Biology subjects

Harada, Y.

Publications and source records attributed to Harada, Y..

At least 19 recordsLinked to original sources

bioRxiv2026

The LudP arrest peptide stalls translation by remodeling the ribosomal A-site

Bacterial ribosome arrest peptides couple translation to secretion and membrane homeostasis, providing an intracellular regulatory feedback system. Although several Sec/YidC-associated arrest peptides have been characterized, how evolutionarily diverse peptides converge on equivalent physiological outcomes remains unclear. Here we identify LudP (Lacrimispora upstream of secDF peptide), a previously unrecognized arrest peptide conserved in the genus Lacrimispora and encoded immediately upstream of secDF. In vitro and in vivo assays show that LudP induces robust Gram-positive-specific translation arrest that is released in a Sec signal-dependent manner. Using systematic mutagenesis and deep mutational scanning, we define a compact 15-residue C-terminal arrest element that governs arrest stability and A-site residue selectivity. Cryo-electron microscopy reveals that a LudP nascent-chain -helix in the exit tunnel remodels the peptidyltransferase center, with Asp133-dependent conformational rearrangements of 23S rRNA, notably U2506, sterically and energetically destabilizing productive A-site aminoacyl-tRNA accommodation. These findings support functional convergent evolution of secretion-coupled arrest peptides across bacterial lineages.

molecular biology↗

bioRxiv2026

Rice blast fungus hijacks a rice RALF peptide to initiate infection

Plant-derived peptides regulate immunity and host-microbe interactions, but whether fungal pathogens exploit endogenous plant peptides for infection-site selection remains largely unknown. Rapid alkalinization factors (RALFs) are a family of plant peptide hormones that contribute to plant development, reproduction, stress responses, and cell-wall organization. Here, we show that OsRALF7, which is an immune-activating peptide in rice (Oryza sativa), is co-opted by the rice blast fungus Magnaporthe oryzae as a host-derived cue for appressorium formation, specialized infection structures required for host penetration. While synthetic OsRALF7 peptide induced defense responses in rice suspension cells, the same peptide induced appressorium formation in M. oryzae. OsRALF7-induced appressorium formation was associated with activation of the cAMP/PKA and Pmk1 MAPK pathways and required a hydrophobic surface. M. oryzae inoculated onto Osralf7 knockout plants formed fewer appressoria, and the positioning of these appressoria relative to the boundaries between two adjacent plant epidermal cells was altered. These findings suggest that OsRALF7 provides positional information for infection initiation for M. oryzae. OsRALF7 localized to the plasma membrane and cell wall in rice, and its appressorium-inducing activity required a conserved YISY motif and cysteine residues. This activity was not conserved in Colletotrichum higginsianum, as RALF23 from Arabidopsis thaliana did not induce appressorium formation but instead showed antifungal activity. Together, these findings uncover a dual function of endogenous plant immune-modulating RALF peptides at the plant-pathogen interface, where they can be exploited by M. oryzae as a host-derived cue for infection initiation while also retaining antifungal activity against other fungal species.

plant biology↗

bioRxiv2026

Intranuclear Quantum Sensing with Fluorescent Nanodiamond Enabled by Electron-Irradiation and Surface-Chemical Optimization for Microinjection

Fluorescent nanodiamonds (FNDs) containing nitrogen-vacancy (NV) centers are promising quantum sensors for intracellular measurements, yet nuclear applications remained out of reach because optically detected magnetic resonance (ODMR) signals are weak and capillary delivery is inefficient. This study addresses both constraints by optimizing the electron irradiation dose to balance NV creation and charge-state stability, and by grafting hyperbranched polyglycerol with terminal carboxyl groups (HPGCOOH) to suppress aggregation and prevent needle clogging. The optimized dose yields strong ODMR contrast while preserving fluorescence suitable for microscopy. HPGCOOH surfaces enable smooth and reproducible microinjection through fine capillaries. Using this strategy, the microinjection of ODMR-active FNDs into the nuclei of living COS7 cells is achieved, and clear intranuclear spectra comparable to cytoplasmic readouts are obtained. Furthermore, field-of-view temperature sensing across multiple cell nuclei is demonstrated, enabling quantitative and spatially resolved thermal mapping within the genomic environment. This methodology provides a practical route to nuclear quantum sensing and opens opportunities for nanoscale physicochemical measurements within the genomic environment.

biophysics↗

bioRxiv2025

Phase-targeting rapid cryofixation of the beating heart and histological analysis unveil contractile state-dependent sarcomere dynamics

The heart is a functional syncytium consisting of numerous cardiomyocytes that repetitively exhibit coordinated contractions/relaxations. However, the extent to which myocyte sarcomere arrangements in the heart differ across beats is unknown. To examine this, we conducted cardiac phase-targeting rapid cryofixation of Langendorff-perfused rat hearts. We adjusted the timepoint of cryogen exposure to the electrically paced heart and observed phase-dependent differences in the sarcomere length (SL) of subepicardial myocytes by -actinin immunohistochemistry, namely a significantly shorter SL during systole than during diastole. We detected spatially inhomogeneous SL distributions by generating a heatmap of the myocardium. For peak systole the SL heatmap exhibited nearly uniform SL shortening within and among the individual myocytes with some myocardia exhibiting nonuniform SLs. During diastole, the heart showed predominant SL elongation, which was also accompanied by patchy distributions of locally short-SL regions, reflecting inhomogeneous SLs. This SL inhomogeneity was attenuated by pharmacological relaxation by 2,3-butanedione monoxime. The heatmap of the rapidly-frozen heart during ventricular fibrillation also revealed inhomogeneous SLs within and among individual myocytes. Overall, cardiac phase-targeting cryofixation unveiled in-depth behaviors on SL in the heart. Our cryofixation strategy will open a new horizon to clarify precise spatiotemporal changes in sarcomere structures and understand cardiac functions.

biophysics↗

bioRxiv2025

Hox - Meis-relayed topographical genetic switch underlies cardiopharyngeal neural crest diversification, revealed by multimodal analysis

Neural crest cells (NCCs) are multipotent migratory cells essential for cardiac development, yet the lineage trajectories and gene regulatory networks (GRNs) underlying their differentiation in the cardiopharyngeal region remain unclear. Here, we integrate single-cell RNA-seq, spatial transcriptomics, and multiomic analyses to construct a comprehensive map of NCC lineages in developing mouse cardiopharyngeal tissues. We identify a transition from Hox-positive pharyngeal NCCs to Hox-negative intracardiac populations associated with the outflow tract cushion, accompanied by a shift in Meis transcription factor binding and GRN architecture. By contrast, NCCs forming the aorticopulmonary septum and great vessel smooth muscle retain distinct Hox-codes. A Meis2-Sox9-Scx GRN defines a skeletogenic progenitor-like intermediate state that gives rise to coronary artery smooth muscle and semilunar valves. Our findings suggest that the loss of Hox-dependent regional identity enables pharyngeal NCCs to acquire new fates upon entering the cardiac cushion, providing insight into the developmental origins of coronary and valvular calcification.

developmental biology↗

bioRxiv2025

Outbreak of leaf spot disease of Cavendish banana caused by Nigrospora spp. in Mindanao Island, Philippines

Banana leaf diseases are a significant threat to Cavendish banana production. In the Philippines, the main disease has been diagnosed as Black sigatoka disease caused by Pseudocercospora fijiensis based on symptoms. However, our study showed that the main pathogen in Mindanao island, the largest banana-producing region in the Philippines, belongs to the genus Nigrospora, contradicting previous assumptions. We clarified the phylogenetic positions of 160 Nigrospora isolates based on molecular phylogenetic analyses using ITS, {beta}-tubulin, and tef1 sequences, and compared their morphology with known species. Molecular phylogenetic and morphological analysis revealed that Nigrospora isolates comprised N. chinensis, N. lacticolonia, N. cf. singularis, N. sphaerica, N. vesicularifera, and a novel species, N. nigrocolonia. Pathogenicity tests on banana leaves confirmed that these species are pathogenic. Species other than N. sphaerica were for the first time reported as pathogens of banana leaf. The results of the fungicide sensitivity test using 14 fungicides, including pyrimethanil, spiroxamine, and tebuconazole, for the Sigatoka disease showed 100% inhibition of all isolates at 100 ppm of active ingredients. However, low-sensitivity isolates were observed for the remaining 11 fungicides. Our findings indicated the need for a comprehensive review of banana leaf disease prevention strategies.

molecular biology↗

bioRxiv2025

Chloroplast- and Mitochondrion-Specific Random C-to-T Mutagenesis for Forward Genetics of Organelle Genomes

Organelle genomes in plastids (including chloroplasts) and mitochondria encode essential genes for photosynthesis, respiration, and agronomic traits, representing promising targets for crop improvement. However, their high copy number and non-Mendelian inheritance have long hindered efficient modification compared to nuclear genomes. Recent advances in organelle base editing (C-to-T and A-to-G) have enabled precise nucleotide substitutions, yet information on useful mutations remains limited. Here, to establish a forward genetics platform for C-to-T substitutions, we developed a method to introduce random C-to-T mutations throughout the entire organelle genomes of Arabidopsis thaliana. We engineered a fusion protein, WHY2-CD mutator, combining cytidine deaminase (CD), uracil glycosylase inhibitor, and sequence-nonspecific DNA-binding protein WHIRLY2 (WHY2), fused to organelle-targeting peptides. This system introduced dispersed C-to-T substitutions specifically within plastid or mitochondrial genomes. In T2 lines, we identified homoplasmic (homozygous) plastid genome mutants, including rpoA knockouts and rbcL variants with an amino acid substitution. Screening T3 populations on spectinomycin revealed plastid genome mutants with resistant traits and their causal mutation. These mutations can be transferred or combined using targeted base editors, such as transcription activator-like effector cytidine deaminase (TALECD). This comprehensive, C-to-T-focused mutagenesis provides a powerful tool for organelle forward genetics and molecular breeding.

plant biology↗

bioRxiv2025

In vitro evaluation of protein-protein interactions in the rice KAI2 ligand signaling complex

KARRIKIN INSENSITIVE 2 (KAI2)/DWARF14-LIKE (D14L) plays key roles in land plant development, environmental responses, and the establishment of arbuscular mycorrhizal symbiosis, likely acting as the receptor for unidentified signaling molecules termed KAI2 ligands (KLs). KL perception by KAI2/D14L promotes DWARF3 (D3)/MORE AXILLARY GROWTH2 (MAX2) F-box protein-mediated ubiquitination of SUPPRESSOR OF MAX2 1 (SMAX1) proteins, thereby transducing the KL signals. Although genetic and in vivo assays have demonstrated the functions of these components, the biochemical details of their interactions remain elusive. Here we investigated physical interactions between rice D14L, D3, and OsSMAX1 in vitro using desmethyl germinone (dMGer), a recently developed KL analog. dMGer elicited KL responses in rice with higher activity and pathway specificity than a widely used KL analog (-)-GR24. dMGer, but not (-)-GR24, directly bound to D14L and promoted the interaction between D14L and D3 in vitro. The interaction between D14L and OsSMAX1 was also enhanced by dMGer. Furthermore, we identified the domain of OsSMAX1 that distinguishes it from its paralog DWARF53 (D53), which is associated with the strigolactone signaling complex. These findings propose a model of the interactions among KL signaling components and highlight the role of the ligand in the signaling complex.

plant biology↗

bioRxiv2025

Development of charged nanobubble-containing media and their effects on iPSC-derived cell cultures

Nanobubbles (NBs) are gas-filled spherical structures less than 1 {micro}m in diameter, characterized by high surface charge, long-term stability, and the ability to generate bactericidal hydroxyl radicals upon collapse. Although these unique properties enable their extensive applications, including wastewater treatment, their cell biological applications at neutral pH remain limited by difficulties in maintaining stability and surface charge. Here, we succeeded in generating both positively and negatively charged NBs, achieving high zeta potentials in human iPSC-derived neural progenitor cell (NPC) and neuron culture media (pH 7.4). Similar to their bactericidal effects, NBs induced cell death in iPSC-derived cells, with positively charged NBs showing stronger cytotoxicity than negatively charged ones. This may be because the cell membrane carries a negative charge, making positively charged NBs more likely to approach and interact with the membrane, or because positively charged nanobubbles exhibit stronger radical generation. In future, we aim to elucidate the molecular biological mechanisms underlying NB-induced cell death and to contribute to the potential application of nanobubbles in regenerative medicine. This study will serve as an initial step toward that goal.

bioengineering↗

bioRxiv2025

Sequential transcriptional gates in the thalamo-cortical circuit coordinate memory stabilization

The molecular mechanisms that enable memories to persist over long time-scales from days to weeks and months are still poorly understood. To develop insights we created a behavioral task where, by varying the frequency of learned associations, mice formed multiple memories but only consolidated some, while forgetting others, over the span of weeks. We then monitored circuit-specific molecular programs that diverge between consolidated and forgotten memories. We identified multiple distinct waves of transcription, i.e., cellular macrostates, specifically in the thalamo-cortical circuit, that defined memory persistence. Notably, a small set of transcriptional regulators orchestrated broad molecular programs that enabled entry into these macrostates. Targeted CRISPR-knockout studies revealed that while these transcriptional regulators had no effects on memory formation, they had prominent, causal, and strikingly time-dependent roles in memory stabilization. In particular, the calmodulin-dependent transcription factor Camta1 was required for initial memory maintenance over days, while Tcf4 and the histone methyl-transferase Ash1l were required later to maintain memory over weeks. These results identify a critical Camta1-Tcf4-Ash1l thalamo-cortical transcriptional cascade required for memory stabilization, and puts forth a model where the sequential, multi-step, recruitment of circuit-specific transcriptional programs enable memory maintenance over progressively longer time-scales.

neuroscience↗

bioRxiv2025

Epigenetic mechanisms governing cell type specific somatic expansion and toxicity in Huntington's disease

Huntingtons disease (HD) is characterized by neuronal dysfunction and degeneration that varies markedly by brain region and cell type. Using high-resolution epigenetic profiling of postmortem human cell types we identify a pathogenic cascade linking cell type specific enhancer activity to somatic CAG expansion, and toxicity to epigenetic dysregulation. Enhancers regulating mismatch-repair (MMR) gene expression explain the specificity of expansion. In the second, toxic phase of HD we identify two distinct epigenetic mechanisms that disrupt regulation of hundreds of genes in the majority of HD MSNs, including several that cause haploinsufficient neurological disorders. Together, these data unify enhancer function, impaired DNA demethylation, and transcriptional dysregulation into a single model highlighting therapeutic opportunities that combine inhibition of somatic CAG expansion with restoration of neuronal DNA demethylation.

neuroscience↗

bioRxiv2025

Turbulence orchestrates actin-mitochondria dynamics to preserve GPIbα and support iPSC-derived platelet biogenesis

The in vitro manufacturing of induced pluripotent stem cell-derived platelets (iPSC-platelets) remains limited by low yield and poor quality, thus impeding practical clinical application. To address this, we developed a turbulence-assisted system using defined physical stimulation to enhance platelet production from immortalized megakaryocyte progenitor cell lines (imMKCLs). This approach significantly increases the yield of glycoprotein Ib (GPIb) functional iPSC-platelets. However, the underlying mechanisms remained unclear. Here, we reveal that GPIb iPSC-platelets retain high mitochondrial membrane potential, low phosphatidylserine (PS) exposure, and active ATP-dependent flippases, preserving mitochondrial integrity and platelet functionality. In contrast, GPIb- platelets exhibit PS exposure, ADAM17 activation, and selective GPIb shedding. Notably, turbulent flow induces late-stage actin depolymerization, promoting even mitochondrial distribution within maturing imMKCLs and enhancing mitochondrial inheritance to iPSC-platelets. These findings highlight the key role of turbulence in regulating actin dynamics and mitochondrial allocation during platelet biogenesis, providing a mechanistic foundation for improving in vitro platelet production for therapeutic use.

cell biology↗

bioRxiv2024

Embryonic diversification of adult neural stem cells and ependymal cells

Both adult neural stem (type B) cells and ependymal (type E) cells in the mouse ventricular-subventricular zone (V-SVZ) are derived from slowly dividing (or quiescent) embryonic neural stem-progenitor cells (NPCs) that are set aside during development. However, it has remained unclear whether fate diversification between adult type B and type E cells actually occurs during embryogenesis. Here we performed single-cell transcriptomic analysis of slowly dividing embryonic NPCs and identified cell subpopulations transcriptionally similar to adult type B or type E cells. The type B- like embryonic cells appeared to emerge before embryonic day (E) 13.5, whereas the type E-like cells became evident between E13.5 and E16.5. Genes differentially expressed in B-like embryonic cells (versus E-like cells) included Tmem100, Cadm2, and bone morphogenetic protein (BMP)-induced genes. Forced expression of an active form of BMP receptor (ALK2QD), TMEM100, or CADM2 in embryonic NPCs resulted in preferential generation of adult type B cells relative to type E cells in the postnatal brain. Moreover, knockdown of TMEM100 resulted in relative enrichment of type E cells over type B cells. Our results indicate that the embryonic origin of adult type B cells and that of type E cells have already diverged molecularly during embryogenesis, and they have identified key molecular players in this fate bifurcation.

developmental biology↗

bioRxiv2023

Raman microscopy of cryofixed biological specimens for high-resolution and high-sensitivity chemical imaging

Raman microscopy is an emerging molecular imaging technology, yet its signal-to-noise-ratio (SNR) in measurements of biological specimens is severely limited due to the small cross-section of Raman scattering. Here, we present Raman imaging techniques of cryofixed specimens to overcome SNR limitations by enabling long exposure of specimens under highly stabilized low-temperature conditions. The observation of frozen specimens in a cryostat at a constant low temperature immediately after rapid freezing enabled the improvement of SNR and significantly enhanced spatial and spectral resolution. We also confirmed that the cryofixation can preserve physicochemical states of specimens by observing alkyne-labeled coenzyme Q in cytosol and hemeproteins in acute ischemic myocardium, which cannot be done by fixation using chemical reagents. Finally, we applied the technique for multiplex Raman imaging of label-free endogenous molecules and alkyne-tagged molecules in cryofixed HeLa cells, demonstrating its capability of high-content imaging of complex biological phenomena while maintaining physiological conditions.

biophysics↗

bioRxiv2023

Time-deterministic cryo-optical microscopy

We developed a technique to rapidly freeze biological systems during observation under an optical microscope, which allows for detailed optical observation at a precise time in the biological dynamics at high spatial resolution and quantifiability under low-temperature conditions. Our study further found that ion distributions and molecular conformations can be fixed in space and time, demonstrated by visualization of calcium waves rapid-frozen at an instant using fluorescent ion indicators. These results show that time-deterministic cryo-optical microscopy can suspend cellular dynamics while maintaining molecular and ionic states, allowing us to view cellular dynamics in a way that has not been possible before. One-Sentence SummaryRapid freezing of dynamic biological systems during optical observation presents time information to the fixed samples

biophysics↗

bioRxiv2022

Inhibition of the BMP pathway suppresses tumor growth via downregulation of EGFR in MEK/ERK-dependent colorectal cancer

The bone morphogenetic protein (BMP) pathway promotes differentiation and induces apoptosis in normal colorectal epithelial cells. However, the effect of the BMP pathway in colorectal cancer (CRC) is controversial; it can either be tumor promoting or tumor suppressing, depending on the study. In this study, we found that CRC cells reside in a BMP-rich environment based on RNA-sequencing database analysis. Suppression of BMP using a specific BMP inhibitor, LDN193189, suppresses the growth of organoids in some CRC cases. CRC organoids treated with LDN193189 exhibited a decrease in epidermal growth factor receptor, which was, at least in part, mediated by protein degradation induced by leucine-rich repeats and immunoglobulin-like domains protein 1 (LRIG1). Among CRC organoid panels from 18 different patients, suppression of organoid growth by BMP inhibition correlated with the induction of LRIG1 gene expression. Notably, knockdown of LRIG1 in organoids diminished the growth-suppressive effect of LDN193189. Furthermore, simultaneous treatment with LDN192189 and trametinib, an FDA-approved MEK inhibitor, resulted in a combination effect in both in vivo and in vitro xenograft tumor treatment in CRC organoids, which are susceptible to growth suppression by LDN193189. Taken together, the simultaneous inhibition of BMP and MEK can be a novel treatment option in CRC cases, and evaluating in vitro growth suppression and LRIG1 induction by BMP inhibition using patient-derived organoids could offer functional biomarkers for predicting potential responders.

cancer biology↗

bioRxiv2022

Metabolic clogging of mannose triggers genomic instability via dNTP loss in human cancer cells

Mannose has anti-cancer activity that inhibits cell proliferation and enhances the efficacy of chemotherapy. How mannose exerts its anti-cancer activity, however, remains poorly understood. Here, using genetically engineered human cancer cells that permit the precise control of mannose metabolic flux, we demonstrate that the large influx of mannose exceeding its metabolic capacity induced metabolic remodeling, leading to the generation of slow-cycling cells with limited deoxyribonucleoside triphosphates (dNTPs). This metabolic remodeling impaired dormant origin firing required to rescue stalled forks by cisplatin, thus exacerbating replication stress. Importantly, pharmacological inhibition of de novo dNTP biosynthesis was sufficient to retard cell cycle progression, sensitize cells to cisplatin, and inhibit dormant origin firing, suggesting dNTP loss-induced genomic instability as a central mechanism for the anti-cancer activity of mannose.

cancer biology↗

bioRxiv2021

Photothermal Dye-based Subcellular-sized Heat Spot Enabling the Modulation of Local Cellular Activities

Thermal engineering at microscale such as the control and measurement of temperature is a key technology in basic biological research and biomaterials development, which remains challenge yet. Here, we engineered the polymeric nanoparticle, in which a fluorescent temperature sensory dye and a photothermal dye were embedded in its polymer matrices, termed nanoHT. When a near infrared laser at 808 nm is illuminated to the particle, it enables to create the subcellular-sized heat spot in a live cell, where fluorescence thermometry allows the read out of the temperature increment concurrently at individual heat spots. Owing to the controlled local heating, we found that the cell death of HeLa cells was induced at the certain temperature at rate of a few seconds. It should be also noted that the cell death was triggered from the very local heat spot at subcellular level. Furthermore, nanoHT was applied for the induction of muscle contraction of the C2C12 myotube by heat. We successfully showed that the heat-induced contraction took place at the limited area of a single myotube according to the alteration of protein-protein interactions related to the contraction event. These studies demonstrated that even a single heat spot provided by a photothermal material could be very effective in altering cellular functions, paving the way for novel photothermal therapies.

bioengineering↗