Biology subjects

Tanaka, J.

Publications and source records attributed to Tanaka, J..

16 recordsLinked to original sources

bioRxiv2026

In-Cell Protein Crystallization via a Locally Flexible 24-mer Assembly Precursor

In-cell protein crystallization (ICPC) produces ordered protein crystals within living cells, but the mechanisms used by proteins to acquire long-range crystalline order in the cellular environment remains poorly understood. Here, we define the assembly pathway of CipB, a crystalline inclusion protein from Photorhabdus luminescens. CipB crystals formed in cells dissolve under mild acidic conditions into a predominant 24-mer species, supporting a model in which an in-cell crystal is built from a discrete 24-mer assembly precursor rather than through direct packing of smaller oligomeric states. Structural analysis of recrystallized CipB shows that the same 24-mer architecture packs into a body-centered cubic lattice, consistent with the lattice observed for the in-cell crystals. Cryo-EM and molecular dynamics analyses indicate that the 24-mer assembly precursor preserves its overall architecture while retaining local conformational flexibility at the N-terminal and surface-loop regions. Mutation analyses further link the N-terminal region to the formation of the 24-mer precursor and surface residues to lattice assembly. These observations support a stepwise crystallization model in which N-terminal flexibility facilitates the formation of an assembly-competent 24-mer precursor, whereas defined hydrophobic surface contacts subsequently organize these precursors into a long-range-ordered lattice.

biophysics↗

bioRxiv2026

BMP-3b suppresses proliferation, migration, invasion, and TGF-β 1/Smad3 signaling in breast cancer cells

ObjectiveBone morphogenetic protein-3b (BMP-3b), also known as growth differentiation factor 10, has been implicated in tumor suppression; however, its role in breast cancer and its interaction with transforming growth factor-{beta}1 (TGF-{beta}1) signaling remain incompletely understood. MethodsPublicly available datasets were used to examine BMP-3b expression in breast lesions and its association with overall survival in patients with stage III or IV breast cancer. Human MCF-7 and murine 4T1 breast cancer cells were treated with recombinant BMP-3b. Cell proliferation, migration, invasion, epithelial-mesenchymal transition-related proteins, and TGF-{beta}1-induced Smad3 phosphorylation were assessed using Cell Counting Kit-8, scratch wound-healing, Transwell invasion, and Western blot assays. ResultsBMP-3b expression was lower in ductal carcinoma in situ than in normal mammary tissue. Low BMP-3b expression was associated with poorer overall survival in patients with stage III or IV breast cancer. BMP-3b reduced proliferation of MCF-7 and 4T1 cells and inhibited migration and invasion of 4T1 cells. BMP-3b increased E-cadherin and decreased vimentin expression in both cell lines. It also attenuated TGF-{beta}1-induced migration, invasion, and Smad3 phosphorylation in 4T1 cells. ConclusionsBMP-3b suppresses malignant phenotypes of breast cancer cells and modulates TGF- {beta}1/Smad3 signaling. These findings identify BMP-3b as a potential endogenous regulator of breast cancer progression.

cell biology↗

bioRxiv2026

Natural variation in rice mitogen-activated protein kinase 4 contributes to increased photosynthetic rate under field conditions

Improving rice (Oryza sativa L.) yield requires a balanced enhancement of both sink size and source capacity. While many QTLs for sink size have been identified, only a few are known for source capacity, which is essential for achieving high yield. Here we identified qHP10 as a major QTL for increased photosynthetic rate by using chromosome segment substitution lines derived from a cross between the high-yielding indica cultivar Takanari and the average-yielding japonica cultivar Koshihikari. High-resolution mapping combined with CRISPR/Cas9-induced mutagenesis revealed that the causative gene underlying qHP10 is Mitogen-Activated Protein Kinase 4 (OsMPK4). A near-isogenic line carrying the OsMPK4Takanari allele (NIL-OsMPK4) had a 15-25% higher photosynthetic rate than Koshihikari. NIL-OsMPK4 also had higher stomatal conductance than Koshihikari but similar stomatal pore size and density, indicating that increased stomatal aperture increases photosynthetic rate. This enhancement is likely attributable to the down-regulation of OsMPK4 expression, which increases stomatal conductance and thus promotes CO2 uptake. Our findings demonstrate that OsMPK4 is a promising genetic target for increasing source capacity and, potentially, rice yield through molecular breeding. (175 words)

plant biology↗

bioRxiv2024

Ephrin Forward Signaling Controls Interspecies Cell Competition in Pluripotent Stem Cells

In the animal kingdom, evolutionarily conserved mechanisms known as cell competition eliminate unfit cells during development. Interestingly, cell competition also leads to apoptosis of donor cells upon direct contact with host cells from a different species during interspecies chimera formation. The mechanisms underlying how host animal cells recognize and transmit cell death signals to adjacent xenogeneic human cells remain incompletely understood. In this study, we developed an interspecies cell contact reporter system to dissect the mechanisms underlying competitive interactions between mouse and human pluripotent stem cells (PSCs). Through single-cell RNA-seq analyses, we discovered that Ephrin A ligands in mouse cells play a crucial role in signaling cell death to adjacent human cells that express EPHA receptors during interspecies PSC co-culture. We also demonstrated that blocking the Ephrin A-EPHA receptor interaction pharmacologically, and inhibiting Ephrin forward signaling genetically in the mouse cells, enhances the survival of human PSCs and promotes chimera formation both in vitro and in vivo. Our findings elucidate key mechanisms of interspecies PSC competition during early embryogenesis and open new avenues for generating humanized tissues or organs in animals, potentially revolutionizing regenerative medicine.

cell biology↗

bioRxiv2024

FGF2 promotes the expansion of parietal mesothelial progenitor pools and inhibits BMP4-mediated smooth muscle cell differentiation

Mesothelial cells, in the outermost layer of internal organs, are essential for both organ development and homeostasis. Although the parietal mesothelial cell is the primary origin of mesothelioma that may highjack developmental signaling, the signaling pathways that orchestrate developing parietal mesothelial progenitor cell (MPC) behaviors, such as MPC pool expansion, maturation, and differentiation, are poorly understood. To address it, we established a robust protocol for culturing WT1+ MPCs isolated from developing pig and mouse parietal thorax. Quantitative qPCR and immunostaining analyses revealed that BMP4 facilitated MPC differentiation into smooth muscle cells (SMCs). In contrast, FGF2 significantly promoted MPC progenitor pool expansion but blocked the SMC differentiation. BMP4 and FGF2 counterbalanced these effects, but FGF2 had the dominant impact in the long-term culture. A Wnt activator, CHIR99021, was pivotal in MPC maturation to CALB2+ mesothelial cells, while BMP4 or FGF2 was limited. Our results demonstrated central pathways critical for mesothelial cell behaviors.

developmental biology↗

bioRxiv2023

Generation of salivary glands derived from pluripotent stem cells via conditional blastocyst complementation

Various patients suffer from dry mouth due to salivary gland dysfunction. Whole salivary gland generation and transplantation is a potential therapy to resolve this issue. However, the lineage permissible to design the entire salivary gland generation has been enigmatic. Here, we discovered Foxa2 as a lineage critical for generating a salivary gland via conditional blastocyst complementation (CBC). Foxa2 linage, but not Shh nor Pitx2, initiated to label between the boundary region of the endodermal and the ectodermal oral mucosa before primordial salivary gland formation, resulting in marking the entire salivary gland. The salivary gland was agenesis by depleting Fgfr2 under the Foxa2 lineage in the mice. We rescued this phenotype by injecting donor pluripotent stem cells into the mouse blastocysts. Those mice survived until adulthood with normal salivary glands compatible in size compared with littermate controls. These results indicated that CBC-based salivary gland generation is promising for next-generation cell-based therapy.

developmental biology↗

bioRxiv2023

Wistar rats raised in an affectionate environment display lifesaving-like behaviors while distinguishing life from death

It is generally believed that humans are the only species that values life. However, it is not well understood whether animals have a nature that values life. In this study, we attempted to determine whether male Wistar rats have this nature. Normally-reared rats did not show lifesaving-like actions towards anesthesia-induced comatose rats, although they seemed to distinguish life from death. Considering the possibility that different rearing conditions may foster a life-valuing nature, male Wistar rat pups were reared in several ways: normal rearing, loving rearing (LR; rats reared as if they were cute pets), rearing in an enriched environment, reared with gentle stroking of the back, and normal rearing of offspring of rats raised under LR conditions. When placed in an anxiety-producing environment, only LR rats escaped into the hands of the person who reared them, indicating attachment. Only the LR rats displayed lifesaving-like actions towards unknown comatose rats or drowning pups. LR rats also stopped attacks by biting ICR mice that were attacking C57BL/6 mice. Thus, rearing in an affectionate environment may foster a life-valuing nature, even in rats, suggesting that the valuing of life may be neither innate nor human-specific.

animal behavior and cognition↗

bioRxiv2022

Foxa2 lineage+ mesendoderm forms a competitive pulmonary mesenchyme niche crucial for generating the entire lungs

Millions suffer from incurable lung diseases, and the donor lung shortage hampers organ transplants. Identifying the crucial lineage and the program for lung organogenesis could facilitate designing whole-lung bioengineering. Using lineage-tracing mice and human iPSC-derived lung-directed differentiation, we revealed that gastrulating Foxa2 lineage contributed to both lung mesenchyme and epithelium formation. Interestingly, Foxa2 lineage-derived cells in the lung mesenchyme progressively increased and occupied more than half of the mesenchyme niche, including endothelial cells, during lung development. Foxa2 promoter-driven, conditional Fgfr2 gene depletion caused the lung agenesis phenotype in mice. Importantly, wild-type donor mouse iPSCs injected into their blastocysts rescued this phenotype by complementing the Fgfr2-defective niche in the lung epithelium and mesenchyme. Donor cell is shown to replace the entire lung epithelial and robust mesenchymal niche during early chimeric lung development, resulting in efficient complementation of the nearly entire lung niche at the late stage of lung development. These results suggest that lung complementation based on the Foxa2 lineage is a unique model for the progressive mobilization of donor cells into both epithelial and mesenchymal lung niches and provides crucial insights for designing new bioengineering strategies to generate whole lungs.

developmental biology↗

bioRxiv2022

A developmental program that regulates mammalian organ size offsets evolutionary distance

Pigs are evolutionarily more distant from humans than mice, but their physiological organs are closest to humans. The molecular program leading to a more than 1,000-fold increase in organ size in pigs and humans over that of mice across evolution has not been elucidated. We generated large-scale transcriptional landscapes throughout swine lung development. Our cross-species single-cell molecular atlas let us discover swine progenitor identities, stage-specific markers, and a core organ-size regulation program (COSRP), well-conserved in swine and humans but less so in mice. Across eight mammalian species, human COSRP promoters showed higher homologies to evolutionary-distant large animals, including pigs, than evolutionary-close small animals. Our study provides a molecular foundation during swine lung development that unveils animal size regulation conserved in the COSRP promoter, independent of genome-wide evolution. COSRP is a critical paradigm for studying thousands-fold changes in biological sizes in evolution, development, cancer, zoology, respirology, organoids, and biotechnology, particularly human-compatible organ generation. One Sentence SummaryA cross-species developmental molecular atlas identified the indicator of lung and animal size beyond evolution

evolutionary biology↗

bioRxiv2022

Dosage differences in 12-OXOPHYTODIENOATE REDUCTASE genes modulate wheat primary root growth

Wheat is an essential crop for global food security and is well adapted to a wide variety of soils1. However, the gene networks regulating different root architectures remain poorly understood. We report here the identification of a cluster of a monocot-specific 12-OXOPHYTODIENOATE REDUCTASE genes from subfamily III (OPRIII) that modulate key differences in wheat root architecture associated with grain yield under water-limited conditions. Wheat plants with a loss-of-function mutation in OPRIII showed longer seminal roots, whereas plants with increased OPRIII dosage or transgenic over-expression showed reduced seminal root growth, precocious development of lateral roots and increased jasmonic acid (JA). A JA-biosynthesis inhibitor eliminated the root differences, confirming a JA-mediated mechanism. Multiple transcriptome analysis of transgenic and wild-type lines revealed significant enriched JA-biosynthetic and reactive oxygen species (ROS) pathways that paralleled changes in ROS distribution. The OPRIII genes provide a useful entry point to engineer root architecture in wheat and other cereals.

plant biology↗

bioRxiv2022

Cell-free Protein Crystallization for Nanocrystal Structure Determination

In-cell protein crystallization (ICPC) has attracted attention as a next-generation structural biology tool because it does not require multistep purification processes and large-scale crystallization screenings. However, significant issues remain to be solved in context of obtaining various protein crystals in sufficient amounts and quality for structure determination by ICPC. Here, we report the development of cell-free protein crystallization (CFPC), a direct protein crystallization technique which uses cell-free protein synthesis. The most crucial advantages of CFPC are that the reaction scale and time can be minimized and that various reagents can be added during the reaction. We obtained high-quality nano-sized polyhedra crystals, which are produced in insect cells by infection with cytoplasmic polyhedrosis virus, at a 200 L reaction scale within 6 h. We applied this technology to structure determination of crystalline inclusion protein A (CipA) by suppressing twin crystal formation with addition of an inhibitor to the reaction solution. We succeeded in determining a 2.11 [A] resolution structure from the nanocrystals of CipA. This technology, which integrates in-cell and in vitro crystallizations significantly expands the tools available for high throughput protein structure determination, particularly in context of unstable, low-yield, or substrate-binding proteins, which are difficult to analyze by conventional methods.

biophysics↗

bioRxiv2022

Improvement of Gene Delivery and Mutation Efficiency in the CRISPR-Cas9 Wheat (Triticum aestivum L.) Genomics System via Biolistics

Discovery of the CRISPR-Cas9 gene editing system revolutionized the field of plant genomics. Despite advantages in ease of designing gRNA and the low cost of the CRISPR-Cas9 system, there are still hurdles to overcome in low mutation efficiencies, specifically in hexaploid wheat. In conjunction with gene delivery and transformation frequency, the mutation rate bottleneck has the potential to slow down advancements in genomic editing of wheat. In this study, nine bombardment parameter combinations using three gold particle sizes and three rupture disk pressures were tested to establish optimal stable transformation frequencies in wheat. Utilizing the best transformation protocol and a knockout cassette of the phytoene desaturase gene, we subjected transformed embryos to four temperature treatments and compared mutation efficiencies. The use of 0.6 m gold particles for bombardment increased transformation frequencies across all delivery pressures. A heat treatment of 34{degrees}C for 24 hours resulted in the highest mutation efficiency with no or minimal reduction in transformation frequency. The 34{degrees}C treatment produced two M0 mutant events with albino phenotypes, requiring biallelic mutations in all three genomes of hexaploid wheat. Utilizing optimal transformation and heat treatment parameters greatly increases mutation efficiency and can help advance research efforts in wheat genomics.

plant biology↗

bioRxiv2022

Impact of temperature and time on DNA-free Cas9-ribonucleoprotein mediated gene editing in wheat protoplasts and immature embryos

The advancement of precision engineering for crop trait improvement is important in the face of rapid population growth, climate change, and disease. To this end, targeted double-stranded break technology using RNA-guided Cas9 has been adopted widely for genome editing in plants. Agrobacterium or particle bombardment-based delivery of plasmids encoding Cas9 and guide RNA (gRNA) is common, but requires optimization of expression and often results in random integration of plasmid DNA into the plant genome. Recent advances have described gene editing by the delivery of Cas9 and gRNA as pre-assembled ribonucleoproteins (RNPs) into various plant tissues, but with moderate efficiency in resulting regenerated plants. In this report we describe significant improvements to Cas9-RNP mediated gene editing in wheat. We demonstrate that Cas9-RNP assays in protoplasts are a fast and effective tool for rational selection of optimal gRNAs for gene editing in regenerable immature embryos (IEs), and that high temperature treatment enhances gene editing rates in both tissue types. We also show that Cas9-mediated editing persists for at least 14 days in gold particle bombarded wheat IEs. The regenerated edited wheat plants in this work are recovered at high rates in the absence of exogenous DNA and selection. With this method, we produce knockouts of a set of three homoeologous genes and two pathogenic effector susceptibility genes that result in insensitivity to corresponding necrotrophic effectors produced by Parastagonospora nodorum. The establishment of highly efficient, DNA-free gene editing technology holds promise for accelerated trait diversity production in an expansive array of crops.

plant biology↗

bioRxiv2021

CRISPR/Cas9 knockout of EPFL10 reduces stomatal density while maintaining photosynthesis and enhancing water conservation in rice

Rice (Oryza sativa) is of paramount importance for global nutrition, supplying at least 20% of global calories. However, water scarcity and increased drought severity are anticipated to reduce rice yields globally. Rice stomatal developmental genetics were explored as a mechanism to improve drought resilience while maintaining yield under climate stress. CRISPR/Cas9-mediated knockouts of EPFL10 and STOMAGEN yielded lines with c. 80% and 25% of wild-type stomatal density, respectively. epfl10 lines with moderate reductions in stomatal densities are able to conserve water to similar extents as stomagen lines, but do not suffer from the concomitant reductions in stomatal conductance, carbon assimilation, or thermoregulation observed in stomagen knockouts. Moderate reductions in stomatal densities achieved by editing EPFL10 may be a climate-adaptive approach in rice that can safeguard yield. Editing the paralog of STOMAGEN in other species may provide a means to tune stomatal density in agriculturally important crops beyond rice.

plant biology↗

bioRxiv2021

Multi-omics prediction of oat agronomic and seed nutritional traits across environments and in distantly related populations

Multi-omics prediction has been shown to be superior to genomic prediction with genome-wide DNA-based genetic markers (G) for predicting phenotypes. However, most of the existing studies were based on historical datasets from one environment; therefore, they were unable to evaluate the efficiency of multi-omics prediction in multi-environment trials and distantly-related populations. To fill those gaps, we designed a systematic experiment to collect omics data and evaluate 17 traits in two oat breeding populations planted in single and multiple environments. In the single-environment trial, transcriptomic BLUP (T), metabolomic BLUP (M), G+T, G+M and G+T+M models showed greater prediction accuracy than GBLUP for 5, 10, 11, 17 and 17 traits, respectively, and metabolites generally performed better than transcripts when combined with SNPs. In the multi-environment trial, multi-trait models with omics data outperformed both counterpart multi-trait GBLUP models and single-environment omics models, and the highest prediction accuracy was achieved when modeling genetic covariance as an unstructured covariance model. We also demonstrated that omics data can be used to prioritize loci from one population with omics data to improve genomic prediction in a distantly-related population using a two-kernel linear model that accommodated both likely casual loci with large-effect and loci that explain little or no phenotypic variance. We propose that the two-kernel linear model is superior to most genomic prediction models that assume each variant is equally likely to affect the trait and can be used to improve prediction accuracy for any trait with prior knowledge of genetic architecture.

genetics↗

bioRxiv2020

Efficient isolation of protoplasts from rice calli with pause points and its application in transient gene expression and genome editing assays

An efficient in vivo transient transfection system using protoplasts is an important tool to study gene expression, metabolic pathways, and multiple mutagenesis parameters in plants. Although rice protoplasts can be isolated from germinated seedlings or cell suspension culture, preparation of those donor tissues can be inefficient, time consuming, and laborious. Additionally, the lengthy process of protoplast isolation and transfection needs to be completed in a single day. Here we report a protocol for isolation of protoplasts directly from rice calli, without using seedlings or suspension culture. The method is developed to employ discretionary pause points during protoplast isolation and prior to transfection. Protoplasts maintained within a sucrose cushion partway through isolation, for completion on a subsequent day, per the first pause point, are referred to as S protoplasts. Fully isolated protoplasts maintained in MMG solution for transfection on a subsequent day, per the second pause point, are referred to as M protoplasts. Both S and M protoplasts, 1 day after initiation of protoplast isolation, had minimal loss of viability and transfection efficiency compared to protoplasts 0 days after isolation. S protoplast viability decreases at a lower rate over time than that of M protoplasts and can be used with added flexibility for transient transfection assays and time-course experiments. The protoplasts produced by this method are competent for transfection of both plasmids and ribonucleoproteins (RNPs). Cas9 RNPs were used to demonstrate the utility of these protoplasts to assay genome editing in vivo. The current study describes a highly effective and accessible method to isolate protoplasts from callus tissue induced from rice seeds. This method utilizes donor materials that are resource-efficient and easy to propagate, permits convenience via pause points, and allows for flexible transfection days after protoplast isolation. It provides an advantageous and useful platform for a variety of in vivo transient transfection studies in rice.

plant biology↗