Biology subjects

Kotani, T.

Publications and source records attributed to Kotani, T..

15 recordsLinked to original sources

bioRxiv2026

Mouse oocytes organize mRNA-protein assemblies and ER-associated translation hubs to drive meiosis

Oocyte meiosis in animals is essential for producing fertilizable eggs. As transcription becomes largely quiescent, meiotic progression depends on temporally regulated translation of accumulated maternal mRNAs. However, the underlying mechanisms remain unclear. Here, we identified diverse and highly organized mRNA-protein assemblies in mouse oocytes that coordinate cytoplasmic mRNA regulation and enable spatiotemporal translation to drive oocyte meiosis. Twelve RNA-binding proteins (RBPs), including DDX4/VASA, HDLBP/Vigilin, and TDRD3, were assembled into widely distributed aggregates in germinal vesicle (GV)-stage oocytes. We depleted DDX4 and HDLBP using Trim-Away and found that these RBPs regulate the precise assembly of RNA granules and protein aggregates through distinct mechanisms, promoting temporal protein synthesis at cortical translation factories with the endoplasmic reticulum, termed CORTE. All 12 RBPs formed distinct mRNA-protein assemblies, whose size and distribution changed upon meiotic initiation. These findings reveal the molecular basis of spatiotemporal mRNA-protein-organelle networks underlying animal reproduction and development.

cell biology↗

bioRxiv2026

Breakdown of Rheological Universality in the Vegetal Hemisphere of Ascidian Embryos Mapped by Atomic Force Microscopy

Mechanical regulation plays an essential role in the organization of early embryogenesis. In ascidian embryos, cells in the animal hemisphere exhibit periodic stiffening-softening cycles, and their cell rheological properties follow a common master curve, indicating a form of universality. In contrast, cells in the vegetal hemisphere show cell-to-cell differences in stiffness even within the same endodermal lineage, but their rheological behavior has not been characterized. Here, using atomic force microscopy (AFM), we investigated the spatiotemporal dynamics of single-cell power-law rheological states in the vegetal hemisphere during early cleavage. We found that both the elastic modulus (stiffness) and the fluidity (power-law exponent) differ among endodermal cells, and that these differences change in a stage-dependent manner. This result indicates that vegetal cells do not exhibit a single common rheological behavior, in contrast to animal hemisphere cells, suggesting that mechanical properties in the vegetal hemisphere are not uniformly regulated but are patterned in space and time during cleavage. Our findings indicate that this mechanical diversification is linked to the progression of early morphogenesis and may contribute to the emergence of distinct cell behaviors during development.

biophysics↗

bioRxiv2025

Two sequential waves of mRNA translation drive embryonic development

Eggs of many species accumulate thousands of dormant mRNAs that are translated after fertilization at specific times and locations to direct development. However, how embryos coordinate translation of these mRNAs remains unclear. In this study, we identified sequential waves of translation critical for proper development progression. The first wave occurred within 1 h and included translation of ewsr1b mRNA that harbored a short 3' untranslated region (UTR) comprising 16 nucleotides. The resulting Ewsr1b protein triggered the second translation wave through binding cytoplasmic mRNAs, including pou5f3, which encodes a transcription factor promoting zygotic genome activation. In contrast, HuR and Syncrip repressed translation until the first and second waves, respectively. ewsr1b mRNA that had a long 3'UTR was translated in the second wave, and the 3'UTRs length determined protein localization and function. Overall, our findings reveal previously unknown molecular principles that coordinate translation timings and protein functions to drive long-term, multilayered processes.

molecular biology↗

bioRxiv2025

Intrathecal administration of palmitoyl-lysophosphatidylethanolamine reduces secondary injury and improves locomotor recovery following spinal cord injury

Spinal cord injury (SCI) triggers secondary pathophysiological cascades, including glutamate excitotoxicity, that result in neuronal loss and impair functional recovery. We have previously shown that lysophosphatidylethanolamine (LPE), a lysophospholipid, promotes neurite outgrowth and protects against glutamate excitotoxicity in cultured cortical neurons. However, whether these effects extend to spinal cord neurons and occur in vivo has remained unclear. In this study, we compared the effects of different LPE species: myristoyl-LPE (14:0 LPE), palmitoyl-LPE (16:0 LPE), stearoyl-LPE (18:0 LPE), and oleoyl-LPE (18:1 LPE) in cultured spinal commissural neurons, and evaluated their effects in vivo using a mouse model of SCI. In cultured neurons, all LPE species promoted neurite outgrowth. Although several species demonstrated a tendency toward neuroprotection, only 16:0 LPE exhibited a statistically significant protective effect against glutamate-induced excitotoxic cell death. Intrathecal administration of 16:0 LPE after SCI reduced TUNEL-positive cells in the acute phase and attenuated lesion expansion at 8 weeks post-injury. Moreover, 5-HT fluorescence intensity was increased in 16:0 LPE-treated mice, suggesting enhanced serotonergic innervation. Furthermore, administration of 16:0 LPE after SCI significantly improved hind-limb motor performance compared with vehicle controls, as assessed by the Basso Mouse Scale. Collectively, these findings suggest that intrathecal administration of 16:0 LPE reduces secondary injury and promotes functional recovery following SCI. Our findings highlight its potential as a therapeutic candidate for SCI.

neuroscience↗

bioRxiv2025

A decade after being listed as Endangered: Japanese eel stock inferred from fishery-dependent and independent monitoring records (Preprint)

This study assesses recent trends in the abundance of Japanese eels (Anguilla japonica), which have been listed as Endangered on the IUCN Red List of Threatened Species since 2014, by updating previously reported coastal fisheries datasets and incorporating new freshwater data and scientific monitoring records for glass eels in Japan and Taiwan. Catch-per-unit-effort (CPUE) data for yellow and silver eels revealed statistically significant declines in seven of eight datasets, with projected reductions over three generations (24 years) ranging from 79.2% to 99.9%. In contrast, no significant temporal trends were detected in the CPUE of glass eels, likely due to high interannual variability driven by oceanic conditions. The integration of freshwater and coastal data, along with fishery-independent monitoring, enhances the reliability of these indicators. The results provided in this study represent the best currently available indicators of Japanese eel population dynamics.

ecology↗

bioRxiv2025

Mapping single-cell mechanics in the early embryogenesis of Xenopus laevis using atomic force microscopy

During early embryonic development, cells undergo rapid cleavage divisions accompanied by morphological changes driven by mechanical cues. However, the spatiotemporal mechanics of regulative embryonic cells remains poorly understood. Here, we use atomic force microscopy (AFM) to map single-cell stiffness of Xenopus laevis embryos, a model of regulative development, from early cleavage (stage 6) to the onset of gastrulation (stage 11). To ensure stable AFM mapping, vitelline membrane-removed embryos were immobilized in custom grooved agarose wells and gently held using a dulled glass pipette. AFM observations revealed marked mechanical heterogeneity within the animal hemisphere: stiffness in apical cytoplasmic regions, which are defined as the central apical surface excluding cell-cell boundaries, varied among cells, regardless of size, indicating intrinsic variability. Cell-cell boundaries consistently showed high stiffness, suggesting strong adhesion, as commonly observed in epithelial monolayers in vitro. In contrast, in the vegetal hemisphere during gastrulation, cell-cell boundaries exhibited low stiffness, indicative of weaker adhesion. Additionally, micron-scale stiff inclusions were detected in the apical vegetal cytoplasm, with sizes comparable to those of yolk platelets. These findings demonstrate the capability of AFM to probe the microscale mechanical architecture of developing regulative embryos and to uncover regional mechanical asymmetries between the animal and vegetal hemispheres. Such asymmetries may contribute to key morphogenetic processes during early vertebrate development.

biophysics↗

bioRxiv2025

Mechanism of bridge-type phospholipid transfer by Atg2 for autophagosome biogenesis

Autophagosome biogenesis requires delivery of millions of phospholipids. In budding yeast, Atg2 and Atg18 couple the ER to the isolation membrane for lipid transport, yet the mechanism is poorly understood. Combining molecular dynamics simulations, in vitro imaging-based lipid transfer assays, and in vivo analyses, we show that Atg1-mediated phosphorylation activates Atg2 as a lipid-transfer bridge. Simulations reveal that Atg2 contains a water-excluding hydrophobic cavity that accommodates [~]25 phospholipids with bilayer-like fluidity. Atg1 phosphorylates an N-terminal helix of Atg2 in vitro and in vivo, thereby opening the ER-facing entrance to the Atg2 cavity and enabling lipid flow between membranes in silico and in vitro. Consistently, phosphodeficient but not phosphomimetic mutations impair autophagy. Cavity narrowing along the conduit disrupts lipid alignment in silico, phosphorylation-activated bridge-type lipid transfer in vitro, and ER-to-isolation-membrane lipophilic dye flow and autophagosome expansion in vivo. These data suggest that Atg1 gates Atg2-mediated bridge-type lipid transport to build autophagosomes.

cell biology↗

bioRxiv2024

Mapping single-cell rheology of ascidian embryos in the cleavage stages using AFM

During early embryo development, cell division is highly organized and synchronized. Understanding the mechanical properties of embryonic cells as a material is crucial for elucidating the physical mechanism underlying embryogenesis. Previous atomic force microscopy (AFM) studies on developing embryos revealed that single cells of ascidian embryos in the cleavage stage stiffened and softened during cell division. However, how embryonic cells, as a compliant material, exhibit viscoelastic properties during the cell cycle remains poorly characterized. In this study, we investigated the rheological properties of embryonic cells in the animal hemisphere in the cleavage stages using stress-relaxation AFM and approach-retraction force curve AFM techniques. The AFM measurements revealed that developing single cells followed a power-law rheology observed in single-cell rheology in vitro. The embryonic cells increased the modulus (stiffness) and decreased the fluidity (the power-law exponent) towards cell division. We found three rheological states in developing embryos during the cell cycle. The correlation between the cell modulus and the fluidity during the cell cycle was collapsed onto a master curve with a negative correlation, indicating that embryonic cells tightly interacting with the neighboring cells conserved the universality of rheological behavior observed in single cells in vitro. SIGNIFICANCEUnderstanding the rheological properties of embryonic cells is crucial to elucidate the origin and mechanism of the functional and morphological changes in cells during embryogenesis. AFM-based microrheology revealed that single cells of ascidian embryos in the animal hemisphere in the cleavage stages followed a single power-law rheology, which has no characteristic time scale. Furthermore, the rheological parameters, such as cell stiffness and fluidity, were collapsed onto a master curve with a negative correlation during the cell cycle. These rheological behaviors are similar to those observed in single cells in vitro, indicating that the embryonic cells tightly interacting with each other conserved the intrinsic rheological behaviors of isolated single cells.

cell biology↗

bioRxiv2024

Phase separation promotes Atg8 lipidation for autophagy progression

Upon starvation, the autophagy-initiating Atg1 complex undergoes phase separation to organize the pre-autophagosomal structure (PAS) in yeast, from which autophagosome formation is considered to proceed. However, the physiological roles of the PAS as a liquid droplet remain unclear. Here we show that core Atg proteins are recruited into early PAS droplets that are formed by phase separation of the Atg1 complex with different efficiencies in vitro. The Atg12-Atg5-Atg16 E3 ligase complex for Atg8 lipidation is the most efficiently condensed in the droplets via specific Atg12-Atg17 interaction, which is also important for the PAS targeting of the E3 complex in vivo. In vitro reconstitution experiments reveal that E3-enriched early PAS droplets promote Atg8 lipidation and incorporate Atg8-coated vesicles to the interior, thereby protecting them from Atg4-mediated delipidation. These data suggest that the PAS utilizes its liquid-like property to function as an efficient production site for lipidated Atg8 and pool membrane seeds to drive autophagosome formation.

cell biology↗

bioRxiv2022

Centrosome Loss And Cell Proliferation Defects Underlie Developmental Failure In Haploid Zebrafish Larvae

Haploid larvae in non-mammalian vertebrates are lethal with characteristic organ growth retardation collectively called "haploid syndrome." In contrast to mammals whose haploid intolerance is attributed to imprinting misregulation, the cellular principle of haploidy-linked defects in non-mammalian vertebrates remains unknown. Here, we investigated cellular defects that disrupt the ontogeny of gynogenetic haploid zebrafish larvae. Unlike diploid control, haploid larvae manifested unscheduled cell death at the organogenesis stage, attributed to haploidy-linked p53 upregulation. Moreover, we found that haploid larvae specifically suffered the gradual aggravation of mitotic spindle monopolarization during 1-3 days post fertilization, causing spindle assembly checkpoint-mediated mitotic arrest throughout the entire body. High-resolution imaging revealed that this mitotic defect accompanied the haploidy-linked centrosome loss occurring concomitantly with the gradual decrease in larval cell size. Either resolution of mitotic arrest or depletion of p53 partially improved organ growth in haploid larvae. Based on these results, we propose that haploidy-linked mitotic defects and cell death are parts of critical cellular causes shared among vertebrates that limit the larval growth in the haploid state, contributing to an evolutionary constraint on allowable ploidy status in the vertebrate life cycle.

developmental biology↗

bioRxiv2021

Transgenerational impact of aberrant inflammation in rat pregnancy

Children of women with pre-eclampsia have increased risk of cardiovascular (CV) and metabolic disease in adult life. Furthermore, the risk of pregnancy complications is higher in daughters born to women affected by pre-eclampsia than in daughters born after uncomplicated pregnancies. While aberrant inflammation contributes to the pathophysiology of pregnancy complications, including pre-eclampsia, the contribution of maternal inflammation to subsequent risk of CV and metabolic disease as well as pregnancy complications in the offspring remains unclear. Here we demonstrate that 24-week-old female rats (F1) born to dams (F0) exposed to lipopolysaccharide (LPS) during pregnancy (to induce inflammation) exhibited mild systolic dysfunction, increased cardiac growth-related gene expression, abnormal glucose tolerance and coagulopathy; whereas male F1 offspring exhibited abnormal glucose tolerance and increased visceral fat accumulation compared with F1 sex-matched offspring born to saline-treated dams. Both male and female F1 offspring born to LPS-treated dams had evidence of anemia. Fetuses (F2) from F1 females born to LPS-treated dams were growth restricted, and this reduction in fetal growth was associated with increased CD68 positivity and decreased expression of glucose transporter-1 in their utero-placental units. These results indicate that abnormal maternal inflammation can contribute to increased risk of CV and metabolic disease in offspring, and that the effects of inflammation may be transgenerational. This study provides evidence in support of early screening for CV and metabolic disease, as well as pregnancy complications in offspring affected by pre-eclampsia or other pregnancy complications associated with aberrant inflammation.

developmental biology↗

bioRxiv2021

Atg39 links and deforms the outer and inner nuclear membranes in selective autophagy of the nucleus

In selective autophagy of the nucleus (hereafter nucleophagy), nucleus-derived double membrane vesicles (NDVs) are formed, sequestered within autophagosomes, and delivered to lysosomes or vacuoles for degradation. In Saccharomyces cerevisiae, the nuclear envelope (NE) protein Atg39 acts as a nucleophagy receptor, which interacts with Atg8 to target NDVs to forming autophagosomal membranes. In this study, we revealed that Atg39 is anchored to the outer nuclear membrane (ONM) via its transmembrane domain and also associated with the inner nuclear membrane (INM) via membrane-binding amphipathic helices (APHs) in its perinuclear space region, thereby linking these membranes. We also revealed that overaccumulation of Atg39 causes the NE to protrude towards the cytoplasm, and the tips of the protrusions are pinched off to generate NDVs. The APHs of Atg39 are crucial for Atg39 assembly in the NE and subsequent NE protrusion. These findings suggest that the nucleophagy receptor Atg39 plays pivotal roles in NE deformation during the generation of NDVs to be degraded by nucleophagy.

cell biology↗

bioRxiv2021

Tdrd3 regulates the progression of meiosis II through translational control of Emi2 mRNA in mouse oocytes

After completion of meiosis I, the oocyte immediately enters meiosis II and forms a metaphase II (MII) spindle without an interphase, which is fundamental for generating a haploid gamete. Here, we identify tudor domain-containing protein 3 (Tdrd3) as a novel regulator of oocyte meiosis. Although early mitotic inhibitor 2 (Emi2) protein has been shown to ensure the meiosis I to II transition and the subsequent MII spindle formation by inhibiting the anaphase-promoting complex/cyclosome (APC/C), how it accumulates after meiosis I has remained unresolved. We isolated Tdrd3 as a protein directly binding to Emi2 mRNA. In GV-stage mouse oocytes, Emi2 mRNA assembled into RNA granules containing Tdrd3, while cyclin B1 mRNA, which was translated in early meiosis I, formed different granules. Knockdown of Tdrd3 attenuated Emi2 synthesis in meiosis II without affecting cyclin B1 synthesis in meiosis I. Moreover, Tdrd3-deficient oocytes entered interphase and failed to form an MII spindle after completion of meiosis I. Taken together, our results indicate the importance of Tdrd3-mediated translational control of Emi2 mRNA, which promotes Emi2 synthesis in meiosis II, for the progression of meiosis.

developmental biology↗

bioRxiv2020

Changes in subcellular structures and states of Pumilio1 regulate the translation of target Mad2 and Cyclin B1 mRNAs

Temporal and spatial control of mRNA translation has emerged as a major mechanism for promoting diverse biological processes. However, the molecular nature of temporal control of translation remains unclear. In oocytes, many mRNAs are deposited as a translationally repressed form and are translated at appropriate timings to promote the progression of meiosis and development. Here, we show that changes in structures and states of the RNA-binding protein Pumilio1 regulate the translation of target mRNAs and progression of oocyte maturation. Pumilio1 was shown to bind to Mad2 and Cyclin B1 mRNAs, assemble highly clustered solid-like aggregates, and surround Mad2 and Cyclin B1 RNA granules in mouse oocytes. These Pumilio1 aggregates were dissolved by phosphorylation prior to the translational activation of target mRNAs. Stabilization of Pumilio1 aggregates prevented the translational activation of target mRNAs and oocyte maturation. Together, our results provide an aggregation-dissolution model for the temporal and spatial control of translation.

cell biology↗

bioRxiv2018

Microglial SIRPα regulates the emergence of CD11c+ microglia and demyelination damage in white matter

A characteristic subset of microglia expressing CD11c appears in response to brain damage. However, the functional role of CD11c+ microglia, as well as the mechanism of its induction, are poorly understood. Here we report that the genetic ablation of signal regulatory protein (SIRP), a membrane protein, induced CD11c+ microglia in the brain white matter. Mice lacking CD47, a physiological ligand of SIRP, and microglia-specific SIRP knockout mice exhibited the same phenotype, suggesting the interaction between microglial SIRP and CD47 on neighbouring cells suppressed the emergence of CD11c+ microglia. A lack of SIRP did not cause detectable damage in the white matter, but resulted in the increased expression of genes characteristic of the repair phase after demyelination. In addition, cuprizone-induced demyelination was alleviated by the microglia-specific ablation of SIRP. Thus, microglial SIRP suppresses the induction of CD11c+ microglia that have the potential to accelerate the repair of damaged white matter.

neuroscience↗