Biology subjects

Murata, S.

Publications and source records attributed to Murata, S..

10 recordsLinked to original sources

bioRxiv2026

A chromatin relay from AIRE to ETS transcription factors sustains peripheral antigen expression in the thymic mimetic cells to ensure central tolerance.

Medullary thymic epithelial cells (mTECs) establish central tolerance by expressing a diverse repertoire of peripheral tissue-specific antigens (TSAs). This diversity is regulated not only by the transcriptional regulator AIRE, but also by lineage-defining factors in tissue-mimetic, AIRE-negative descendants of Aire mTECs (post-Aire mimetic TECs). However, whether and how prior AIRE activity contributes to TSA expression in post-AIRE mimetic TECs remains unclear. Here, we identify the ETS transcription factors, EHF and ELF3, as key regulators that sustain AIRE-primed gene expression in these cells. EHF and ELF3 preferentially bind distal genomic regions which are rendered accessible in advance by AIRE. Combined loss of EHF and ELF3 disrupts expression of AIRE-regulated genes expressed in mimetic TECs, leading to tissue-selective autoimmunity. Our findings reveal a relay mechanism in which AIRE primes chromatin for ETS factors to maintain TSA expression in post-AIRE mimetic TECs, thereby safeguarding self-tolerance.

immunology↗

bioRxiv2025

Aggregation of HAPLN2, a component of the perinodal extracellular matrix, is a hallmark of physiological brain aging in mice

Protein aggregation is a hallmark of neurodegenerative diseases and is also observed in the brains of elderly individuals without such conditions, suggesting that aging drives the accumulation of protein aggregates. However, the comprehensive understanding of age-dependent protein aggregates involved in brain aging remains unclear. Here, we investigated proteins that become sarkosyl-insoluble with age and identified hyaluronan and proteoglycan link protein 2 (HAPLN2), a hyaluronic acid-binding protein of the extracellular matrix at the nodes of Ranvier, as an age-dependent aggregating protein in mouse brains. Elevated hyaluronic acid levels and impaired microglial function reduced the clearance of HAPLN2, leading to its accumulation. HAPLN2 oligomers induced microglial inflammatory responses both in vitro and in vivo. Furthermore, age-associated HAPLN2 aggregation was also observed in the human cerebellum. These findings suggest that HAPLN2 aggregation results from age-related decline in brain homeostasis and may exacerbate the brain environment by activating microglia. This study provides new insights into the mechanisms underlying cerebellar aging and highlights the role of HAPLN2 in age-associated changes in the brain. Author SummaryTo identify age-dependent protein aggregates unrelated to disease, we analyzed the sarkosyl-insoluble proteome of young and aged mouse brains. We discovered that hyaluronan and proteoglycan link protein 2 (HAPLN2), a hyaluronic acid-binding protein existing in the nodal extracellular matrix, accumulated with age. Age-dependently accumulated HAPLN2 formed large protein aggregates that cannot be solubilized by the anionic detergent sarkosyl or by hyaluronidase digestion. In addition, HAPLN2 formed irregularly shaped puncta that were mislocalized from the nodes of Ranvier in the cerebellar white matter of not only aged mice but also aged human brains. Oligomers of full-length HAPLN2 specifically induced microglial activation in vitro and in vivo. Our findings suggest that the accumulation of HAPLN2 aggregates is a new hallmark of brain aging and a possible factor contributing to brain inflammation.

biochemistry↗

bioRxiv2024

Light-guided actin polymerization drives directed motility in protocells

Motility is a hallmark of lifes dynamic processes, enabling cells to actively chase prey, repair wounds, and shape organs. Recreating these intricate behaviors using well-defined molecules remains a major challenge at the intersection of biology, physics, and molecular engineering. Although the polymerization force of the actin cytoskeleton is characterized as a primary driver of cell motility, building a minimal platform to test this process in protocellular systems has proven elusive. The difficulty lies in the daunting task of distilling key components from motile cells and integrating them into model membranes in a physiologically relevant manner. To address this, we developed a method to optically control actin polymerization with high spatiotemporal precision within cell-mimetic lipid vesicles known as giant unilamellar vesicles (GUVs). Within these active protocells, the reorganization of actin networks triggered outward membrane extensions as well as the unidirectional movement of GUVs at speeds of up to 0.43 {micro}m/min, within the range of adherent mammalian cells. Notably, our findings reveal the requirements of both branched and linear actin networks for efficient membrane protrusions. This approach offers a powerful platform for unraveling the intricacies of cell migration, designing synthetic cells with active morphodynamics, and advancing bioengineering applications, such as self-propelled delivery systems and autonomous tissue-like materials.

synthetic biology↗

bioRxiv2023

Proteasomal regulation of ASK family kinases dictates cell fate under hyperosmotic stress

The proteasome has an essential role in proteostasis maintenance and is critical for cell survival under proteotoxic conditions including hyperosmotic stress. However, it is unknown how proteasome activity is linked to cell survival/death under hyperosmotic stress. We have previously reported that apoptosis signal-regulating kinase 3 (ASK3) contributes to cell survival through its inactivation under hyperosmotic stress. 19S regulatory particle subunits of the proteasome were enriched in the ASK3 inactivator candidates identified through our genome-wide small interfering RNA (siRNA) screen. In this study, we demonstrate that the proteasome regulates ASK3 inactivation through its proteolytic activity. Intriguingly, the proteasome inactivates ASK3 via degradation of not ASK3 per se but another ASK family member ASK1 which activates ASK3 in a kinase activity-dependent manner. Furthermore, the elevated ASK1 level under proteasome inhibition sensitizes cells to hyperosmotic stress. These findings suggest that ASK family maintenance links proteasome capacity to cell fate bifurcation under hyperosmotic stress.

molecular biology↗

bioRxiv2023

Acute irradiation causes persistently altered heterogeneity among medullary thymic epithelial cells

The thymus has the ability to regenerate from acute injury caused by radiation, infection, and stressors. In addition to thymocytes, thymic epithelial cells in the medulla (mTECs), which are crucial for T cell self-tolerance by ectopically expressing and presenting thousands of tissue-specific antigens (TSAs), are damaged by these insults and recover thereafter. However, given recent discoveries on the high heterogeneity of mTECs, it remains to be determined whether the frequency and properties of mTEC subsets are restored during thymic recovery from radiation damage. Here we demonstrate that acute total body irradiation with a sublethal dose induces aftereffects on heterogeneity and gene expression of mTECs. Single-cell RNA-sequencing (scRNA-seq) analysis showed that irradiation reduces the frequency of mTECs expressing AIRE, which is a critical regulator of TSA expression, 15 days after irradiation. In contrast, transit-amplifying mTECs (TA-mTECs), which are progenitors of AIRE-expressing mTECs, and Ccl21a-expressing mTECs, were less affected. Interestingly, detailed analysis of scRNA-seq data suggested that the proportion of a unique mTEC cluster expressing Ccl25 and high level of TSAs was severely decreased by irradiation. Overall, we propose that acute irradiation disturbs the heterogeneity and properties of mTECs, and may thereby impair TSA expression for thymic T cell selection.

immunology↗

bioRxiv2022

Diagnosis and early prediction of lymphoma using high-throughput clonality analysis of bovine leukemia virus-infected cells

Bovine leukemia virus (BLV), a retrovirus, infects into B cells of ruminants and causes aggressive leukemia or lymphoma in cattle, enzootic bovine leukosis (EBL). Clonal expansion of BLV-infected cells is a promising marker for early detection and diagnosis of EBL. Recently, we developed rapid amplification of the integration site without interference by genomic DNA contamination (RAISING) and CLOVA, a software to analyze clonality. RAISING-CLOVA could assess the risk of adult T-cell leukemia/lymphoma development in human T-cell leukemia virus-I-infected individuals through its clonality analysis. Thus, we herein examined the performance of RAISING-CLOVA for the clonality analysis of BLV-infected cells and conducted a comprehensive clonality analysis by RAISING-CLOVA in EBL and non-EBL cattle. RAISING-CLOVA successfully distinguished EBL from non-EBL cattle with high sensitivity and specificity. A longitudinal clonality analysis in BLV-infected sheep, an EBL model, also confirmed the effectiveness of BLV clonality analysis with RAISING-CLOVA for early detection of EBL development. Therefore, our study emphasizes the usefulness of RAISING-CLOVA as a routine clinical test for monitoring virus-related cancers.

microbiology↗

bioRxiv2022

Extension of bacterial rDNA sequencing to concurrent epigenetic analysis and its application to 16S meta-epigenetics

Although polymerase chain reaction (PCR) amplification and sequencing of the 16S rDNA region has been used in a wide range of scientific fields, it does not provide DNA methylation information. We describe a simple add-on method to investigate 5-methylcytosine residues in the bacterial 16S rDNA region from clinical samples or flora. Single-stranded bacterial DNA after bisulfite conversion was preferentially amplified with multiple displacement amplification (MDA) at pH neutral, and the 16S rDNA region was analyzed using nested bisulfite PCR and sequencing. 16S rDNA bisulfite sequencing can provide clinically important bacterial DNA methylation status concurrently with intact 16S rDNA sequence information. We used this approach to identify novel methylation sites and a methyltransferase (M. MmnI) in Morganella morganii. Next, we analyzed bacterial flora from clinical specimens of small amount and identified different methylation motifs among Enterococcus faecalis strains. The method developed here, referred to as "add-on" to the conventional 16S rDNA analysis, is the most clinically used bacterial identification genetic test, which provides additional information that could not be obtained with the conventional method. Since the relationship between drug resistance in bacteria and DNA methylation status has been reported, bacterial epigenetic information would be useful in clinical testing as well. Our analysis suggests that M. MmnI has a promotive effect on erythromycin resistance. 16S rDNA bisulfite PCR and sequencing coupled with MDA at pH neutral is a useful add-on tool for analyzing 16S meta-epigenetics.

microbiology↗

bioRxiv2020

PD-L1 expression in equine malignant melanoma and functional effects of PD-L1 blockade

Programmed death-1 (PD-1) is an immunoinhibitory receptor expressed on exhausted T cells during chronic illness. Interaction of PD-1 with its ligand PD-ligand 1 (PD-L1) delivers inhibitory signals and impairs proliferation, cytokine production, and cytotoxicity of T cells. We reported that the PD-1/PD-L1 pathway is closely associated with T-cell exhaustion and disease progression in bovine chronic infections and canine tumors. Moreover, we found that blocking antibodies targeting PD-1 and PD-L1 restore T-cell functions and may be used in immunotherapy in cattle and dogs. However, the immunological role of the PD-1/PD-L1 pathway remains unclear for chronic equine diseases, including tumors. In this study, we identified nucleotide sequences of equine PD-1 (EqPD-1) and PD-L1 (EqPD-L1) and investigated the role of anti-bovine PD-L1 monoclonal antibodies (mAbs) against EqPD-L1 using in vitro assays. We also evaluated the expression of PD-L1 in tumor tissues of equine malignant melanoma (EMM). The amino acid sequences of EqPD-1 and EqPD-L1 share a high identity and similarity with homologs from other mammalian species. Two clones of the anti-bovine PD-L1 mAbs recognized EqPD-L1 in flow cytometry, and one of these cross-reactive mAbs blocked the binding of equine PD-1/PD-L1. Importantly, PD-L1 expression was confirmed in EMM tumor tissues by immunohistochemistry. A cultivation assay revealed that PD-L1 blockade enhanced the production of Th1 cytokines in equine immune cells. These results suggest that our anti-PD-L1 mAbs may be useful for investigating the expression and role of the equine PD-1/PD-L1 pathway. Further research is required to discover the immunological role of PD-1/PD-L1 in chronic equine diseases and elucidate a future application in immunotherapy for horse.

immunology↗

bioRxiv2019

Complement- and inflammasome-mediated autoinflammation-paroxysmal nocturnal hemoglobinuria

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hematopoietic stem cell disorder characterized by complement-mediated hemolysis and thrombosis, and bone marrow failure. Affected cells harbor somatic mutation in X-linked PIGA gene, essential for the initial step in glycosylphosphatidylinositol (GPI) biosynthesis. Loss of GPI biosynthesis results in defective cell-surface expression of GPI-anchored complement regulators CD59 and DAF. The affected stem cells generate many abnormal blood cells after clonal expansion, which occurs under bone marrow failure. Here, we report the mechanistic basis of a disease entity, autoinflammation-paroxysmal nocturnal hemoglobinuria (AIF-PNH), caused by germline mutation plus somatic loss of PIGT on chromosome 20q. A region containing maternally imprinted genes implicated in clonal expansion in 20q-myeloproliferative syndromes was lost together with normal PIGT from paternal chromosome 20. Taking these findings together with a lack of bone marrow failure, the mechanisms of clonal expansion in AIF-PNH appear to differ from those in PNH. AIF-PNH is characterized by intravascular hemolysis and recurrent autoinflammation, such as urticaria, arthralgia, fever and aseptic meningitis. Consistent with PIGTs essential role in synthesized GPIs attachment to precursor proteins, non-protein-linked free GPIs appeared on the surface of PIGT-defective cells. PIGT-defective THP-1 cells accumulated higher levels of C3 fragments and C5b-9 complexes, and secreted more IL-1{beta} than PIGA-defective cells after activation of the complement alternative pathway. IL-1{beta} secretion was dependent upon C5b-9 complexes, accounting for the effectiveness of the anti-C5 drug eculizumab for both intravascular hemolysis and autoinflammation. These results suggest that free GPIs enhance complement activation and inflammasome-mediated IL-1{beta} secretion.

immunology↗

bioRxiv2017

Sugar intake elicits a small-scale search behavior in flies and honey bees that involves capabilities found in large-scale navigation

Social insects, particularly bees and ants, show exceptional large-scale navigational skills to find and carry back food to their nests. Honey bees further evolved a symbolic communication to direct nest mates to attractive food sources. Till now it is unclear how these capabilities evolved. Sixty years ago, Vincent Dethier demonstrated that a small-scale sugar-elicited search behavior identified in flies shows remarkable similarities with honey bee dance behavior. Those findings suggested that both behaviors are based on common mechanisms and are likely evolutionary related. We now present for the first time a detailed comparison of the sugar-elicited search behavior in Drosophila melanogaster and Apis mellifera. In both species, intake of sugar elicits a complex of searching responses. The most obvious response was an increase in turning frequency, but more importantly we found that flies and honey bees returned to the location of the sugar drop. They even returned to the food location when we prevented them from using visual and chemosensory cues indicating that this small scale local search involves path integration mechanisms. Finally, we show that visual landmarks presented in the vicinity of the sugar drop affected the search trajectory and in honey bees the sugar intake induced learning of landmarks. Together, our experiments indicate that the sugar-elicited local search exhibits two major behavioral capabilities of large-scale navigation, path integration and landmark orientation.\n\nSignificance StatementTo search for food social insects evolved sophisticated strategies of spatial orientation and large-scale navigation. We now show that even a small-scale local search behavior in solitary flies and social honey bees involves path integration and landmark learning two major mechanisms of large-scale navigation. We propose that in the future sugar-elicited local search can be used to identify neural circuits involved in navigation, path integration, and landmark learning.

animal behavior and cognition↗