Biology subjects

Higashi, T.

Publications and source records attributed to Higashi, T..

11 recordsLinked to original sources

bioRxiv2026

A novel metalloprotease family discovered through a tardigrade

Tardigrades possess numerous proteins with poorly understood functions, including candidates proposed to have been acquired through horizontal gene transfer. Here, we characterized RvY_14254 from Ramazzottius varieornatus, previously designated ATPM5, whose homologs are widely distributed across bacteria and eukaryotes but remain largely uncharacterized. We determined the crystal structure of the protein, hereafter termed RvPeti, at 1.35 [A] resolution. RvPeti adopts a compact, C-shaped architecture with no close structural homologs in the Protein Data Bank and contains a conserved His-Asp-Asp (HDD) metal-binding motif within a deep surface pocket. Structural comparison revealed partial similarity of the active-site architecture to that of metalloproteases, including pseudoalterin. Anomalous diffraction analyses demonstrated that the HDD site can accommodate Zn2+ and Mn2+. Biochemical assays showed that RvPeti exhibits Zn2+ dependent proteolytic activity, which was strongly reduced by a metal chelating reagent and by alanine substitutions of the metal-coordinating residues. In contrast, only weak esterase activity was detected, with the substrates tested. These results identify RvPeti as a founding member of a previously uncharacterized family of metalloproteases and provide a structural framework for investigating the physiological and evolutionary roles of this widespread protein family.

biochemistry↗

bioRxiv2026

BMP4 Restricts Axonal Ramification and Bouton Density in Cholinergic Projections from the Basal Forebrain

Axonal projections and precise synapse formation are essential for the establishment of functional neural circuits, and their dysregulation contributes to various psychiatric and neurodegenerative disorders. Although numerous extracellular cues secreted from surrounding tissues regulate axon guidance and synapse formation, the mechanisms by which neurons locally regulate terminal patterning within their projection fields remain poorly understood. Bone morphogenetic protein 4 (BMP4) is a classical morphogen that is involved in early neural tube patterning during development. However, its postnatal function remains unclear. Previous in vitro studies using hippocampal glutamatergic neurons demonstrated that BMP4 can act locally on axons to suppress synapse formation, raising the possibility that BMP4 regulates terminal differentiation after target innervation. Here, we investigated the role of BMP4 in postnatal cholinergic circuit development using cholinergic neuron-specific Bmp4 conditional knockout (Bmp4 cKO) mice, with adult analyses performed in males. Basal forebrain cholinergic neurons project extensively throughout the neocortex and are essential for higher brain functions, including arousal and attention. Bmp4 cKO mice exhibited increased cholinergic axon terminals in the neocortex, hippocampus, and amygdala, as well as increased axonal ramification in the neocortex without affecting cytoarchitectonic organization or cholinergic neuron numbers in the basal forebrain. These findings demonstrate that BMP4 functions as a negative regulator of cholinergic terminal formation and axonal arborization, suggesting that BMP4 may act locally to restrict cholinergic axonal expansion during neural circuit maturation.

neuroscience↗

bioRxiv2026

Symmetry and force response of cohesin loop extrusion are determined by diffusion of its motor and anchor domains

Cohesin, a Structural Maintenance of Chromosomes (SMC) complex is thought to organize genomes by generating DNA loops, yet the molecular basis of loop extrusion remains incompletely understood. Here, we show that a single S. pombe cohesin complex extrudes DNA loops both symmetrically or asymmetrically, depending on the external force applied to DNA and that the mode and speed are controlled by stochastic switching of the cohesin motor between driving and diffusive states. We directly measured the weak forces generated during cohesin-mediated loop extrusion while simultaneously visualizing DNA loops and found that forces as low as 0.05 pN pull DNA out of cohesin loop without disrupting cohesin-DNA interaction. We further identified the Scc3 subunit as a diffusive DNA anchor required for loop extrusion and showed that its physical separation from the motor domain by unstructured regions of Scc1 promotes loop-extrusion initiation. Together, our results support a model in which cohesin loop extrusion is limited and tuned by diffusive motion of both anchor and motor modules, providing a mechanistic framework for how weak and diffusive cohesin-DNA interactions could modulate cohesin function in chromatin organization.

biophysics↗

bioRxiv2026

Sidekick2 facilitates multiciliated cell penetration through multicellular adherens junctions

Multicellular vertices are important cellular interfaces for mediating tissue dynamics across the epithelium. Here, we report that expression of the vertebrate adhesion protein, Sidekick2 (Sdk2), localizes to vertebrate multicellular adherens junctions in the embryonic Xenopus laevis epithelium. Using the developmental process of radial intercalation, we find Sdk2 facilitates multiciliated cell penetrance into the outer epithelium at multicellular junctions. With fluorescence recovery after photobleaching, we characterize Sdk2 dynamics at multicellular and bicellular junctions and interrogate how the intracellular and extracellular domains of Sdk2 contribute to these dynamics. We observe that overexpression of the highly conserved C terminal intracellular domain alone but not the extracellular domain alone is sufficient to act as a dominant negative, delaying radial intercalation. These results implicate Sdk2s intracellular interactions as critical for regulating the junctional remodeling required during radial intercalation. Ultimately, our findings implicate Sdk2 as a component of multicellular vertices that aids the governance of epithelial integrity.

cell biology↗

bioRxiv2025

PAK4 promotes vertex remodeling to maintain epithelial integrity and barrier function

Cell-cell junctions are essential for epithelial integrity and barrier function, but the mechanisms regulating their remodeling remain unclear. Here, we investigated the role of the junctional kinase PAK4 in vertex remodeling. PAK4 localized to apical junctions and accumulated at multicellular vertices in MCDK cells and Xenopus embryos. Inhibition or knockout of PAK4 increased higher-order vertices, caused junctional discontinuities, and impaired barrier function in MDCK cells. PAK4 recruitment required the scaffolding protein Afadin. Afdn-KO cells exhibited severe junctional defects and reduced barrier function. Artificial targeting of PAK4 in Afdn-KO cells partially restored junctional continuity and barrier function. In Xenopus embryos, live imaging showed dynamic PAK4 accumulation at remodeling vertices, and PAK4 inhibition hindered resolution of multicellular vertices. Expression of an amino-terminal fragment (PAK4-NT) impaired remodeling and induced cytokinetic failure. Live imaging in Xenopus revealed barrier leakage at multicellular vertices upon PAK4 inhibition. These findings indicate that PAK4 and Afadin cooperate to maintain epithelial integrity and barrier function by promoting vertex remodeling. SummaryAdhikary et al. show that PAK4, recruited to epithelial junctions by Afadin, drives dynamic vertex remodeling in cells and embryos. Inhibiting PAK4 disrupts tissue packing and compromises junctional continuity and barrier function, implicating PAK4 and Afadin as critical partners in vertex remodeling to preserve tissue integrity.

cell biology↗

bioRxiv2025

Class I myosins direct circumferential F-actin flows to define cell chirality

Eukaryotic cells possess intrinsic chirality in their structure, motility, and intracellular dynamics, which are designated cell chirality. Cell chirality participates in the left-right asymmetric morphogenesis and tissue integrity. However, the mechanisms of cell chirality formation remain elusive. In Drosophila, two evolutionarily conserved myosin I genes, Myosin 1D (Myo1D) and Myosin 1C (Myo1C), respectively, dictate the dextral and sinistral chirality of the cells and body. Here, we reported that Myo1D and Myo1C respectively directed the clockwise and counterclockwise circumferential flow of F-actin in Drosophila macrophages. Both induced the corresponding circular cytoplasm flows and depended on Myosin2 (Myo2). In a modified in vitro motility assay using near-physiological actin concentrations, Myo1D triggered the self-organization of the F-actin ring (chiral F-actin ring) that rotated clockwise; conversely, Myo1C induced the random flow of F-actin. The chiral F-actin ring implied that the F-actin bundle was parallelly and annularly polarized concerning its barbed pointed end. Considering that Myo1D and Myo1C are localized to the dorsal plasma membrane of macrophages, Myo1D and Myo1C might organize the parallelly polarized F-actin in macrophages. Our results suggest that Myo2 might drive the clockwise circumferential flow of F-actin along its parallel and annular polarity induced by Myo1D, which may be a molecular basis of cell and organ chirality.

cell biology↗

bioRxiv2023

Distinctive cellular and junctional dynamics independently regulate the rotation and elongation of the internal organ

Complex structures of organs are formed at high reproducibility, and deformations of epithelia play major roles in these processes. To acquire the intricate morphology, an epithelium simultaneously suffers multiple structural changes. For example, to form the left-right asymmetric structure of Drosophila embryonic hindgut, its epithelial tube concurrently rotates and elongates, which are driven by cell sliding and convergent extension, respectively. However, how an epithelium simultaneously accomplishes multiple structural changes remains unclear. To address this issue, we here studied the relevancy between these two mechanisms in the hindgut morphogenesis. Our live imaging analysis revealed that Myosin1D and E-cadherin, or Par-3 are required only for cell sliding or convergent extension, respectively, while Myosin II is essential for both. Mathematical models showed that these cellular dynamics share a single mechanical system in the same time window. Such specificity and universality of the machineries controlling epithelial dynamics might be a general strategy adopted in complex tissue morphogenesis.

developmental biology↗

bioRxiv2022

Zinc-based Ultrasensitive Microscopic Barrier Assay (ZnUMBA): a live-imaging method for detecting epithelial barrier breaches with spatiotemporal precision

Epithelial barrier function is commonly analyzed using transepithelial electrical resistance (TER), which measures the ion flux across epithelia, or by adding traceable macromolecules to one side of the epithelium and monitoring their passage to the other side. While these methods effectively measure changes to global barrier function, they are not sensitive enough to detect local or transient disruptions in the barrier, and they do not reveal the location of barrier breaches within the context of cell or tissue morphology. Therefore, we developed a method that we named Zinc-based Ultrasensitive Microscopic Barrier Assay (ZnUMBA), which overcomes these limitations, allowing for detection of local tight junction (TJ) leaks with high spatial and temporal resolution (Stephenson et al., 2019; Varadarajan et al., 2021). Here, we present expanded applications for ZnUMBA. First, we show that ZnUMBA can be used in Xenopus embryos to measure the dynamics of barrier restoration and actin dynamics following laser injury of the junction. We also demonstrate that ZnUMBA can be effectively utilized in developing zebrafish embryos as well as cultured monolayers of Madin-Darby Canine Kidney II (MDCK II) epithelial cells. ZnUMBA is a powerful and flexible method that, with some optimization, can be applied to multiple systems to measure dynamic changes in barrier function with spatiotemporal precision.

cell biology↗

bioRxiv2021

Quantitative Measurement of Diffusion-weighted Imaging Signal Using Expression-controlled Aquaporin-4 Cells: Comparative Study of 2-compartment and Diffusion Kurtosis Imaging Models

PurposeThe purpose of this study was to compare parameter estimates for the 2-compartment (2Comp) and diffusion kurtosis imaging (DKm) models obtained from diffusion-weighted imaging (DWI) of aquaporin-4 (AQP4) expression-controlled cells, and to look for biomarkers that indicate differences in the cell membrane water permeability. MethodsDWI was performed on AQP4-expressing and non-expressing cells and the signal was analyzed with the 2Comp and DKm models. For the 2Comp model, the diffusion coefficients (Df, Ds) and volume fractions (Ff, Fs, Ff=1-Fs) of the fast and slow compartments were estimated. For the DKm model, estimates of the diffusion kurtosis (K) and corrected diffusion coefficient (D) were obtained. ResultsFor the 2Comp model, Ds and Fs showed clear differences between AQP4-expressing and non-expressing cells. Fs was also sensitive to cell density. There was no clear relationship with the cell type for the DKm parameters. ConclusionsChanges to cell membrane water permeability due to AQP4 expression affected DWI of cell suspensions. For the 2Comp and DKm models, Ds was the parameter most sensitive to differences in AQP4 expression.

cell biology↗

bioRxiv2020

Robotic Search for Optimal Cell Culture in Regenerative Medicine

Induced differentiation is one of the most experience- and skill-dependent experimental processes in regenerative medicine, and establishing optimal conditions often takes years. We developed a robotic AI system with a batch Bayesian optimization algorithm that autonomously induces the differentiation of induced pluripotent stem cell-derived retinal pigment epithelial (iPSC-RPE) cells. The system performed 216 forty-day cell culture experiments, with a total experimentation time of 8,640 days. From 200 million possible parameter combinations, the system performed cell culture in 143 different conditions in 111 days, resulting in 88% better iPSC-RPE production than that by the pre-optimized culture in terms of pigmented scores. Our work demonstrates that the use of autonomous robotic AI systems drastically accelerates systematic and unbiased exploration of experimental search space, suggesting immense use in medicine and research.

systems biology↗

bioRxiv2020

SARS-CoV-2 infects human pluripotent stem cell-derived cardiomyocytes, impairing electrical and mechanical function

Global health has been threatened by the COVID-19 pandemic, caused by the novel severe acute respiratory syndrome coronavirus (SARS-CoV-2)1. Although considered primarily a respiratory infection, many COVID-19 patients also suffer severe cardiovascular disease2-4. Improving patient care critically relies on understanding if cardiovascular pathology is caused directly by viral infection of cardiac cells or indirectly via systemic inflammation and/or coagulation abnormalities3,5-9. Here we examine the cardiac tropism of SARS-CoV-2 using human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) and three-dimensional engineered heart tissues (3D-EHTs). We observe that hPSC-CMs express the viral receptor ACE2 and other viral processing factors, and that SARS-CoV-2 readily infects and replicates within hPSC-CMs, resulting in rapid cell death. Moreover, infected hPSC-CMs show a progressive impairment in both electrophysiological and contractile properties. Thus, COVID-19-related cardiac symptoms likely result from a direct cardiotoxic effect of SARS-CoV-2. Long-term cardiac complications might be possible sequelae in patients who recover from this illness.

pathology↗