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Sato, M.

Publications and source records attributed to Sato, M..

At least 55 records · Page 3Linked to original sources
bioRxiv2024

Selective Translation Orchestrates Key Signaling Pathways in Primed Pluripotency

Pluripotent stem cell (PSC) identities, such as differentiation and infinite proliferation, have long been understood within the frameworks of transcription factor networks, epigenomes, and signal transduction, yet remain unclear and fragmented. Directing attention toward translational regulation, as a bridge between these events, promises to yield new insights into previously unexplained mechanisms. Our functional CRISPR interference screening-based approach revealed that EIF3D maintains primed pluripotency through selective translational regulation. The loss of EIF3D disrupts the balance of pluripotency-associated signaling pathways, impairing primed pluripotency. Moreover, we discovered that EIF3D ensures robust proliferation by controlling the translation of various p53 regulators, which maintain low p53 activity in the undifferentiated state. In this way, selective translation by EIF3D tunes the homeostasis of the primed pluripotency networks, ensuring the maintenance of an undifferentiated state with high proliferative potential. Therefore, this study establishes a paradigm for selective translational regulation as a defining feature of primed PSC identity.

cell biology↗
bioRxiv2024

Functional Modification of Cyanobacterial Phycobiliprotein and Phycobilisomes through Bilin Metabolism Control

The light-harvesting antenna complexes in cyanobacteria are known as phycobilisomes (PBSs), and they can adapt to a diverse range of light environments owing to the deployment of different phycobiliproteins within the PBS structures. Freshwater cyanobacteria such as Synechococcus elongatus PCC 7942 thrive under red light due to phycocyanin (PC), along with phycocyanobilin (PCB), in PBS. Conversely, cyanobacteria in shorter-wavelength light environments, such as green light, employ phycoerythrin paired with phycoerythrobilin (PEB) alongside PC in PBS. Synthetic biology studies have shown that PEB production can be enhanced by additional expression of the heterologous PEB synthases, namely 15,16-dihydrobiliverdin:ferredoxin oxidoreductase and PEB:ferredoxin oxidoreductase (PebAB), leading to cellular browning due to PEB accumulation. This approach is genetically unstable, and the properties of the resulting PEB-bound PBS complexes remain uncharacterized. Herein, we engineered a novel strain of Synechococcus 7942 PEB1, with finely tuned control of PEB metabolism. PEB1 exhibited a remarkable and reversible color shift from green to brown and pink, based on the PebAB induction levels. High induction led to a complete PCB-to-PEB substitution, causing the disassembly of the PBS rod complex. In contrast, low PebAB levels resulted in the formation of a stable chimeric PBS complex with partial PCB-to-PEB substitution. This adaptation enabled efficient light harvesting in the green spectrum and energy transfer to the photosynthetic reaction center. These findings, which improve our understanding of PBS and highlight the structural importance of the bilin composition, lay the foundation for future PBS adaptation studies in bioengineering, synthetic biology, and renewable energy.

synthetic biology↗
bioRxiv2023

Chromosomal DNA sequences of the Pacific saury genome: versatile resources for fishery science and comparative biology

Pacific saury (Cololabis saira) is a commercially important small pelagic fish species in Asian. In this study, we conducted the first-ever whole genome sequencing of this species, with single molecule, real-time (SMRT) sequencing technology. The obtained high-fidelity (HiFi) long-read sequence data, which amount to approximately 30 folds of its haploid genome size that was measured with quantitative PCR (1.17 Gb), were assembled into contigs. Scaffolding with Hi-C reads yielded a whole genome assembly containing 24 chromosome-scale sequences, with a scaffold N50 length of 47.7 Mb. Screening of repetitive elements including telomeric repeats was performed to characterize possible factors that need to be resolved towards telomere-to-telomere sequencing. The larger genome size than in medaka, a close relative in Beloniformes, is at least partly explained by larger repetitive element quantity, which is reflected in more abundant tRNAs, in the Pacific saury genome. Protein-coding regions was predicted using transcriptome data, which resulted in 22,274 components. Retrieval of Pacific saury homologs of aquaporin (AQP) genes known from other teleost fishes validated high completeness and continuity of the genome assembly. These resources are available at https://treethinkers.nig.ac.jp/saira/ and will assist various molecular-level studies in fishery science and comparative biology.

genomics↗
bioRxiv2023

Src/Fas2-dependent Ephrin phosphorylation initiates Eph/Ephrin reverse signaling through Rac1 to shape columnar units in the fly brain

Columns are the morphological and functional units containing multiple neurons in the brain. The molecular mechanisms of column formation are largely unknown. Ephrin/Eph signaling mediates a variety of developmental processes. Ephrin acts as a ligand for Eph to regulate forward signaling, while Eph acts as a ligand for Ephrin to regulate reverse signaling. However, whether and how the uni- or bi-directional Ephrin/Eph signaling is involved in column formation remains elusive. In this study, we show that Ephrin and Eph regulate the morphology and location of columnar neurons through bi-directional repulsive signaling. Furthermore, Eph ligand triggers cytoplasmic tyrosine phosphorylation of Ephrin under the control of Src kinases and Fasciclin II (Fas2), forming Ephrin/Src/Fas2 complex to promote reverse signaling through a downstream regulator, Rac1. This study presents for the first time a unified picture of the molecular interactions in the defined context of column formation using the fly brain as a model.

developmental biology↗
bioRxiv2023

Visualizing hemoglobin oxygen saturation distribution in small animals: an in vivo application of a 3D photoacoustic imaging scanner with a hemispherical detector array

AbstractO_ST_ABSSignificanceC_ST_ABSPhotoacoustic (PA) imaging has garnered considerable attention due to its capability to render vascular images in a label-free manner. Specifically, devices employing a hemispherical detector array (HDA) have been heralded for various clinical applications, owing to their potential to yield high reproducibility three-dimensional images. While high-resolution models utilizing high-frequency sensors have been introduced for animal experimentation, their evaluation has been constrained to a single wavelength. In this study, we demonstrate the applicability of in vivo mouse models for visualizing body oxygen saturation distribution using dual wavelengths. AimWith the aid of our uniquely developed device and analysis software, our primary objective is to map the spatial distribution of the hemoglobin oxygen saturation coefficient (S-factor) through non-invasive in vivo imaging. Subsequently, we aim to observe the temporal alterations within this distribution, specifically assessing changes in hemoglobin oxygen saturation in both normal and tumor vessels over time. ApproachHigh-quality S-factor images were obtained by integrating a newly developed scanning sequence for high contrast with alternate two-wavelength irradiation. Following validation with phantoms, in vivo images were procured in mice. Sequential scanning of the same mouse yielded information about temporal changes. S-factor evaluation was conducted with our photoacoustic image viewer to analyze trends in hemoglobin oxygen saturation. ResultsHigh-contrast images were achieved by increasing the number of integrations during scanning. S-factor images were acquired using both healthy and tumor-bearing mice. Vessels within the liver and kidneys were distinctly reconstructed, and differences in oxygen saturation discriminated between arteries and veins. Repeated measurements on the same mice, both live and post-euthanasia, provided spatiotemporal information, such as a decrease in oxygen saturation after euthanasia or a precipitous drop in oxygen saturation inside the tumor nine days post-cell line transplantation. ConclusionsBy analyzing S-factor images using a photoacoustic imaging system designed for animal experiments, we succeeded in discerning variations in in vivo oxygen saturation. The custom-built system holds promise as a versatile tool for diverse basic research endeavors, as it can seamlessly interface with human clinical applications.

bioengineering↗
bioRxiv2023

Intronic TNR-retained ISOPROPYLMALATE ISOMERASE LARGE SUBUNIT1 transcripts impair leaf development in Arabidopsis

Intronic trinucleotide repeat (TNR) is widely distributed in plant genomes. In Arabidopsis accession Bur-0, abnormally expanded TTC repeat in intron-3 of the ISOPROPYLMALATE ISOMERASE LARGE SUBUNIT1 (IIL1) gene causes growth defects called the irregularly impaired leaves (iil) phenotype, triggered by DNA methylation-mediated IIL1 gene silencing at elevated temperature. However, little is known about how the reduced expression of IIL1 causes the iil phenotype. We demonstrated that the iil phenotype was resulted from the relative increase of intron-3-retained IIL1 transcripts through the experiments where the iil phenotype was reproduced by introducing the IIL1 gene harboring 100 copies of TTC repeat into Col-0. The iil phenotype appeared when the total amount of the IIL1 transcripts was decreased by co-suppression and the percentage of intron-3-retained IIL1 transcripts was increased. The IIL1 gene encodes an isopropylmalate isomerase large subunit, forming heterodimers with small subunits (AtLeuD1, AtLeuD2, or AtLeuD3). In the myb28 myb29 mutant lacking AtLeuD1 and AtLeuD2, the iil phenotype was almost completely suppressed regardless of higher percentage of intron-3-retained IIL1 transcripts. The results indicated that the iil phenotype was associated with interaction with AtLeuDs, suggesting that intronic TNR-containing transcripts were translated into abnormal proteins and perturbed the metabolic pathway supporting the leaf development.

plant biology↗
bioRxiv2022

Molecular engineering of emicizumab to improve hemophilia A treatment

Emicizumab, a factor (F)VIIIa-function mimetic bispecific antibody (BsAb) to FIXa and FX, has become an indispensable treatment for people with hemophilia A (PwHA). Although emicizumab is very potent, long-term outcomes from the clinical studies suggest that a small proportion of PwHA still experiences bleeds. Additionally, non-clinical studies indicate that the maximum cofactor activity of emicizumab is lower than international standard activity (100 IU/dL of FVIII). An increased cofactor activity BsAb would benefit such patients. Here, we report NXT007, a BsAb binding FIXa and FX developed through further engineering of emicizumab. Emicizumab has a common light chain, but through advances in antibody engineering, we were able to create a more potent BsAb with two new non-common light chains. After extensive optimization of the heavy and light chains, the resulting BsAb, NXT007, exerted in vitro thrombin generation (TG) activity in hemophilia A plasma equivalent to 100 IU/dL of FVIII when triggered by tissue factor. NXT007 demonstrated potent hemostatic activity in an acquired hemophilia A model in non-human primates at a much lower dosage than emicizumab, consistent with an around 30-fold dose shift in the in vitro TG activity between NXT007 and emicizumab. Moreover, together with Fc engineering that enhanced FcRn binding and reduced in vivo clearance, we demonstrate that NXT007 could be effective at a much lower dosage with a longer dosing interval compared to emicizumab. These non-clinical results suggest that NXT007 could maintain a non-hemophilic range of coagulation potential in PwHA and provides a rationale for its clinical testing.

bioengineering↗
bioRxiv2022

VR-based real-time imaging reveals abnormal cortical dynamics during behavioral transitions in a mouse model of autism

Functional connectivity (FC) can provide insight into cortical circuit dysfunction in neuropsychiatric disorders. However, dynamic changes in FC related to locomotion with sensory feedback remain unexplored. To investigate FC dynamics in locomoting mice, we developed mesoscopic Ca2+ imaging with a virtual reality (VR) environment. We find rapid reorganization of cortical FC in response to changing behavioral states. Using machine learning classification, behavioral states are accurately decoded. We then use our VR-based imaging system to study cortical FC in a mouse model of autism and find that locomotion states are associated with altered FC dynamics. Furthermore, we identify FC patterns involving the motor area as the most distinguishing features of the autism mice from wild-type mice during behavioral transitions, which might correlate with motor clumsiness in patients with autism. Our VR-based real-time imaging system provides invaluable information to understand FC dynamics linked to a behavioral abnormality of neuropsychiatric disorders.

neuroscience↗
bioRxiv2022

SEN1 is responsible for molybdate transport into nodule symbiosomes for nitrogen fixation in Lotus japonicus

Symbiotic nitrogen fixation (SNF) in legume root nodules requires a steady supply of molybdenum (Mo) for synthesis of the iron-Mo cofactor for nitrogenase in bacteroids. For this nutrient to be exported by the host plant it must cross the peribacteroid membrane (PBM), however, the molybdate transporter responsible has not yet been identified. A Lotus japonicus symbiotic mutant, sen1, forms nodules that do not fix nitrogen; it has nodule defects and bacteroid degradation. The biochemical function and subcellular localization of SEN1 protein remains to be elucidated. Here, we found a new phenotype in which the SEN1 mutation resulted in increased Mo accumulation in the nodule host fractions but decreased Mo accumulation in the bacteroids at 10 days post inoculation. We identified the molybdate efflux transport activity of SEN1 via heterologous expression in yeast. SEN1 was expressed exclusively in nodules, and its expression was stable in response to varying Mo supply in nutrient solution. In situ immunostaining verified that the SEN1 protein is localized, in part, to the PBM in the rhizobium-infected cells. Taken together, these results confirmed that SEN1 is responsible for mediating molybdate efflux from the cytosol of nodule host cells to the symbiosomes for SNF. Furthermore, SEN1 mutation reduced the expression of nifD and nifK, suggesting that SEN1 may be pertinent to iron-Mo-cofactor assembly. This work fills the knowledge gap regarding how molybdate is allocated from the host plant to the bacteroids; such knowledge is critical for developing new SNF biological systems in non-legume plants. One-sentence summarySEN1 is localized partly in the peribacteroid membrane of nodule cells and mediates the molybdate exportation from the host plant cytosol to the symbiosomes for symbiotic nitrogen fixation.

plant biology↗
bioRxiv2022

Fission yeast Dis1 is an unconventional TOG/XMAP215 that induces microtubule catastrophe to drive chromosome pulling

The shortening of microtubules attached to kinetochores is the driving force of chromosome movement during cell division. Specific kinesins are believed to shorten microtubules but are dispensable for viability in yeast, implying the existence of additional factors responsible for microtubule shortening. Here, we demonstrate that Dis1, a TOG/XMAP215 ortholog in fission yeast, promotes microtubule shortening to carry attached chromosomes. Although TOG/XMAP215 orthologs are generally accepted as microtubule polymerases, Dis1 promoted microtubule catastrophe in vitro and in vivo. Notably, microtubule catastrophe was promoted when the tip was attached to kinetochores, as they steadily anchored Dis1 at the kinetochore-microtubule interface. Engineered Dis1 oligomers artificially tethered at a chromosome arm region induced the shortening of microtubules in contact, frequently pulling the chromosome arm towards the vicinity of spindle poles in meiocytes. Thus, unlike Alp14 and other TOG/XMAP215 orthologs, Dis1 plays an unconventional role in promoting microtubule catastrophe, thereby driving chromosome movement.

cell biology↗
bioRxiv2022

Relationship of Rickettsia infection rates and population genetic structure of ixodid ticks along an altitudinal gradient

Ixodid ticks, such as Ixodes ovatus and Haemaphysalis flava, are important vectors of tick-borne diseases in Japan, such as Japanese spotted fever caused by Rickettsia japonica. This study describes the population genetic structure and gene flow of I. ovatus and H. flava and their Rickettsia infection rates along an altitudinal gradient. A total of 346 adult I. ovatus and 243 H. flava were analyzed for the presence of Rickettsia by nested PCR targeting the 17kDA, gltA, rOmpA, and rOmpB genes. Population genetic structure was performed using the mitochondrial cytochrome oxidase 1 (cox1) marker. The Rickettsia infection rates were 13.26% in I. ovatus and 6.17% in H. flava. For I. ovatus, the global FST value revealed significant genetic differentiation among the different populations, whereas H. flava showed non-significant genetic differentiation. The cox1 I. ovatus cluster dendrogram showed two cluster groups, while the haplotype network and phylogenetic tree showed three genetic groups. The two cluster groups and the three genetic groups found in I. ovatus showed a significant difference in their Rickettsia infection rates and mean altitude per group. No significant differences were found in the mean altitude or Rickettsia infection rates of H. flava. Our results suggest that low gene flow in the I. ovatus populations has caused spatially heterogenous Rickettsia infection rates along the altitudinal gradient. This information can be used in understanding the relationship between the tick vector, its pathogen, and environmental factors, such as altitude, and for the control of tick-borne diseases in Japan.

ecology↗
bioRxiv2022

Obox4 secures zygotic genome activation upon loss of Dux

Once fertilized, mouse zygotes rapidly proceed to zygotic genome activation (ZGA), during which long terminal repeats (LTRs) of murine endogenous retroviruses with leucine tRNA primer (MERVL) are activated by a conserved homeodomain-containing transcription factor, DUX. However, Dux-knockout embryos produce fertile mice, suggesting that ZGA is redundantly driven by an unknown factor(s). Here we present multiple lines of evidence that the multicopy homeobox gene, Obox4, encodes a transcription factor that is highly expressed in mouse 2-cell embryos and redundantly drives ZGA. Genome-wide profiling revealed that OBOX4 specifically binds and activates MERVL LTRs as well as a subset of murine endogenous retroviruses with lysine tRNA primer (MERVK) LTRs. Depletion of Obox4 is tolerated by embryogenesis, whereas concomitant Obox4/Dux depletion markedly compromises embryonic development. Our study identified OBOX4 as a transcription factor that provides genetic redundancy to pre-implantation development.

genetics↗
bioRxiv2022

A novel Smg6 mouse model reveals regulation of circadian period and daily CRY2 accumulation through the nonsense-mediated mRNA decay pathway

Nonsense-mediated mRNA decay (NMD) has been intensively studied as a surveillance pathway that degrades erroneous transcripts arising from mutations or RNA processing errors. While additional roles in controlling regular mRNA stability have emerged, possible functions in mammalian physiology in vivo have remained unclear. Here, we report a novel conditional mouse allele that allows converting the NMD effector nuclease SMG6 from wild-type to nuclease domain-mutant protein. We analyzed how NMD downregulation affects the function of the circadian clock, a system known to require rapid mRNA turnover. We uncover strong lengthening of free-running circadian periods for liver and fibroblast clocks, and direct NMD regulation of Cry2 mRNA, encoding a key transcriptional repressor within the rhythm-generating feedback loop. In the entrained livers of Smg6 mutant animals we reveal transcriptome-wide alterations in daily mRNA accumulation patterns, altogether expanding the known scope of NMD regulation in mammalian gene expression and physiology.

genetics↗
bioRxiv2022

Hydrodynamics shapes self-recruitment in anemonefishes

Many marine species have a pelagic larval phase that undergo dispersal among habitats. Studies on marine larval dispersal have revealed a large variation in the spatial scale of dispersal, and accumulated evidence has shown that seascape patchiness is the major determinant for variation in self-recruitment. However, few studies have investigated the influence of geographic settings on marine larval dispersal. Bays or lagoons generally enhance the retention of larvae, while larvae are more likely to be flushed by strong currents in open coasts. To examine associations between larval dispersal, geographic setting, and hydrodynamics, we compared fin-scale dispersal patterns, self-recruitment, and local retention of two anemonefishes (Amphiprion frenatus and A. perideraion) between a semi-enclosed bay and an open coast in the Philippines combining genetic parentage analysis and biophysical dispersal modelling. Contrary to our expectations, parentage analysis revealed lower estimates of self-recruitment in the semi-closed bay (0-2%) than in the open coast (14-15%). The result was consistent with dispersal simulations predicting lower local retention and self-recruitment in the former (0.4% and 19%) than in the latter (2.9% and 38%). Dispersal modelling also showed that cross-shore currents toward offshore were much stronger around the semi-closed bay and were negatively correlated with local retention and self-recruitment. These results suggest that stronger cross-shore currents around the semi-closed bay transport anemonefish larvae to the offshore and mainly contributed to the lower self-recruitment. Our results highlight difficulty in predicting self-recruitment from geographic setting alone and importance of hydrodynamics on it.

ecology↗
bioRxiv2022

Fission yeast Smi1p participates in the synthesis of the primary septum by regulating β-1,3-glucan synthase Bgs1p function

Cytokinesis is the concluding step of the cell cycle. Coordination between multiple cellular processes is essential for the success of cytokinesis. The fission yeast, Schizosaccharomyces pombe, like other fungal cells is contained within a cell wall. During cell division, the external cell wall is extended inwards to form a special septum wall structure in continuity with the cell wall. The primary septum, the central component of the three-layered division septum, is enriched with linear {beta}-1,3-glucan formed by Bgs1p, a {beta}-1,3-glucan synthase. In this study we uncover a novel essential protein, Smi1p, that functions as a suppressor of the Bgs1p temperature-sensitive mutant, cps1-191. We observe a rescue in the cell wall composition and ultrastructure and also in actomyosin ring dynamics. Further, we identify a colocalization and physical association between Bgs1p and Smi1p. Altogether, our results indicate that Smi1p regulates the function of Bgs1p during cytokinesis.

cell biology↗
bioRxiv2022

HOTARU: Automatic sorting system for large scale calcium imaging data

Currently, calcium imaging allows long-term recording of large-scale neuronal activity in diverse states. However, it remains difficult to extract neuronal dynamics from recorded imaging data. In this study, we propose an improved constrained nonnegative matrix factorization (CNMF)-based algorithm and an effective method to extract cell shapes with fewer false positives and false negatives through image processing. We also show that the evaluation metrics obtained during image and signal processing can be combined and used for false-positive cell determination. For the CNMF algorithm, we combined cell-by-cell regularization and baseline shrinkage estimation, which greatly improved its stability and robustness. We applied these methods to real data and confirmed their effectiveness. Our method is simpler and faster, detects more cells with lower firing rates and signal-to-noise ratios, and enhances the quality of the extracted cell signals. These advances can improve the standard of downstream analysis and contribute to progress in neuroscience.

neuroscience↗
bioRxiv2022

Tiling mechanisms of the compound eye through geometrical tessellation

Tilling patterns are observed in many biological structures. Hexagonal tilling, commonly observed in the compound eyes of wild-type Drosophila, is dominant in nature; this dominance can probably be attributed to physical restrictions such as structural robustness, minimal boundary length, and space filling efficiency. Surprisingly, tetragonal tiling patterns are also observed in some Drosophila small eye mutants and aquatic crustaceans. Herein, geometrical tessellation is shown to determine the ommatidial tiling patterns. In small eye mutants, the hexagonal pattern is transformed into a tetragonal pattern as the relative positions of neighboring ommatidia are stretched along the dorsal-ventral axis. Hence, the regular distribution of ommatidia and their uniform growth collectively play an essential role in the establishment of tetragonal and hexagonal tiling patterns in compound eyes.

developmental biology↗