Biology subjects

Miyata, Y.

Publications and source records attributed to Miyata, Y..

12 recordsLinked to original sources

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

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

Early-peaking caspase-7 activity at the plasma membrane drives apoptotic phosphatidylserine exposure

Apoptosis is an immunologically silent form of regulated cell death executed by caspase1. Caspase cleaves hundreds of substrates throughout the cell to regulate apoptotic processes2, including phosphatidylserine (PS) externalization3-5. However, spatio-temporal regulation of caspase activity in dying cells remains unclear. Here, we show that caspase activity peaks earlier at the plasma membrane (PM) during apoptosis by establishing a Dual Forster resonance energy transfer (Dual FRET) imaging system that combines subcellularly targeted FRET-based caspase biosensors with a cytosolic reference counterpart6,7. Genetic analysis identified caspase-7, an executioner caspase considered an inefficient backup for caspase-3, the major executioner caspase8,9, as a caspase responsible for the Early-Peaking Caspase Activity at the PM (EP-CAP). Mechanistically, EP-CAP is mediated via electrostatic interactions between PS in the inner leaflet of the PM and polybasic residues in the N-terminal intrinsically disordered region (IDR) of caspase-7, which are liberated by the caspase-mediated removal of polyacidic residues. Physiologically, EP-CAP facilitates the efficient cleavage of phospholipid scramblases for the rapid externalization of PS and subsequent efferocytosis. Accordingly, we propose that caspase-7, but not caspase-3, is a bona fide immunologically silent death caspase reinforcing the non-inflammatory nature of apoptosis via EP-CAP.

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↗

bioRxiv2023

Hepatocyte-Targeted siTAZ Therapy Lowers Liver Fibrosis in NASH Diet-Fed Chimeric Mice with Hepatocyte-Humanized Livers

Nonalcoholic steatohepatitis (NASH) is emerging as the most common cause of liver disease. Although many studies in mouse NASH models have suggested therapies, translation to humans is poor, with no approved drugs for NASH. One explanation may lie in inherent differences between mouse and human hepatocytes. We used NASH diet-fed chimeric mice reconstituted with human hepatocytes (hu-liver mice) to test a mechanism-based hepatocyte-targeted siRNA, GalNAc-siTaz, shown previously to block the progression to fibrotic NASH in mice. Mice were reconstituted with human hepatocytes following ablation of endogenous hepatocytes, resulting in ~95% human hepatocyte reconstitution. The mice were then fed a high-fat choline-deficient Lamino acid-defined diet for 6 weeks to induce NASH, followed by six weekly injections of GalNAc-siTAZ to silence hepatocyte-TAZ or control GalNAc-siRNA (GalNAc-control) while still on the NASH diet. The results revealed that GalNAc-siTAZ lowered human hepatic TAZ and IHH, the major TAZ target that promotes liver fibrosis in NASH. Most importantly, GalNAc-siTAZ decreased liver inflammation, hepatocellular injury, hepatic fibrosis, and profibrogenic mediator expression, and profibrotic NOTCH vs. GalNAc-control, indicating that GalNAc-siTAZ decreased the progression of NASH in mice reconstituted with human hepatocytes. In conclusion, silencing TAZ in human hepatocytes suppresses liver fibrosis in a hu-liver model of NASH. Impact and ImplicationsNo drugs have yet been approved for NASH, which is a leading cause of liver disease worldwide. The findings here provide support for this therapeutic strategy of using hepatocyte-targeted siTAZ to decrease NASH progression. More generally, the study illustrates how hu-liver NASH mice can be used to evaluate therapeutic hepatocyte-targeted siRNAs to help prioritize future testing in human NASH.

pharmacology and toxicology↗

bioRxiv2023

A Sodium-dependent Trehalose Transporter Contributes to Anhydrobiosis in Insect Cell Line, Pv11

Pv11 is the only animal cell culture that, when preconditioned with a high concentration of trehalose, can be preserved in the dry state at room temperature for more than one year while retaining the ability to resume proliferation. This extreme desiccation tolerance is referred to as anhydrobiosis. Here we identified a novel transporter that contributes to the recovery of Pv11 cells from anhydrobiosis. In general, the SLC5 family of secondary active transporters co-transport Na+ and carbohydrates including glucose. Here we identified a novel transporter STRT1 (sodium-ion trehalose transporter 1) belonging to the SLC5 family that is highly expressed in Pv11 cells and transports trehalose with Na+ dependency. This is the first report of an SLC5 family member that transports a naturally occurring disaccharide, such as trehalose. Knockout of the Strt1 gene significantly reduced the viability of Pv11 cells upon rehydration after desiccation. During rehydration, when intracellular trehalose is no longer needed, Strt1-knockout cells released the disaccharide more slowly than the parent cell line. During rehydration, Pv11 cells became roughly spherical due to osmotic pressure changes, but then returned to their original spindle shape after about 30 min. Strt1-knockout cells, however, required about 50 min to adopt their normal morphology. STRT1 probably regulates intracellular osmolality by releasing unwanted intracellular trehalose with Na+, thereby facilitating the recovery of normal cell morphology during rehydration. STRT1 likely improves the viability of dried Pv11 cells by rapidly alleviating the significant physical stresses that arise during rehydration. Significance StatementThis is the first report of an SLC5 family member, STRT1 (sodium ion trehalose transporter 1), with Na+-dependent trehalose transport activity. A Strt1-knockout cell line revealed that STRT1 likely plays an important role during anhydrobiosis in Pv11 cells: it efficiently discharges unwanted trehalose in the presence of Na+ during rehydration of dried Pv11 cells, effectively reducing intracellular osmolality and thereby restoring cell morphology to a normal state.

biochemistry↗

bioRxiv2023

Identification of FAM53C as a suppressive binding protein of a neurodevelopmental disorders-related kinase DYRK1A

The protein kinase DYRK1A encoded in human chromosome 21 is the major contributor to the multiple symptoms observed in Down syndrome patients. In addition, DYRK1A malfunction is associated with various other neurodevelopmental disorders such as autism spectrum disorder. Here we identified FAM53C with no hitherto known biological function as a novel suppressive binding partner of DYRK1A. FAM53C bound to the catalytic protein kinase domain of DYRK1A, whereas DCAF7/WDR68, the major DYRK1A-binding protein, binds to the N-terminal domain of DYRK1A. The binding of FAM53C inhibited autophosphorylation activity of DYRK1A and its kinase activity to an exogenous substrate MAPT/Tau. FAM53C did not bind directly to DCAF7/WDR68, whereas DYRK1A tethered FAM53C and DCAF7/WDR68 by binding concurrently to both of them, forming a tri-protein complex. DYRK1A possesses a nuclear localization signal and accumulates in the nucleus when overexpressed in cells. Co-expression of FAM53C induced cytoplasmic re-localization of DYRK1A, revealing the cytoplasmic anchoring function of FAM53C to DYRK1A. Moreover, the binding of FAM53C to DYRK1A suppressed the DYRK1A-dependent nuclear localization of DCAF7/WDR68. All the results show that FAM53C binds to DYRK1A, suppresses its kinase activity, and anchors it in the cytoplasm. In addition, FAM53C bound to the DYRK1A-related kinase DYRK1B with an Hsp90/Cdc37-independent manner. The results explain for the first time why endogenous DYRK1A is distributed in the cytoplasm in normal brain tissue. FAM53C-dependent regulation of the kinase activity and intracellular localization of DYRK1A may play a significant role in gene expression regulation caused by normal and aberrant levels of DYRK1A.

cell biology↗

bioRxiv2023

MAPK/MAK/MRK overlapping kinase (MOK) controls microglial inflammatory/type-I IFN responses via Brd4 and is involved in ALS pathophysiology

Amyotrophic lateral sclerosis (ALS) is a fatal and incurable neurodegenerative disease affecting motor neurons and characterized by microglia-mediated neurotoxic inflammation whose underlying mechanisms remain incompletely understood. In this work we reveal that MAPK/MAK/MRK overlapping kinase (MOK), with unknown physiological substrate, displays an immune function by controlling inflammatory and type-I IFN responses in microglia which are detrimental to primary motor neurons. Moreover, we uncover the epigenetic reader bromodomain-containing protein 4 (Brd4) as the first molecule regulated by MOK, by promoting Ser492-phospho-Brd4 levels. We further demonstrate that MOK regulates Brd4 functions by supporting its binding to cytokine gene promoters, therefore enabling innate immune responses. Remarkably, we show that MOK levels are increased in ALS spinal cord, particularly in microglial cells, and that administration of a chemical MOK-inhibitor to ALS model mice is able to modulate Ser492-phospho-Brd4 levels, suppress microglial activation and modify disease course, indicating a pathophysiological role of MOK kinase in ALS and neuroinflammation.

neuroscience↗

bioRxiv2020

Cas9-mediated Genome Editing Reveals a Significant Contribution of Calcium Signaling Pathways to Anhydrobiosis in Pv11

Pv11 is an insect cell line established from the midge Polypedilum vanderplanki that exhibits an extreme desiccation tolerance known as anhydrobiosis. Pv11 has also an anhydrobiotic ability which is induced by trehalose treatment. Here we report the successful construction of the genome editing system for Pv11 cells and its application for identifying the signaling pathways in the anhydrobiosis. Using the Cas9-mediated gene knock-in system, we established GCaMP3-stably expressing Pv11 cells to monitor intracellular Ca2+ mobilization. Intriguingly, trehalose treatment evoked a transient increase of cytosolic Ca2+ concentration, and further experiments indicated the contribution of the calmodulin - calcineurin - NFAT pathway to the tolerance for trehalose treatment as well as the desiccation tolerance, while the calmodulin - calmodulin Kinase - CREB pathway conferred only the desiccation tolerance on Pv11 cells. Thus, our results show the critical contribution of the trehalose-induced Ca2+ surge to the anhydrobiosis and the temporal different roles of each signaling pathway.

molecular biology↗

bioRxiv2020

Development of a Tet-On inducible expression system for the anhydrobiotic cell line, Pv11

The Pv11 cell line established from an African chironomid, Polypedilum vanderplanki, is the only cell line tolerant to complete desiccation. In Pv11 cells, a constitutive expression system for Pv11 cells was previously exploited and several reporter genes were successfully expressed. Here we report the identification of an effective minimal promoter for Pv11 cells and its application to the Tet-On inducible expression system. First, using a luciferase reporter assay, we showed that a 202 bp deletion fragment derived from the constitutively active 121-promoter functions in Pv11 cells as an appropriate minimal promoter with the Tet-On inducible expression system. The AcGFP1 protein was also successfully expressed in Pv11 cells using the inducible system. In addition to these reporter genes, avian myeloblastosis virus reverse transcriptase subunit (AMV RT), which is one of the most widely commercially available RNA-dependent DNA polymerases, was successfully expressed through the inducible expression system and its catalytic activity was verified. These results demonstrate the establishment of an inducible expression system in cells that can be preserved in the dry state and highlight a possible application to the production of large and complex proteins. HighlightsO_LIA 202 bp-deletion fragment derived from a constitutively active promoter was identified as a minimal promoter in Pv11 cells. C_LIO_LIA Tet-On inducible expression system was developed for Pv11 cells using the minimal promoter. C_LIO_LITypical reporter genes (GFP and luciferase) and an enzyme with complex structure, i.e. a viral reverse transcriptase, were successfully and inducibly expressed in Pv11 cells using the Tet-On system. C_LI

bioengineering↗

bioRxiv2020

Safety and Efficacy of Avaren-Fc Lectibody Targeting HCV High-Mannose Glycans in a Human Liver Chimeric Mouse Model

Infection with hepatitis C virus (HCV) remains to be a major cause of morbidity and mortality worldwide despite the recent advent of highly effective direct-acting antivirals. The envelope glycoproteins of HCV are heavily glycosylated with a high proportion of high-mannose glycans (HMGs), which serve as a shield against neutralizing antibodies and assist in the interaction with cell-entry receptors. However, currently there is no approved therapeutic targeting this potentially druggable biomarker. Here, we investigated the therapeutic potential of the lectibody Avaren-Fc (AvFc), a HMG-binding lectin-Fc fusion protein. In vitro assays showed AvFcs capacity to neutralize cell culture-derived HCV in a genotype independent manner with IC50 values in the low nanomolar range. A histidine buffer-based AvFc formulation was developed for in vivo studies using the PXB human liver chimeric mouse model. Systemic administration of AvFc was well tolerated; after 11 consecutive doses every other day at 25 mg/kg, there were no significant changes in body or liver weights, nor any impact noted in blood human albumin levels or serum alanine aminotransferase activity. Gross necropsy and liver pathology further confirmed the lack of discernible toxicity. This treatment regimen successfully prevented genotype 1a HCV infection in all animals, while an AvFc mutant lacking HMG binding activity failed to block the infection. These results suggest that targeting envelope HMGs is a promising therapeutic approach against HCV infection. In particular, AvFc may provide a safe and efficacious means to prevent recurrent infection upon liver transplantation in HCV-related end-stage liver disease patients.

pharmacology and toxicology↗

bioRxiv2019

Intracellular localization and gene expression analysis provide new insights on LEA proteins diversity in anhydrobiotic midge

Anhydrobiosis, an adaptive ability to withstand complete desiccation, is in insects limited to a single species: the nonbiting midge Polypedilum vanderplanki (the sleeping chironomid). Evolution of anhydrobiosis in a single representative of a large genus is associated with drastic changes in genome structure, including the emergence of new multimember gene families directly involved in desiccation tolerance. Among them, Late Embryogenesis Abundant (LEA) proteins, which protect other proteins from aggregation caused by desiccation, are believed to originate via horizontal gene transfer from a bacterial donor. To obtain new insights on the biological background of the expanded 27-member LEA protein group in P. vanderplanki, we investigated the expression of corresponding genes in a P. vanderplanki-derived cell line, capable of anhydrobiosis, in a normal state and during induction of desiccation tolerance. We found that all LEA proteins genes identified in P. vanderplankis genome except PvLea16 and PvLea17 are also expressed in Pv11 cells. Their expression was elevated in response to anhydrobiosis-inducing trehalose treatment. Expression patterns of PvLea genes were well preserved in Pv11 cells in comparison to P. vanderplankis larvae both in the control group and during the anhydrobiosis cycle. We also investigated localization of LEA proteins in Pv11 cells and Sf9 cells and found a different level of conservation in intracellular localization of the protein expressed in mammalian and insect cells.

molecular biology↗