Biology subjects

Hirano, K.

Publications and source records attributed to Hirano, K..

12 recordsLinked to original sources

bioRxiv2026

NLRX1 is an essential, druggable regulator of mitochondrial permeability transition

The molecular composition of the mitochondrial permeability transition pore (mPTP) remains contested, and several efficacious mPTP inhibitors act through undefined, cyclophilin D (CypD)-independent targets. Using two structurally distinct chemotypes of optimised, brain-penetrant mPTP inhibitors as chemical probes, we applied affinity-based chemoproteomics to identify the mitochondrial NOD-like receptor NLRX1 as their shared target. Both chemotypes bind NLRX1, and binding potency across a compound series tracks mPTP-inhibitory activity. Using CRISPR-Cas9-edited human cells and Nlrx1-/- mouse tissues, we show that NLRX1 is required for normal calcium-induced mPTP opening: its loss raises the calcium threshold for pore opening and its overexpression lowers it, independently of CypD. NLRX1 associates with postulated mPTP components, including ATP synthase and the adenine nucleotide translocase, in a compound-sensitive manner, and sustains mitochondrial protein homeostasis over longer timescales. The lead compound, GSK900, is orally bioavailable, brain-penetrant, and active in an mPTP-sensitive neurological injury model. These findings, converging with recent genetic studies, establish NLRX1 as an essential, CypD-independent regulator of mitochondrial permeability transition and provide brain-penetrant chemical tools to interrogate this biology.

cell biology↗

bioRxiv2026

Biomechanical regulation of Ca2+ dynamics during muscle stem cell activation

Muscle satellite cells (MuSCs) are muscle-resident stem cells that are responsible for myofiber regeneration. Although the importance of calcium ions (Ca2+) in muscle physiology has been well established, the mechanism by which Ca2+ mobilization governs MuSC function remains poorly understood. In this study, we aimed to systematically characterize Ca2+ dynamics in MuSCs and to define the mechanisms regulating these signals during muscle regeneration. By employing modified protocols for mouse MuSC isolation and Ca2+ measurement, we observed spontaneous Ca2+ fluctuations in MuSCs isolated from regenerating muscle after cardiotoxin-induced myofiber injury. Our detailed analysis using chemical Ca2+ indicators and a genetically encoded Ca2+ indicator revealed that the frequency and amplitude of Ca2+ fluctuations increased significantly during the activated and proliferative stages of MuSCs in muscle regeneration. This effect was more pronounced in MuSCs isolated from dystrophic and aged mice. Mechanistically, these Ca2+ fluctuations were at least partially mediated by mechanosensitive ion channels, including PIEZO1 and TRPM7, which promote MuSC migration. Collectively, our findings demonstrate that Ca2+ fluctuations through mechanosensitive ion channels act as a key regulator of MuSC activation during muscle regeneration and may provide new insights into the role of Ca2+ influx in muscle biology and the pathogenesis of muscle diseases.

cell biology↗

bioRxiv2026

A pistil peptide toxin-pollen antidote system for reproductive barrier

Summary paragraphPrezygotic reproductive barriers in angiosperms are crucial for female-mediated selection of compatible male gametes. In this study, we identified a biological activity within the stylar tissues of Arabidopsis thaliana pistils that inhibits the elongation of heterospecific pollen tubes. Using genome-wide association studies, we identified a cysteine-rich peptide predominantly expressed in the style as the responsible barrier. This secreted peptide, named Femme fatale (FEM), inhibited pollen tube elongation of heterospecific species and some conspecific strains, both in vivo and in vitro. Importantly, FEMs function was independent of the known reproductive barrier component SPRI1 that functions in the stigmatic papilla cell surface. This means two factors act spatially in sequence to create a multilayered reproductive barrier system against pollen in pistils. We also identified a secreted protein expressed in pollen, Homme capable (HOM), which is encoded by a gene next to FEM. HOM interacts with FEM to counteract its barrier effect and allow pollen tube elongation. The FEM-HOM locus has undergone frequent tandem duplications during Brassicaceae evolution. Dosage differences between the two factors can lead to reproductive incompatibility among lineages. Together, our findings suggest that FEM and HOM form a toxin-antidote system that helps establish reproductive barriers.

plant biology↗

bioRxiv2026

Expanding and Decoding the Chemistry of Phospholipid Headgroup in Eukaryotes

Cellular membranes have diverse phospholipids, chemical differences in whose headgroups impact many biological processes. Phosphatidylcholine is an essential phospholipid for human health, but not universally required for life. The evolutionary mechanisms underlying phospholipid preferences remain poorly understood, due to the difficulty of investigating metabolite structure-activity relationships in a cellular context. Here, we developed a generalizable metabolic-rewiring method to manipulate phospholipid headgroups together and their biological effects. This approach utilizes synthetic media to hijack evolutionarily conserved phosphatidylcholine biosynthesis, leveraging xenobiotics as principal precursors for scalable headgroup transformations. By identifying over 100 artificial headgroups, we expanded the chemical diversity of xenobiotic phospholipids. Unexpectedly, we discovered that subtle headgroup alterations produced distinct mammalian cellular activities. We demonstrated that chemical headgroup modifications differentially elicited structure-dependent effects on phospholipid-protein interactions, calcium dynamics, transcriptomic profiles, and stem cell differentiation. Notably, cross-species comparison revealed that human and yeast cells have different headgroup preferences critical for cell life and death. As proof-of-concept, interactome analysis identified headgroup-sensitive human microproteins vital for mitochondrial respiration, but non-conserved in yeast. These results exemplify the evolutionary diversity of key phospholipid-protein interactions, illustrating why humans depend on phosphatidylcholine. Overall, our findings establish a programmable platform for elucidating and engineering phospholipid-driven cellular functions.

cell biology↗

bioRxiv2025

Baculovirus-mediated gene transfer enables functional expression of PIEZO1 ion channel in isolated muscle satellite cells

Primary tissue stem cells are useful not only for basic cell biological research but also for therapeutic applications: however, their broader utility is often limited by technical challenges, such as a low efficiency of exogenous gene expression. PIEZO1 is a large mechanosensitive ion channel that plays an important role in muscle-resident stem cells, known as muscle satellite cells (MuSCs), during muscle regeneration. In this study, we developed a method for the ectopic expression of PIEZO1 in isolated MuSCs. Using a baculovirus vector system, we expressed PIEZO1 in myoblast C2C12 cells. Following optimization of the infection condition, we achieved robust PIEZO1 expression in isolated MuSCs during activated and differentiated states, with appropriate subcellular localization and ion channel activity. Importantly, the baculovirus-mediated PIEZO1 expression restored the reduced proliferative capacity of Piezo1-deficient MuSCs to a level comparable to wild-type cells, indicating that the exogenously expressed PIEZO1 is functionally equivalent to the endogenous protein. Overall, we established an efficient method for the transfer of the Piezo1 gene into isolated MuSCs, which should provide a versatile platform to study other large proteins in MuSCs. Summary StatementA baculovirus-based method was developed, enabling robust and functional PIEZO1 expression in isolated muscle satellite cells and providing a platform to study large proteins in isolated stem cells.

cell biology↗

bioRxiv2025

Actin Painting: a multicolor fluorescence staining method highlighting cell type-specific differences in the composition of filamentous actin architectures.

Actin is a major structural component of the cytoskeleton in eukaryotic cells, and filamentous actin (F-actin) forms a variety of types of cellular structures. Many actin probes have been developed to visualize F-actin architectures in eukaryotic cells. However, it is known that double-stained images of F-actin obtained by two different types of actin probes often show partial inconsistencies. While developing actin probes, we observed that the merged images of three distinct actin probes, each labeled with a different fluorescent dye (red, green and blue), enabled various F-actin architectures to be distinguished based on color. This differentiation arises from slight variations in the distribution of each actin probe, and results in a unique color combination of actin probes that reflects the relative intensities of the three fluorescent dyes. In this report, we introduce a new cell staining method, named Actin Painting, which exploits the unique affinity of actin-binding molecules in each actin probe to F-actin architectures. This technique allows the classification of various cell types based on their specific actin cytoskeleton.

cell biology↗

bioRxiv2024

Self-organization of spermatogenic wave coordinates sustained sperm production in the mouse testis

Spermatogenesis takes place in the testis, relying on the ordered turnover of differentiating cells supplied from stem cells. Classic histological analyses have revealed that this process shows hierarchical spatiotemporal patterning known as the spermatogenic cycle, wave, and descent of segmental order, indicative of currently underexplored mechanisms of tissue- and organ-scale homeostasis. Here, using mice, we conducted high-resolution, wide-field, and ultra long-term live imaging studies in vivo and ex vivo, combined with whole-organ mapping of differentiation stages. Such trans-scale measures demonstrate how stereotypic local cell turnover is coordinated into characteristic phase waves propagating along the seminiferous tubules, further organized into organ-scale patterning over the tubule loops. Minimal mathematical modeling shows that such higher-order dynamics can emerge from the local coupling of autonomous oscillators, which are rooted in delayed feedback interplay between stem and differentiating cells via retinoic acid signaling. These findings highlight a self-organization mechanism underpinning organ-scale homeostasis and constant sperm production.

developmental biology↗

bioRxiv2024

Engineering of highly active and diverse nuclease enzymes by combining machine learning and ultra-high-throughput screening

Optimizing enzymes to function in novel chemical environments is a central goal of synthetic biology, but optimization is often hindered by a rugged, expansive protein search space and costly experiments. In this work, we present TeleProt, an ML framework that blends evolutionary and experimental data to design diverse protein variant libraries, and employ it to improve the catalytic activity of a nuclease enzyme that degrades biofilms that accumulate on chronic wounds. After multiple rounds of high-throughput experiments using both TeleProt and standard directed evolution (DE) approaches in parallel, we find that our approach found a significantly better top-performing enzyme variant than DE, had a better hit rate at finding diverse, high-activity variants, and was even able to design a high-performance initial library using no prior experimental data. We have released a dataset of 55K nuclease variants, one of the most extensive genotype-phenotype enzyme activity landscapes to date, to drive further progress in ML-guided design.

bioinformatics↗

bioRxiv2023

Wnt4a is indispensable for genital duct elongation but not for gonadal sex differentiation in the medaka Oryzias latipes

In most vertebrates, oviducts are derived from Mullerian ducts and sperm ducts from Wolffian ducts. In teleosts, however, Mullerian ducts are absent. Wolffian ducts function as nephric ducts in both sexes, and genital ducts are formed by posterior extension of either ovaries or testes. Whether genital ducts of teleosts are newly evolved organs or are a variant of the Mullerian ducts is an important question for evolutionary mechanisms of morphogenesis. One of the genes essential for Mullerian duct formation in mice, wnt4, is expressed in the mesenchyme and induces invagination of the coelomic epithelium and its posterior elongation. Here, we approached to the above question by examining genital duct development in mutants of two Wnt4 genes in medaka (wnt4a is orthologous to mouse wnt4 and wnt4b paralogous). The wnt4b mutants had short body but were fertile with normal genital ducts. In contrast, both male and female wnt4a mutants had posterior elongation of the gonads stopped within or just outside the coelom, depending on the mutated alleles. Genetic females of the scl mutants (unable to synthesize testosterone or estrogens) have gonads containing both previtellogenic oocytes and spermatogenic cysts. Their gonads do not have ovarian cavities or sperm duct primordia and are lacking genital ducts completely. The results suggest Wnt4a target organs are posterior parts of the ovarian cavities or the sperm duct primordia. Medaka wnt4a was expressed in the mesenchyme ventral to the genital ducts in both sexes. Thus, the aborted elongation of genital ducts in the wnt4a mutants, the ortholog of mouse wnt4, suggests strongly that mouse Mullerian ducts and teleost genital ducts share homologous developmental processes. To further demonstrate this possible homology, mechanisms of genital duct formation and possible roles of Wnt4 should be compared before and after the appearance of Mullerian ducts in vertebrate phylogeny, namely jawless fish and cartilaginous fish. Additionally, wnt4a and wnt4b single mutants or double mutants did not show sex-reversal, suggesting both genes are dispensable for gonadal sex differentiation in medaka. This is in contrast to indispensable function of Wnt4 in mammalian ovarian differentiation.

developmental biology↗

bioRxiv2021

The mechanosensitive Ca2+-permeable ion channel PIEZO1 promotes satellite cell function in skeletal muscle regeneration

Muscle satellite cells (MuSCs), myogenic stem cells in skeletal muscle, play an essential role in muscle regeneration. During the regeneration process, cues from the surrounding microenvironment are critical for the proliferation and function of MuSCs. However, the mechanism by which mechanical stimuli from the MuSCs niche is converted into biochemical signals to promote muscle regeneration is yet to be determined. Here, we show that PIEZO1, a calcium ion (Ca2+)-permeable cation channel that is activated by membrane tension, mediates the spontaneous Ca2+ influx to controls the regenerative function of MuSCs. Our genetically engineering approach in mice revealed that PIEZO1 is functionally expressed in MuSCs, and the conditional deletion of Piezo1 in MuSCs delays myofiber regeneration after myofiber injury, which is at least in part due to the growth defect in MuSCs via the reduction in RhoA-mediated actomyosin formation. Thus, we provide the first evidence in MuSCs that PIEZO1, a bona fide mechanosensitive ion channel, promotes the proliferative and regenerative function during skeletal muscle regeneration.

cell biology↗

bioRxiv2021

Role of the N- and C-terminal regions of FliF, the MS ring component in Vibrio flagellar basal body

The MS ring is a part of the flagellar basal body and formed by 34 subunits of FliF, which consists of a large periplasmic region and two transmembrane segments connected to the N- and C-terminal regions facing the cytoplasm. A cytoplasmic protein, FlhF, which determines the position and number of the basal body, supports MS ring formation in the membrane. In this study, we constructed FliF deletion mutants that lack 30 or 50 residues at the N-terminus ({Delta}N30 and {Delta}N50), and 83 ({Delta}C83) or 110 residues ({Delta}C110) at the C-terminus. The N-terminal deletions were functional and conferred motility of Vibrio cells, whereas the C-terminal deletions were nonfunctional. The mutants were expressed in Escherichia coli to determine whether an MS ring could still be assembled. When co-expressing {Delta}N30FliF or {Delta}N50FliF with FlhF, fewer MS rings were observed than with the expression of wild-type FliF, in the MS ring fraction, suggesting that the N-terminus interacts with FlhF. MS ring formation is probably inefficient without an additional factor or FlhF. The deletion of the C-terminal cytoplasmic region did not affect the ability of FliF to form an MS ring because a similar number of MS rings were observed for {Delta}C83FliF as with wild-type FliF, although further deletion of the second transmembrane segment ({Delta}C110FliF) abolished it. These results suggest that the terminal regions of FliF have distinct roles; the N-terminal region for efficient MS ring formation and the C-terminal region for MS ring function. The second transmembrane segment is indispensable for MS ring assembly. ImportanceThe bacterial flagellum is a supramolecular architecture involved in cell motility. At the base of the flagella, a rotary motor that begins to construct an MS ring in the cytoplasmic membrane comprises 34 transmembrane proteins (FliF). Here, we investigated the roles of the N and C terminal regions of FliF, which are MS rings. Unexpectedly, the cytoplasmic regions of FliF are not indispensable for the formation of the MS ring, but the N-terminus appears to assist in ring formation through recruitment of FlhF, which is essential for flagellar formation. The C-terminus is essential for motor formation or function.

microbiology↗

bioRxiv2019

Prior physical synchrony enhances rapport and inter-brain synchronization during subsequent educational communication

Physical synchrony has been suggested to have positive effects on not only concurrent but also subsequent communication, but the underlying neural processes are unclear. Using functional near-infrared spectroscopy (fNIRS) hyperscanning, we tested the effects of preceding physical synchrony on subsequent dyadic teaching-learning communication. Thirty-two pairs of participants performed two experimental sessions. In each session, they underwent a rhythmic arm movement block with synchronous or asynchronous conditions, and then taught/learned unknown words to/from each other according to a given scenario. Neural activities in their medial and left lateral prefrontal cortex (PFC) were measured and inter-brain synchronization (IBS) during the teaching-learning blocks was evaluated. Participants rated their subjective rapport during the teaching-learning blocks, and took a word memory test. The analyses revealed that (1) prior physical synchrony enhanced teacher-learner rapport; (2) prior physical synchrony also enhanced IBS in the lateral PFC; and (3) IBS changes correlated positively with rapport changes. Physical synchrony did however not affect word memory performance. These results suggest that IBS can be useful to measure the effects of social-bonding facilitation activities for educational communication.

neuroscience↗