratin network reorganization. Here, we investigated the role of Solo in regulating keratin-DSM interactions in response to cell density. The number of radial keratin spokes at cell-cell adhesion sites was markedly reduced at high cell density, concomitant with increased Solo localization at these sites. Conversely, Solo knockout (KO) increased the number of radial keratin spokes. Expression of wild-type Solo, but not a GEF-inactive Solo mutant, rescued the increase in radial keratin spokes in confluent Solo-KO cells, indicating that Solo RhoGEF activity is required for this regulation. Inhibition of actin polymerization or ROCK activity also suppressed the reduction in radial spoke density. Furthermore, expansion microscopy and electron microscopy revealed that DSMs in Solo-KO cells remained small and immature even under confluent conditions. Solo was also partially required for the localization of plakoglobin (PG), a core DSM protein, whereas PG-KO markedly increased the number of radial keratin spokes. These findings suggest that increased cell density promotes DSM maturation accompanied by a reduction in radial keratin spokes, and Solo contributes to both processes, potentially through facilitating PG localization at DSMs and regulating Rho-ROCK-dependent actin cytoskeletal remodeling.

cell biology↗

bioRxiv2026

An ancient polymorphism in myosin I a/b determines the left-right asymmetry of Japanese snails

Left-right (LR) asymmetry is a fundamental feature of animal development, yet progress in understanding its establishment has been limited by the fact that almost all animals exhibit invariant chirality. Snails are the exception because some wild-living species show abundant chiral variation, yet the existing knowledge is from snails in which the chirality mutation is rare and causes pathology. Here, we show that a chiral polymorphism in Japanese Euhadra snails is due to functional variation in an unconventional myosin I a/b, an isoform not previously implicated in LR specification in any animal. Both the sinistral and dextral gene variants are ancient, impart minimal transcriptional differences in the single-cell embryo and are not pathological. Phylogenetic and structural modelling suggests that dominant-acting amino acid substitutions in the myosin actin-binding domain and/or motor-level junction were enabled by relaxed selection. These results broaden the known molecular repertoire underlying LR asymmetry, suggest key mutations and positions that should be explored in other model animals, and highlight snails as a powerful model for understanding the origins of animal LR asymmetry.

evolutionary biology↗

bioRxiv2026

eSkip2 prioritizes exon-skipping antisense oligonucleotide target regions across exon--intron contexts

Exon-skipping antisense oligonucleotides (ASOs) can restore productive transcripts, but identifying effective binding regions remains difficult because splicing regulation extends across exons, introns and splice junctions. Here we develop eSkip2, a genome-informed framework that ranks target regions within a unified exon-intron sequence context. eSkip2 combines a genome-pretrained sequence model with ASO-induced exon-skipping data and single-nucleotide-variant splicing perturbations, followed by target-locus adaptation that requires no experimental ASO labels from the locus being designed. Across benchmarks comprising canonical exons and pseudoexons, multiple cell types and chemistries, and exonic, intronic and exon-intron-spanning targets, eSkip2 prioritized active regions and showed a higher median AUROC than applicable exon-restricted models. Prospective application to the combinatorial design of dual-targeting ASOs for DMD exon 46 enriched active candidates near the top of the ranking: the two most active new ASOs ranked within the top three and produced dose-dependent dystrophin restoration in patient-derived cells. These results support eSkip2 as a practical first-pass strategy for reducing experimental search space in exon-skipping ASO discovery.

bioinformatics↗

bioRxiv2026

Density-Dependent Rearrangement of Desmoplakin in Epithelial Junctions: Insights from Expansion Microscopy

Desmosomes mechanically couple neighboring epithelial cells to the intermediate filament cytoskeleton. However, how the nanoscale organization changes with epithelial cell density remains incompletely understood. Here, we used expansion microscopy (ExM) to examine Madin-Darby canine kidney epithelial monolayers. ExM revealed density-associated differences in the relative apicobasal organization of epithelial junctions, with increased separation between occludin-positive tight junctions and desmoglein 2-positive desmosomes in confluent monolayers. A comparison of subconfluent and confluent cultures revealed a density-associated reorganization of desmoplakin (DSP) within opposing desmosomal plaques, with a more pronounced change in C-terminal spacing than in N-terminal spacing. Independent STED analysis of non-expanded samples confirmed the density-associated increase in DSP C-terminal spacing. In keratin filament-deficient cells, this spacing was elevated in subconfluent cultures and did not increase with cell density. Pharmacological inhibition of myosin II or ROCK reduced this elevated spacing, indicating that keratin loss-associated reorganization of DSP is sensitive to actomyosin contractility. These findings show that epithelial cell density is associated with the remodeling of epithelial junction organization and DSP plaque architecture, demonstrating the utility of ExM for quantitative analysis of junctional architecture.

cell biology↗

bioRxiv2025

Foliar application of mixed DAMP- and MAMP-oligosaccharides enhances root growth and fruit yield in okra and tomato in greenhouse experiments

Plants perceive diverse endogenous and microbial molecular patterns that shape their defense, growth, and productivity. Damage-associated molecular patterns (DAMPs) such as cello- and xylo-oligosaccharides, and microbe-associated molecular patterns (MAMPs) such as chitin-oligosaccharides, are known to promote both development and immunity. Here, we evaluated the combined application of these three oligosaccharides (Oligo-Mix) in okra and cherry tomato under greenhouse conditions. Regular foliar treatment with Oligo-Mix enhanced root elongation and biomass accumulation, and increased fruit number and yield in both crops. RNA-seq analysis of Oligo-Mix-treated tomato leaves showed coordinated induction of photosynthetic genes, components of the translational machinery, cell-wall remodeling factors, and enzymes involved in flavonoid/anthocyanin biosynthesis, indicating broad enhancement of metabolic capacity and protective secondary pathways. Together, these results demonstrate that Oligo-Mix functions as a biostimulant that supports both vegetative growth and fruit production, offering a sustainable approach to improving crop performance.

plant biology↗

bioRxiv2025

intDesc-AbMut: Describing and understanding how antibody mutations impact their environmental interactions

Previously, we proposed a double-point mutation (DPM) strategy involving the simultaneous substitution of two amino acids to optimize antibodies. By selecting mutants based on the criterion that favorable interactions between mutated residues and their local environments are preserved or enhanced, improvements in antibody affinity were achieved. Nonetheless, manual extraction of these interactions proved to be time-consuming and labor-intensive. Thus, to streamline this process, we developed intDesc-AbMut, an automated software tool designed to extract interactions between designated mutant residues and their surroundings from three-dimensional antigen-antibody complex structures. intDesc-AbMut identifies and classifies 36 distinct types of interactions, enabling visualization of changes before and after mutation. Additionally, the effects of mutations can be represented by interaction descriptors generated from the extracted interactions. As a practical application, we constructed a machine learning model using these descriptors combined with side-chain rotamer frequencies to determine whether the environment around mutated residues in antigen-antibody complexes is crystal-like, achieving a Matthews correlation coefficient of 0.505 and an accuracy of 0.855. The intDesc-AbMut software is available at: https://github.com/riken-yokohama-AI-drug/intDesc/tree/intDesc-AbMut.

bioengineering↗

bioRxiv2025

A First-in-Class Therapy Targeting Transcription-Replication Conflicts Drives Synthetic Lethality in Homologous Recombination Deficient Cancers

Hidden beneath the layer of tumor malignancy lies unchecked proliferation driven by hyperactive transcription, which fosters the formation of RNA:DNA hybrids. These structural intermediates precipitate collisions between the transcription and replication machineries. Central to mitigating this genomic threat is interleukin enhancer-binding factor 2 (ILF2), which orchestrates the recruitment of RNA:DNA helicases to resolve R-loops and, in doing so, reveals cancer cells dependency on stress-mitigating mechanisms to avert genome catastrophe. Our discovery of Molephantin and its lead derivative, NYH0002 is the outcome of delineating ILF2s function. NYH002 exerts direct binding to the ILF2 complex and disrupts RNA:DNA helicase activity to elicit genome-wide DNA breakages. Tumors with elevated cyclin E and E2F1 expression are sensitive to NYH002, while those deficient in homologous recombination repair exhibit lethality. Collectively, these findings position ILF2 as a key regulator of R-loop homeostasis and reveal a therapeutic vulnerability that NYH002 exploits to suppress tumor malignancy.

cancer biology↗

bioRxiv2025

Actin cytoskeletal remodeling requires the interaction between Solo and LARG in response to substrate stiffness

In response to external mechanical stimuli, cells remodel their actin cytoskeleton. Solo, a Rho guanine nucleotide exchange factor (RhoGEF), is involved in mechanical stress responses. Using BioID, we identified PDZ-RhoGEF (PRG), a member of the RGS-RhoGEF family (regulator of G protein signaling domain-containing RhoGEFs, as a Solo-interacting protein. Moreover, we found that Solo regulates PRG during the mechanical stress response. Furthermore, we identified leukemia-associated RhoGEF (LARG), another RGS-RhoGEF member, as a Solo-interacting protein; however, the functional role of this interaction remains unknown. Therefore, in this study, we investigated the interaction between Solo and LARG and found that LARG localizes to Solo accumulation sites at the basal plane and that LARG is required for Solo-induced actin polymerization. Additionally, Solo is required to maintain LARG activity in cells, and this interaction is related to actin regulation in response to substrate stiffness. We further investigated the relationship between LARG and PRG as a function of Solo. We noted that although they did not competitively localize at Solo accumulation sites, knockdown of either PRG or LARG suppressed Solo-induced actin polymerization to the same extent as double knockdown, indicating that these signaling pathways cooperatively regulate Solo-induced actin polymerization.

cell biology↗

bioRxiv2024

Mixed DAMP/MAMP oligosaccharides promote both growth and defense against fungal pathogens of cucumber

Plants recognize a variety of environmental molecules, thereby triggering appropriate responses to biotic or abiotic stresses. Substances containing microbes-associated molecular patterns (MAMPs) and damage-associated molecular patterns (DAMPs) are representative inducers of pathogen resistance and damage repair, thus treatment of healthy plants with such substances can pre-activate plant immunity and cell repair functions. In this study, the effects of DAMP/MAMP oligosaccharides mixture (Oligo-Mix) derived from plant cell wall (cello-oligosaccharide and xylo-oligosaccharide), and fungal cell wall (chitin-oligosaccharide) were examined in cucumber. Treatment of cucumber with Oligo-Mix promoted root germination and plant growth, along with increased chlorophyll contents in the leaves. Oligo-Mix treatment also induced typical defense responses such as MAP kinase activation and callose deposition in leaves. Pretreatment of Oligo-Mix enhanced disease resistance of cucumber leaves against pathogenic fungi Podosphaera xanthii (powdery mildew) and Colletotrichum orbiculare (anthracnose). Oligo-Mix treatment increased the induction of hypersensitive cell death around the infection site of pathogens, which inhibited further infection and the conidial formation of pathogens on the cucumber leaves. RNA-seq analysis revealed that Oligo-Mix treatment upregulated genes associated with plant structural reinforcement, responses to abiotic stresses and plant defense. These results suggested that Oligo-Mix has beneficial effects on growth and disease resistance in cucumber, making it a promising biostimulant for agricultural application.

plant biology↗

bioRxiv2024

Chromosome-level genome assembly of the Asian tramp snail Bradybaena similaris (Stylommatophora: Camaenidae)

While terrestrial land snails have long been subjected to evolutionary research, a lack of high-quality genomic resources has impeded recent progress. Bradybaena snails in particular have numerous intriguing traits that make them a good model for studying evolution, including shell pattern polymorphism and convergent evolution. They are also introduced and invasive across the world. In this study, we present a chromosome-level genome assembly of the Asian tramp snail Bradybaena similaris utilizing 88-fold Illumina short-read sequences, 125-fold Nanopore long-read sequences, 63-fold PacBio HiFi sequences, and 47-fold Hi-C sequences. The assembled genome of 2.18 Gb is anchored to 28 chromosomes, and exhibits high completeness (98.8% metazoan BUSCO completeness) and contiguity (N50 of 75.6 Mb). Additionally, we also obtained a high-quality transcriptome for annotation. This resource represents the first chromosome-level assembly for snails in the superfamily Helicoidea, which includes more than 5,000 species of terrestrial snails, and will facilitate genomic study in Bradybaena and, more broadly, in the superfamily Helicoidea. SIGNIFICANCE STATEMENTWhile the Helicoidea is the largest land snail superfamily, consisting of more than 5,000 species, many of interest to evolutionary studies, no chromosome-level assembly has been available for any species. Previously, the genus Bradybaena in the Helicoidea has been studied in the context of speciation, adaptation and invasive biology, and thereby has high potential for further research. In this study, we present a chromosome-level assembly and transcriptome of the Asian tramp snail Bradybaena similaris. These high-quality genomic resources will facilitate research on related species, and eventually enhance our understanding of many areas of evolutionary biology.

genomics↗

bioRxiv2024

Self-cleaving ribozymes conserved in RNA viruses unveil a new role in protein translation

Small self-cleaving ribozymes are catalytic RNAs originally discovered in viroid-like agents, which are replicating circular RNAs (circRNAs) postulated as relics of a prebiotic RNA world. In the last decade, however, small ribozymes have also been detected across the tree of life, from bacterial to human genomes, and more recently, in unusual circRNA viruses. Here we report the conserved occurrence of diverse small ribozymes within the linear genomes of typical double- and single-stranded RNA virus families from fungi and plants. Type I hammerhead ribozyme motifs occur in the 5-UTR regions of chrysovirids and fusarivirids, displaying self-cleaving activity in vitro and in vivo. Similar hammerhead, as well as hepatitis delta and twister ribozymes, are also found in diverse megabirna-, hypo-, fusagra-, toti-or tombus-like viruses among others. The ribozymes occur not only as isolated motifs within UTRs but also as tandem pairs that encompass small RNA segments (186-399 nt) resembling Zetavirus-like sequences. In vivo characterization of the 5-UTR with a ribozyme from a chrysovirid revealed that the RNA-cleaving activity is essential for protein translation initiation in fungi. Analogous experiments in plants with diverse ribozyme motifs indicated that just the presence of a self-cleaving activity can induce cap-independent translation. We conclude that RNA self-cleaving activity, historically linked to the rolling circle replication of viroid-like circRNAs, appears to be co-opted by linear RNA viruses for translational roles.

microbiology↗

bioRxiv2024

Botrytis cinerea detoxifies the sesquiterpenoid phytoalexin rishitin through multiple metabolizing pathways

Botrytis cinerea is a necrotrophic pathogen that infects across a broad range of plant hosts, including high-impact crop species. Its generalist necrotrophic behavior stems from its ability to detoxify structurally diverse phytoalexins. The current study aims to provide evidence of the ability of B. cinerea to tolerate the sesquiterpenoid phytoalexin rishitin, which is produced by potato and tomato. While the growth of potato pathogens Phytophthora infestans (late blight) and Alternaria solani (early blight) was severely inhibited by rishitin, B. cinerea was tolerant to rishitin. After incubation of rishitin with the mycelia of B. cinerea, it was metabolized to at least six oxidized forms. Structural analysis of these purified rishitin metabolites revealed a variety of oxidative metabolism including hydroxylation at C7 or C12, ketone formation at C5, and dihydroxylation at the 10,11-olefin. Six rishitin metabolites showed reduced toxicity to P. infestans and A. solani, indicating that B. cinerea has at least 5 distinct enzymatic reactions to detoxify rishitin. Four host-specialized phytopathogenic Botrytis species, namely B. elliptica, B. allii, B. squamosa, and B. tulipae also had at least a partial ability to metabolize rishitin as B. cinerea, but their metabolic capacity was significantly weaker than that of B. cinerea. These results suggest that the ability of B. cinerea to rapidly metabolize rishitin through multiple detoxification mechanisms could be critical for its pathogenicity in potato and tomato.

microbiology↗

bioRxiv2024

A role for the S4-domain containing protein YlmH in ribosome-associated quality control in Bacillus subtilis

Ribosomes trapped on mRNAs during protein synthesis need to be rescued for the cell to survive. The most ubiquitous bacterial ribosome rescue pathway is trans-translation mediated by tmRNA and SmpB. Genetic inactivation of trans-translation can be lethal, unless the ribosomes are rescued by ArfA or ArfB alternative rescue factors or the ribosome-associated quality control (RQC) system, which in B. subtilis involves MutS2, RqcH, RqcP and Pth. Using transposon sequencing in a trans-translation-incompetent B. subtilis strain we identify a poorly characterized S4-domain-containing protein YlmH as a novel potential RQC factor. Cryo-EM structures reveal that YlmH binds peptidyl-tRNA-50S complexes in an position analogous to that of S4-domain-containing RqcP, and that, similarly to RqcP, YlmH can co-habit with RqcH. Consistently, we show that YlmH can assume the role of RqcP in RQC in facilitating the addition of polyalanine tails to the truncated nascent polypeptides. While in B. subtilis the function of YlmH is redundant with RqcP, our taxonomic analysis reveals that in multiple bacterial phyla RqcP is absent, while YlmH and RqcH are present, suggesting that in these species the YlmH plays a central role in the RQC.

microbiology↗

bioRxiv2024

Resolution of ribosomal stalling by ABCF ATPases YfmR and YkpA/YbiT

Efficiency of protein synthesis on the ribosome is strongly affected by the amino acid composition of the assembled amino acid chain. Challenging sequences include proline-rich motifs as well as highly positively and negatively charged amino acid stretches. Members of the F subfamily of ABC ATPases (ABCFs) have been long hypothesised to promote translation of such problematic motifs. In this study we have applied genetics and reporter-based assays to characterise the four housekeeping ABCF ATPases of Bacillus subtilis: YdiF, YfmM, YfmR/Uup and YkpA/YbiT. We show that YfmR cooperates with the translation factor EF-P that promotes translation of Pro-rich motifs. Simultaneous loss of both YfmR and EF-P results in a dramatic growth defect. Surprisingly, this growth defect can be largely suppressed though overexpression of an EF-P variant lacking the otherwise crucial 5-amino-pentanolylated residue K32. Using in vivo reporter assays, we show that overexpression of YfmR can alleviate ribosomal stalling on Asp-Pro motifs. Finally, we demonstrate that YkpA/YbiT promotes translation of positively and negatively charged motifs but is inactive in resolving ribosomal stalls on proline-rich stretches. Collectively, our results provide insights into the function of ABCF translation factors in modulating protein synthesis in B. subtilis.

microbiology↗

bioRxiv2023

Restoration of the Functional nif Gene Cluster by Seven Excision and Two Inversion Events during Heterocyst Development in the Nitrogen-Fixing Cyanobacterium Calothrix sp. NIES-4101

In the genome of the heterocystous cyanobacterium Calothrix sp. NIES-4101 (NIES-4101), the four genes essential for nitrogen fixation (nifB, nifH, nifD, and nifK) are highly fragmented into 13 parts in a 350-kb chromosomal region, and four of these parts are encoded in the reverse strand. Such a complex fragmentation feature makes it difficult to restore the intact nifBHDK genes by the excision mechanism found in the nifD gene of the Anabaena sp. PCC 7120 heterocyst. To examine the nitrogen-fixing ability of NIES-4101, we confirmed that NIES-4101 grew well on combined nitrogen-free medium and showed high nitrogenase activity, which strongly suggested that the complete nifBHDK genes are restored by a complex recombination process in heterocysts. Next, we resequenced the genome prepared from cells grown under nitrogen-fixing conditions. Two contigs covering the complete nifHDK and nifB genes were found by de novo assembly of the sequencing reads. In addition, DNA fragments covering the nifBHDK operon were successfully amplified by PCR. We propose that the process of nifBHDK restoration occurs as follows. First, the nifD-nifK genes are restored by four excision events. Then the complete nifH and nifB genes are restored by two excision events followed by two successive inversion events between the inverted repeat sequences and one excision event, forming the functional nif gene cluster, nifB-fdxN-nifS-nifU-nifH-nifD-nifK. All genes coding recombinases responsible for these nine recombination events are located close to the terminal repeat sequences. The restoration of the nifBHDK genes in NIES-4101 is the most complex genome reorganization reported in heterocystous cyanobacteria.

microbiology↗

bioRxiv2023

Interaction between Solo and PDZ-RhoGEF is involved in actin cytoskeletal remodeling and response to substrate stiffness

Recent findings indicate that Solo, a RhoGEF, is involved in cellular mechanical stress responses. However, the mechanism of actin cytoskeletal remodeling via Solo remains unclear. Therefore, this study was aimed at identifying Solo-interacting proteins using the BioID, a proximal-dependent labeling method and elucidating the molecular mechanisms of function of Solo. We identified PDZ-RhoGEF (PRG) as a Solo-interacting protein. PRG co-localized with Solo in the basal area of cells, depending on Solo localization, and enhanced actin polymerization at Solo accumulation sites. Additionally, Solo and PRG interaction was necessary for actin cytoskeletal remodeling and RhoA activation. Moreover, overexpression of the binding domains of Solo and PRG had a dominant-negative effect on actin polymerization and actin stress fiber formation in response to substrate stiffness. Therefore, Solo restricts the localization of PRG and regulates actin cytoskeletal remodeling in synergy with PRG in response to the surrounding mechanical environment.

cell biology↗

bioRxiv2023

Patchy and widespread distribution of bacterial translation arrest peptides associated with the protein localization machinery

Regulatory arrest peptides exert cellular functions via mechanisms involving regulated translational arrest. Monitoring substrates, a class of arrest peptides, feedback-regulate the expression of the Sec or YidC protein localization machinery. Previously, only a limited number of monitoring substrates were identified. In this study, we performed a bacterial domain-wide search, followed by in vivo and in vitro analyses, leading to a comprehensive identification of many novel Sec/YidC-related arrest peptides that showed patchy, but widespread, phylogenetic distribution throughout the bacterial domain. Identification of five novel arrest-inducing sequences suggests that bacteria have evolved various arrest-inducing mechanisms. We also identified many arrest peptides that share an R-A-P-P like sequence, suggesting that this sequence could serve as a common evolutionary seed that could overcome the species-specific structures of ribosomes, to evolve arrest peptides. Our comprehensive phylogenetic study revealed that arrest peptide is a prevalent mechanism for the gene regulation of the protein localization machinery.

molecular biology↗

bioRxiv2023

Characterization of an EG.5.1 clinical isolate in vitro and in vivo

EG.5.1 is a subvariant of the SARS-CoV-2 Omicron XBB variant that is rapidly increasing in prevalence worldwide. EG.5.1 has additional substitutions in its spike protein (namely, Q52H and F456L) compared with XBB.1.5. However, the pathogenicity, transmissibility, and immune evasion properties of clinical isolates of EG.5.1 are largely unknown. In this study, we used wild-type Syrian hamsters to investigate the replicative ability, pathogenicity, and transmissibility of a clinical EG.5.1 isolate. Our data show that there are no obvious differences in growth ability and pathogenicity between EG.5.1 and XBB.1.5, and both EG.5.1 and XBB.1.5 are attenuated compared to a Delta variant isolate. We also found that EG.5.1 is transmitted more efficiently between hamsters compared with XBB.1.5. In addition, unlike XBB.1.5, we detected EG.5.1 virus in the lungs of four of six exposed hamsters, suggesting that the virus tropism of EG.5.1 is different from that of XBB.1.5 after airborne transmission. Finally, we assessed the neutralizing ability of plasma from convalescent individuals and found that the neutralizing activity against EG.5.1 was slightly, but significantly, lower than that against XBB.1.5 or XBB.1.9.2. This suggests that EG.5.1 effectively evades humoral immunity and that the amino acid differences in the S protein of EG.5.1 compared with that of XBB.1.5 or XBB.1.9.2 (i.e., Q52H, R158G, and F456L) alter the antigenicity of EG.5.1. Our data suggest that the increased transmissibility and altered antigenicity of EG.5.1 may be driving its increasing prevalence over XBB.1.5 in the human population.

microbiology↗