Biology subjects

Kubota, M.

Publications and source records attributed to Kubota, M..

8 recordsLinked to original sources

bioRxiv2026

Galectin and Myc enable cochlear progenitor expansion in vitro and in vivo

The neonatal cochlear epithelium harbors regenerative capacity, attributable to the transient greater epithelial ridge (GER). After injury, GER cells re-enter the cell cycle, migrate into the damaged organ of Corti, and differentiate into sensory or supporting cells in vivo; in culture, they proliferate to form inner ear organoids. The mechanisms underlying this competence remain unclear. Here, we generated organoids from mouse GER cells and performed single-cell transcriptomics at organoid initiation. Analysis revealed extracellular matrix reorganization with prominent involvement of galectins. Pharmacological inhibition reduced organoid growth, whereas overexpression of galectin-1 or Myc enhanced growth, identifying both factors as regulators of cochlear progenitor expansion. Moreover, Myc overexpression conferred organoid-forming capacity in post-neonatal cochlear epithelial cells after hearing onset, thereby extending the time window for regenerative competence. In vivo, organ of Corti ablation induced galectin-1 upregulation and GER proliferation, suppressed by galectin-1 blockade, establishing a model to rekindle proliferative potential in mature cochlear cells.

neuroscience↗

bioRxiv2026

A reabsorption mechanism in Drosophila renal tubules maintains amino acid homeostasis

The kidney plays a central role in maintaining systemic homeostasis by regulating the excretion of metabolic compounds. In mammals, this is achieved through filtration followed by selective reabsorption, whereas insect Malpighian tubules (MTs) are thought to function primarily through active secretion. Although reabsorption of ions and water has been described in MTs, whether these tissues regulate metabolite balance through reabsorption remains unclear. Here, we uncover an amino acid (AA) reabsorption mechanism in Drosophila MTs. Quantification of AAs in excreta reveals that AA excretion changes rapidly and markedly in response to dietary protein levels, while systemic AA levels remain stable. Using ex vivo assays, we show that MTs actively transport AAs. We identify the AA transporter Slimfast (Slif), expressed in the lower tubule, as a key mediator required to maintain systemic levels of basic AAs. These findings reveal a previously unrecognised reabsorption-based strategy in insect renal tubules that functionally parallels mammalian kidney physiology, despite their distinct organisation. Our study demonstrates that AA excretion forms an additional layer to achieve metabolic homeostasis.

physiology↗

bioRxiv2025

Mitotic phosphorylation of ADAR1 regulates its centromeric localization and is required for faithful mitotic progression

Adenosine Deaminase Acting on RNA 1 p110 (ADAR1p110), the constitutively expressed nuclear isoform of the RNA-editing enzyme ADAR1, plays well-established roles in adenosine-to-inosine RNA editing, but its functions during mitosis remain poorly defined. Here, we identify ADAR1p110 as a chromatin-associated factor essential for mitotic chromosome segregation and cell viability. Depletion of ADAR1 caused metaphase arrest, DNA damage accumulation, and apoptosis. Co-immunoprecipitation experiments revealed that ADAR1p110 physically interacts with Structural Maintenance of Chromosome 3 (SMC3), a core component of the cohesin complex. Genome-wide DNA immunoprecipitation sequencing (DIP-seq) showed that ADAR1p110 selectively binds centromeric -satellite DNA during mitosis, a finding validated by DNA immunoprecipitation quantitative polymerase chain reaction (DIP-qPCR). Complementary DNA-RNA immunoprecipitation sequencing (DRIP-seq) revealed that RNA:DNA hybrids (R-loops) are enriched in centromeric regions during mitosis and are further augmented by ADAR1 overexpression. We identified serine 614 (S614) as a key mitosis-specific phosphorylation site on ADAR1p110, and demonstrated that this post-translational modification is essential for its chromatin recruitment, stability, and mitotic function. Rescue experiments using phospho-mimetic mutants (S614D and 3xD) successfully restored mitotic progression in ADAR1-deficient cells, whereas non-phosphorylatable variants failed to do so. These results reveal that ADAR1p110 is phosphorylated in a cell cycle-dependent manner and functions at the intersection of post-transcriptional and post-translational regulation. By coordinating R-loop recognition at centromeres with chromosome cohesion, ADAR1p110 safeguards genome integrity during mitosis. Our findings uncover a previously uncharacterized mechanism through which a canonical RNA-editing enzyme contributes to chromosomal dynamics independent of its deaminase activity.

molecular biology↗

bioRxiv2025

Transcriptional control of T cell tissue adaptation and effector function in infants and adults

The first years of life are essential for the development of memory T cells, which rapidly populate the bodys diverse tissue sites during infancy. However, the degree to which tissue memory T cell responses in early life reflect those during adulthood is unclear. Here, we use single cell RNA-sequencing of resting and ex vivo activated T cells from lymphoid and mucosal tissues of infant (aged 2-9 months) and adult (aged 40-65 years) human organ donors to dissect the transcriptional programming of memory T cells over age. Infant memory T cells demonstrate a unique stem-like transcriptional profile and tissue adaptation program, yet exhibit reduced activation capacity and effector function relative to adults. Using CRISPR-Cas9 knockdown, we define Helios (IKZF2) as a critical transcriptional regulator of the infant-specific tissue adaptation program and restricted effector state. Our findings reveal key transcriptional mechanisms that control tissue T cell fate and function in early life.

immunology↗

bioRxiv2025

Structural insights into heterohexameric assembly of epilepsy-related ligand-receptor complex LGI1-ADAM22

Leucine-rich glioma-inactivated 1 protein (LGI1) is a secreted neuronal protein consisting of the N-terminal leucine-rich repeat (LRR) and C-terminal epitempin repeat (EPTP) domains. LGI1 is linked to epilepsy, a neurological disorder that can be caused by genetic mutations of genes regulating neuronal excitability (e.g., voltage- or ligand-gated ion channels). ADAM22 is a membrane receptor that binds to LGI1 extracellularly and interacts with AMPA-type glutamate receptors via PSD-95 intracellularly to maintain normal synaptic signal transmission. Structural analysis of the LGI1-ADAM22 complex is important for understanding the molecular mechanism of epileptogenesis and developing new therapies against epilepsy. We previously reported the crystal structure of a 2:2 complex consisting of two molecules of LGI1 and two molecules of the ADAM22 ectodomain (ECD), which is suggested to bridge neurons across the synaptic cleft. On the other hand, multiangle light scattering, small-angle X-ray scattering, and cryo-EM analyses have suggested the existence of a 3:3 complex consisting of three molecules of LGI1 and three molecules of ADAM22. In the previous cryo-EM analysis, many observed particles were in a dissociated state, making it difficult to determine the three-dimensional (3D) structure of the 3:3 complex. In this study, we stabilized the 3:3 LGI1-ADAM22ECD complex using chemical crosslinking and determined the cryo-EM structures of the LGI1LRR-LGI1EPTP- ADAM22ECD and 3:3 LGI1-ADAM22ECD complexes at 2.78 [A] and 3.79 [A] resolutions, respectively. Furthermore, high-speed atomic force microscopy (HS-AFM) visualized the structural features and flexibility of the 3:3 LGI1-ADAM22ECD complex in solution. We discuss new insights into the interaction modes of the LGI1-ADAM22 higher-order complex and the structural properties of the 3:3 LGI1-ADAM22 complex. SignificanceThe neuronal secretory protein Leucine-rich glioma-inactivated 1 (LGI1) and its receptor protein ADAM22 play a critical role in maintaining normal synaptic signal transmission. Genetic mutations in LGI1 are linked to epilepsy. Structural analysis of the LGI1-ADAM22 complex is crucial for understanding the molecular mechanisms of epileptogenesis and for developing targeted treatments. In this study, we determined the cryo-EM structure of the heterohexameric LGI1-ADAM22 complex and visualized the dynamics of this complex by high-speed atomic force microscopy.

biochemistry↗

bioRxiv2024

Multimodal profiling reveals tissue-directed signatures of human immune cells altered with age

The immune system comprises multiple cell lineages and heterogeneous subsets found in blood and tissues throughout the body. While human immune responses differ between sites and over age, the underlying sources of variation remain unclear as most studies are limited to peripheral blood. Here, we took a systems approach to comprehensively profile RNA and surface protein expression of over 1.25 million immune cells isolated from blood, lymphoid organs, and mucosal tissues of 24 organ donors aged 20-75 years. We applied a multimodal classifier to annotate the major immune cell lineages (T cells, B cells, innate lymphoid cells, and myeloid cells) and their corresponding subsets across the body, leveraging probabilistic modeling to define bases for immune variations across donors, tissue, and age. We identified dominant tissue-specific effects on immune cell composition and function across lineages for lymphoid sites, intestines, and blood-rich tissues. Age-associated effects were intrinsic to both lineage and site as manifested by macrophages in mucosal sites, B cells in lymphoid organs, and T and NK cells in blood-rich sites. Our results reveal tissue-specific signatures of immune homeostasis throughout the body and across different ages. This information provides a basis for defining the transcriptional underpinnings of immune variation and potential associations with disease-associated immune pathologies across the human lifespan.

immunology↗

bioRxiv2021

Molecular programs of fibrotic change in aging human lung

Aging is associated with both overt and subclinical lung fibrosis, which increases risk for mortality from viruses and other respiratory pathogens. The molecular programs that induce fibrosis in the aging lung are not well understood. To overcome this knowledge gap, we undertook multimodal profiling of distal lung samples from healthy human donors across the lifespan. Telomere shortening, a cause of cell senescence and fibrosis, was progressive with age in a sample of 86 lungs and was associated with foci of DNA damage. Bulk RNA sequencing confirmed activation of cellular senescence and pro-fibrotic pathways as well as genes necessary for collagen processing with increasing age. These findings were validated in independent datasets for lung and sun-exposed skin, but not other organs including heart, liver and kidney. Cell type deconvolution analysis revealed a progressive loss of lung epithelial cells and an increasing proportion of fibroblasts. Consistent with the observed pro-fibrotic transcriptional profile, second harmonic imaging demonstrated increased density of interstitial collagen in aged human lungs. Furthermore, regions of parenchymal fibrosis were associated with decreased alveolar expansion and surfactant secretion. These findings reveal the transcriptional and structural features of fibrosis and associated physiologic impairments in normal lung aging.

genomics↗

bioRxiv2018

Overlapping but asymmetrical relationships between schizophrenia and autism revealed by brain connectivity

(Abstract included 248 words)Although the relationship between schizophrenia spectrum disorder (SSD) and autism spectrum disorder (ASD) has long been debated, it has not yet been fully elucidated. To address this issue, we took advantage of dual (ASD and SSD) classifiers that discriminate patients from their controls based on resting state brain functional connectivity. An SSD classifier using sophisticated machine-learning algorithms that automatically selected SSD- specific functional connections was applied to Japanese datasets including adult patients with SSD in a chronic stage. We demonstrated good performance of the SSD classification for independent validation cohorts. The generalizability was tested by USA and European cohorts in a chronic stage, and one USA cohort including first episode schizophrenia. The specificity was tested by two adult Japanese cohorts of ASD and major depressive disorder, and one European cohort of attention-deficit hyperactivity disorder. The weighted linear summation of the classifiers functional connections constituted the biological dimensions representing neural liability to the disorders. Our previously developed robust ASD classifier constituted the ASD dimension. Distributions of individuals with SSD, ASD and healthy controls were examined on the SSD and ASD biological dimensions. The SSD and ASD populations exhibited overlapping but asymmetrical patterns on the two biological dimensions. That is, the SSD population showed increased liability on the ASD dimension, but not vice versa. Furthermore, the two dimensions were correlated within the ASD population but not the SSD population. Using the two biological dimensions based on resting-state functional connectivity enabled us to quantify and visualize the relationships between SSD and ASD.

neuroscience↗