Biology subjects

Miyoshi, T.

Publications and source records attributed to Miyoshi, T..

9 recordsLinked to original sources

bioRxiv2026

Dual-color fluorescent reporters resolve Alu and LINE-1 retrotransposition in single cells

LINE-1 (L1) is transcribed by RNA polymerase (Pol) II, whereas the non-coding Short INterspersed Element (SINE) Alu is transcribed by Pol III and exploits L1 ORF2p for mobilization. Conventional L1 retrotransposition reporters rely on spliceosome-dependent introns and cannot directly monitor Alu retrotransposition. Here, we developed EGFPTet, a fluorescent SINE reporter in which EGFP is interrupted by a Tetrahymena thermophila group I self-splicing intron. EGFPTet enabled flow cytometry, fluorescence microscopy, cell sorting, and time-course analysis of Alu mobilization without colony formation. Reporter activation required ORF2p reverse transcriptase activity, and recovered Alu-EGFPTet insertions displayed retrotransposition hallmarks. EGFPTet quantified Alu retrotransposition across cell lines and host-factor perturbations and was repurposed to detect mobilization of the phylogenetically distinct mouse B2 SINE. Defined amino acid substitutions generated an mTurquoise2 derivative without redesigning the self-splicing junctions, allowing dual-color analysis of Alu trans-mobilization and L1 cis retrotransposition in individual cells. In this assay, wild-type L1 supported a higher Alu retrotransposition frequency than the ORF1p-mutant L1. Despite their shared dependence on ORF2p, Alu co-expression did not significantly reduce L1 retrotransposition, and Alu/L1 dual-positive counts exceeded independence-based expectations, indicating preferential co-occurrence of the two reporter signals. EGFPTet provides a fluorescent platform for investigating the shared and distinct cellular conditions that support SINE and L1 mobilization.

molecular biology↗

bioRxiv2026

The Ets family transcription factor EHF suppresses senescence-associated inflammatory responses

Cellular senescence is a tumor-suppressive program characterized by irreversible growth arrest; however, senescent cells can also promote inflammation and alter the tumor microenvironment through the senescence-associated secretory phenotype (SASP). Although SASP induction is regulated by pathways such as p38/NF-{kappa}B/I{kappa}B{zeta}, the mechanisms that restrain excessive or persistent SASP remain largely unknown. Here, we investigated the role of the Ets family transcription factor EHF in SASP regulation during cellular senescence. In IMR-90 human fibroblasts undergoing oncogene-induced senescence, EHF expression was upregulated after the onset of canonical senescence phenotypes. EHF knockdown did not substantially affect senescence establishment but increased SASP-related gene expression. Conversely, overexpression of full-length EHF suppressed SASP-related gene induction during senescence, whereas an ETS-domain-deficient EHF mutant failed to do so, suggesting that this EHF-mediated SASP suppression requires its DNA-binding domain. Furthermore, knockdown of NFKBIZ, which encodes I{kappa}B{zeta} and is induced downstream of NF-{kappa}B signaling, reduced EHF expression during senescence; however, NFKBIZ overexpression increased EHF and SASP-related gene expression. These results link EHF induction to the p38/NF-{kappa}B/I{kappa}B{zeta} inflammatory axis and support a model in which the inflammatory pathway that induces SASP also engages EHF as a negative regulator of SASP. Finally, conditioned medium from senescent cells promoted HCT116 cancer cell migration, and this activity showed a further increase after EHF knockdown. These findings suggest that EHF suppresses senescence-associated inflammatory responses and may function as a senomorphic effector that attenuates SASP-related inflammation without substantially affecting senescence establishment.

molecular biology↗

bioRxiv2025

Kalman-filter Force Inference: an estimation framework for cellular forces from temporal evolution of epithelial morphogenesis

Epithelial morphogenesis is orchestrated by cellular forces, such as cell junctional tension and cellular pressure. Therefore, elucidating the spatiotemporal distribution of these forces and their dynamics is paramount to understand morphogenesis during development. Over the past decade, various force inference methods have been proposed to estimate cellular forces based on the shape and geometry of cells within epithelial tissues. Most of these methods were developed under the assumption of static force balance, which neglects the cell deformation, thereby limiting their applicability to tissue undergoing dynamic deformation. To address this issue, we develop a novel method to accurately infer cellular forces from time-lapse imaging data of epithelial deformation, without relying on the static force balance assumption. Our method is based on two fundamental assumptions. First, the forces exerted by cells are balanced with dissipative forces (such as viscous and frictional forces) at each vertex arising from cell deformation, referred to as the dynamic force balance. Second, cellular forces vary smoothly over time. By formalizing these assumptions using a Bayesian framework, we construct a new approach of force inference, named Kalman-filter Force Inference (KFI). The effectiveness of our method is evaluated using synthetic data of a deforming tissue generated by numerical simulations of the cell vertex model. The results demonstrate the accurate estimation of cellular force dynamics. Furthermore, our systematic evaluation reveals the capability of our method to accurately estimate cellular forces across tissues with diverse mechanical parameters. Finally, we assessed the robustness of our method to observation noise. We anticipate that Kalman-filter Force Inference will broaden the applicability of force inference techniques and significantly contribute to our understanding of epithelial mechanics.

biophysics↗

bioRxiv2025

The interferon-stimulated gene product HERC5 inhibits human LINE-1 retrotransposition with an ISGylation-independent mechanism

Text AbstractMobilization of long interspersed element-1 (LINE-1 or L1) compromises genome stability and can cause sporadic genetic disease. Accordingly, cells have evolved multiple mechanisms to restrict L1 retrotransposition. Several interferon-stimulated genes (ISGs) that interact with cytoplasmic L1 ribonucleoproteins (RNPs), which contain the L1-encoded proteins ORF1p and ORF2p, have been identified as suppressors of L1 retrotransposition. We previously reported that the ISG protein HECT and RLD domain containing E3 ubiquitin-protein ligase 5 (HERC5) efficiently inhibits L1 retrotransposition. While HERC5 is known to restrict numerous viruses through ISGylation, how HERC5 inhibits L1 remains to be elucidated. Here, we show that HERC5 inhibits L1 retrotransposition through an ISGylation-independent mechanism. HERC5 interacts with L1 RNA and reduces ORF1p levels, a function that requires the ORF1 coding sequence. We further demonstrate that HERC5 decreases L1 translation efficiency and prevents the assembly of L1 RNPs. Our comparative analysis suggests that HERC5 may have acquired its L1-inhibitory function during the evolution of the small HERC family. These findings uncover a previously unidentified mechanism by which an ISG protein recognizes and inhibits L1 and suggest a role for HERC5 as an evolutionarily adapted restriction factor that expands the repertoire of cellular defenses against retrotransposons.

molecular biology↗

bioRxiv2024

Dynamic Kidney Organoid Microphysiological Analysis Platform

Kidney organoids, replicating human development, pathology, and drug responses, are a promising model for advancing bioscience and pharmaceutical innovation. However, reproducibility, accuracy, and quantification challenges hinder their broader utility for advanced biological and pharmaceutical applications. Herein, we present a dynamic kidney organoid microphysiological analysis platform (MAP), designed to enhance organoid modeling and assays within physiologically relevant environments, thereby expanding their utility in advancing kidney physiology and pathology research. First, precise control of the dynamic microenvironment in MAP enhances the ability to fine-tune nephrogenic intricacies, facilitating high-throughput and reproducible human kidney organoid development. Also, MAPs miniaturization of kidney organoids significantly advances pharmaceutical research by allowing for detailed analysis of entire nephron segments, which is crucial for assessing the nephrotoxicity and safety of drugs. Furthermore, the MAPs application in disease modeling faithfully recapitulates pathological development and functions as a valuable testbed for therapeutic exploration in polycystic kidney diseases. We envision the kidney organoid MAP enhancing pharmaceutical research, standardizing processes, and improving analytics, thereby elevating the quality and utility of organoids in biology, pharmacology, precision medicine, and education.

bioengineering↗

bioRxiv2024

Live-cell single-molecule fluorescence microscopy for protruding organelles reveals regulatory mechanisms of MYO7A-driven cargo transport in stereocilia of inner ear hair cells

Stereocilia are unidirectional F-actin-based cylindrical protrusions on the apical surface of inner ear hair cells and function as biological mechanosensors of sound and acceleration. Development of functional stereocilia requires motor activities of unconventional myosins to transport proteins necessary for elongating the F-actin cores and to assemble the mechanoelectrical transduction (MET) channel complex. However, how each myosin localizes in stereocilia using the energy from ATP hydrolysis is only partially understood. In this study, we develop a methodology for live-cell single-molecule fluorescence microscopy of organelles protruding from the apical surface using a dual-view light-sheet microscope, diSPIM. We demonstrate that MYO7A, a component of the MET machinery, traffics as a dimer in stereocilia. Movements of MYO7A are restricted when scaffolded by the plasma membrane and F-actin as mediated by MYO7As interacting partners. Here, we discuss the technical details of our methodology and its future applications including analyses of cargo transportation in various organelles.

cell biology↗

bioRxiv2022

The interferon stimulated gene-encoded protein HELZ2 inhibits human LINE-1 retrotransposition and LINE-1 RNA-mediated type I interferon induction

Some interferon stimulated genes (ISGs) encode proteins that inhibit LINE-1 (L1) retrotransposition. Here, we used immunoprecipitation followed by liquid chromatography-tandem mass spectrometry to identify proteins that associate with the L1 ORF1-encoded protein (ORF1p) in ribonucleoprotein particles. Three ISG proteins that interact with ORF1p inhibit retrotransposition: HECT and RLD domain containing E3 ubiquitin-protein ligase 5 (HERC5); 2-5-oligoadenylate synthetase-like (OASL); and helicase with zinc finger 2 (HELZ2). HERC5 destabilizes ORF1p, but does not affect its cellular localization. OASL impairs ORF1p cytoplasmic foci formation. HELZ2 recognizes sequences and/or structures within the L1 5UTR to reduce L1 RNA, ORF1p, and ORF1p cytoplasmic foci levels. Overexpression of WT or reverse transcriptase-deficient L1s led to a modest induction of IFN- expression, which was abrogated upon HELZ2 overexpression. Notably, IFN- expression was enhanced upon overexpression of an ORF1p RNA binding mutant, suggesting ORF1p binding might protect L1 RNA from "triggering" IFN- induction. Thus, ISG proteins can inhibit retrotransposition by different mechanisms.

molecular biology↗

bioRxiv2020

Epigenetic transcriptional reprogramming by WT1 mediates a repair response during podocyte injury

In the context of human disease, the mechanisms whereby transcription factors reprogram gene expression in response to injury are not well understood. This is particularly true in kidney podocytes, injury to which is the common and initial event in many processes that lead End Stage Kidney Disease. WT1 is a master regulator of gene expression in podocytes, binding nearly all genes known to be crucial for maintenance of the glomerular filtration barrier. Here, using purified populations of podocytes and glomeruli, we investigated WT1-mediated transcriptional reprogramming during the course of podocyte injury. Using the Adriamycin murine model of Focal Segmental Glomerulosclerosis, we discovered that podocyte injury led to increased intensity of WT1 binding and to the acquisition of new WT1 binding sites, both at previously identified target genes and at newly bound target genes, providing mechanistic insight on the transcriptional response to injury. We also observed a previously unrecognized transient increase in expression of WT1 target genes in both mice and human kidney organoids. Together, these features appear to constitute an attempt to repair the glomerular filtration barrier after podocyte injury. At later stages of injury, when proteinuria became severe, there was greatly decreased WT1 binding to most target genes. Furthermore, WT1 appeared to be required to maintain active chromatin marks at its target genes. These active marks were converted to repressive marks after loss of WT1 or Adriamycin-induced injury. This response to injury suggests that there may be a potential window of opportunity for repairing podocyte injury as a treatment for glomerular disease in humans.

molecular biology↗

bioRxiv2019

Short-term effects on brain functional network caused by focused-attention meditation revealed by Tucker3 clustering on graph theoretical metrics

This study examines the short-term effects of focused-attention meditation on functional brain state in novice meditators. There are a number of feature metrics for functional brain states, such as functional connectivity, graph theoretical metrics, and amplitude of low frequency fluctuation (ALFF). It is necessary to choose appropriate metrics and also to specify the region of interests (ROIs) from a number of brain regions. Here, we use a Tucker3 clustering method, which simultaneously selects the feature vectors (graph theoretical metrics and fractional ALFF) and the ROIs that can discriminate between resting and meditative states based on the characteristics of the given data. In this study, breath-counting meditation, one of the most popular forms of focused-attention meditation, was used and brain activities during resting and meditation states were measured by functional magnetic resonance imaging. The results indicated that the clustering coefficients of eight brain regions tended to increase through the meditation. Our results reveal that short-term effects of breath-counting meditation can be explained by network density changes in these eight brain regions.

neuroscience↗