Biology subjects

Hosokawa, Y.

Publications and source records attributed to Hosokawa, Y..

10 recordsLinked to original sources

bioRxiv2025

Early pathogenesis of spinal and bulbar muscular atrophy uncovered by human iPSC-derived motor neurons highlights pathogenic neuropeptides as therapeutic targets

Spinal and bulbar muscular atrophy (SBMA) is a neuromuscular disorder caused by the expansion of the polyglutamine tract in the androgen receptor (AR). Motor neurons (MNs) derived from patient-specific induced pluripotent stem cells (iPSCs) robustl y recapitulated early SBMA phenotypes driven by endogenous mutant AR in the absence of testosterone (dihydrotestosterone) and detectable mutant AR aggregation. Notably, endoplasmic reticulum stress markedly exacerbated SBMA pathology. Cross-species integrative analyses of patient-derived neurons and spinal cords of transgenic mouse models revealed high expression of multiple disease-associated neuropeptides, including urotensin II (UTS2), in patient spinal MNs that was correlated with disease onset and progression in iPSC-derived MNs. Downstream signaling analyses of these neuropeptides revealed convergent molecular pathways whose pharmacological inhibition rescued cellular phenotypes. Together, these results establish a human disease model harboring endogenous mutant AR that closely reproduces early SBMA pathology and provides molecular leads for elucidating disease mechanisms and biomarkers and developing therapeutic targets.

neuroscience↗

bioRxiv2025

A skeletal muscle-sympathetic nerve-intestine network underlies muscle inflammation and atrophy induced by immobilization

Immobility is a common cause of muscle atrophy, but the underlying mechanisms have remained unclear. Here we show that limb immobilization in mice elicits inflammation and atrophy of skeletal muscle that are preventable by neutralizing antibodies to the chemokine CXCL10. Limb immobilization also induced changes to the gut microbiota and intestinal inflammation, and either sterilization of the intestine with antibiotics or administration of 10-hydroxy-cis-12-octadecenoic acid--a linoleic acid-derived gut microbial metabolite--prevented intestinal and muscle inflammation as well as muscle atrophy induced by immobilization, implicating intestinal inflammation in muscle inflammation and atrophy. Limb immobilization activated sympathetic nerves and increased {beta}2-adrenergic receptor gene (Adrb2) expression in the intestine. Single-cell RNA-sequencing analysis revealed that, among cells expressing Adrb2 in the intestine, immobilization increased only the population of macrophages. Pharmacological inhibition or macrophage-specific ablation of Adrb2 prevented immobilization-induced intestinal and muscle inflammation. Our results thus implicate a previously unrecognized muscle-nerve-intestine network in immobilization-induced muscle atrophy.

molecular biology↗

bioRxiv2025

Differential O-Glc elongation on the specific EGF repeat within a ligand binding domain regulates NOTCH1 signaling

Three types of O-linked glycosylation - O-glucose, O-fucose, and O-N-acetylglucosamine - are crucial for the function of Notch receptors, which regulates critical cell fate determination processes in a wide variety of contexts. O-Glucose glycans are added to serine residues located between the first and second conserved cysteines within the epidermal growth factor (EGF) -like repeats in the Notch extracellular domain. Previously, O-glucose glycans were shown to be extended to a trisaccharide structure with two xyloses via an 1-3 linkage. Our recent studies, however, indicated that the O-glucose glycan on NOTCH1 EGF10 can be extended by hexose and Neu5Ac. Here, we demonstrated that this hexose- and Neu5Ac-extended glycan has a 3-sialyllactose-like structure synthesized by specific members of two isoenzyme families, B4GALT1 and ST3GAL4. Using mass spectrometry, we identified this modification exclusively on NOTCH1 EGF10 and NOTCH3 EGF9 (equivalent to NOTCH1 EGF10), with no detection in any other EGF domains in NOTCH1-3. Sequence comparison and mutagenesis experiments identified one amino acid at position -2 of the fourth cysteine in the EGF domain as crucial for the galactose elongation of O-glucose glycans. We further demonstrated that this site-specific elongation of O-glucose on NOTCH1 EGF10 significantly impact ligand binding and signal transduction of NOTCH1. Our findings contribute to the understanding of the intricate regulatory mechanisms of Notch receptor function mediated by distinct positions and structures of O-glycans. Significance StatementNotch, a signaling receptor critical for development, differentiation, and homeostasis in multicellular organisms, undergoes multiple post-translational glycan modifications to regulate its function. While O-glucose glycan elongation by xylose was previously known, we identified an O-glucose glycan extended by galactose and Neu5Ac on specific epidermal growth factor (EGF) -like repeats of certain Notch paralogs, NOTCH1 and NOTCH3. As far as we know, this structure has not been reported on any other proteins. We identify the enzymes involved in the biosynthesis of this glycan and demonstrate that modifications at a specific site on Notch direct receptor functions, including ligand binding and Notch signaling.

biochemistry↗

bioRxiv2024

Cell morphogenesis via shape-guided self-propelled treadmilling actin waves

Cells frequently undergo spontaneous morphogenesis, yet the underlying mechanisms remain incompletely understood. While actin filaments are central to cell morphogenesis and are typically regulated by biochemical signaling, cells can form protrusions even without clear external cues, suggesting the existence of intrinsic physical mechanisms. Here, we report that actin filaments undergo directional movement driven by treadmilling, an ATP-fueled polymerization-disassembly cycle intrinsic to actin. These Self-propelled Treadmilling Actin filaments (SpTAs), exhibit stochastic yet directional motion, in a manner similar to self-propelled "particles" rather than the previously reported reaction-diffusion "waves". SpTA arrival at the cell periphery drives membrane protrusion by orienting their polymerizing ends outwards. This SpTA accumulation, guided by nascent membrane curvature, further amplifies protrusion growth and expansion, driving cellular polarization for migration. Our findings establish actin filament as a novel class of active particle, providing a fundamental physical framework for understanding how molecular-scale motion leads to higher-order organization in living systems.

cell biology↗

bioRxiv2022

LUZP1 regulates the constriction velocity of the contractile ring during cytokinesis

There has been a great deal of research on cell division and its mechanisms; however, its processes have not yet been entirely elucidated. To find novel proteins that regulate cell division, we performed the screening using siRNAs and/or the expression plasmid of the target genes and identified leucine zipper protein 1 (LUZP1). Recent studies have shown that LUZP1 interacts with various proteins and stabilizes the actin cytoskeleton; however, the function of LUZP1 in mitosis is not known. In this study, we found that LUZP1 colocalized with the chromosomal passenger complex (CPC) at the centromere in metaphase and at the central spindle in anaphase and that these LUZP1 localizations were regulated by CPC activity and kinesin family member 20A (KIF20A). Mass spectrometry analysis identified that LUZP1 interacted with death-associated protein kinase 3 (DAPK3), one regulator of the cleavage furrow ingression in cytokinesis. In addition, we found that LUZP1 also interacted with myosin light chain 9 (MYL9), a substrate of DAPK3, and comprehensively inhibited MYL9 phosphorylation by DAPK3. In line with a known role for MYL9 in the actin-myosin contraction, LUZP1 suppression accelerated the constriction velocity at the division plane in our timelapse analysis. Our study indicates that LUZP1 is a novel regulator for cytokinesis that regulates the constriction velocity of the contractile ring.

cell biology↗

bioRxiv2022

Cell viability assessment associated with a contact of gas bubbles produced by femtosecond laser breakdown in cell culture media

High intensity near infrared femtosecond laser is a promising tool for three-dimensional processing of biological materials. During the processing of cells and tissues, long lasting gas bubbles randomly appeared around the laser focal point, however physicochemical and mechanical effects of the gas bubbles has not been emphasized. This paper presents characteristic behaviors of the gas gabbles and their contact effects on cell viability. High-speed imaging of the gas bubble formation with various additives in physiological medium confirms that the gas bubble consists of dissolved air, and amphipathic proteins stabilize the bubble surface. This surface protective layer reduces interactions of gas bubbles and cell membranes. Consequently, the gas bubble contact does not cause critical effects on cell viability. On the other hands, burst of gas bubbles stimulated by an impact of femtosecond laser induced cavitation can lead to liquid jet flow that might cause serious mechanical damages on cells. These results provide insights for the parameter of biological tissue processing with intense fs laser pulses.

bioengineering↗

bioRxiv2021

Guided Axon Outgrowth of Neurons by Real-Time Femtosecond Laser Penetration on a 4 μm Thick Thin-Glass Sheet

Transplantation of scaffold-embedded guided neurons has been reported to increase neuronal regeneration following brain injury. However, precise axonal integration between host and transplant neurons to form functional synapses remains a major problem. This study aims to develop a real-time femtosecond (fs) laser penetration on a 4 m thick thin-glass sheet to promote guided axon outgrowth influenced by molecular gradients in a microfluidic device. The device enables the introduction of the guidance molecule (i.e., netrin-1), neuronal culture, and manipulation by fs laser. After fabricating multiple micro-holes on the thin-glass sheet using fs laser, netrin-1 gradients with radial concentrations are generated in the chamber, affecting axon outgrowth and guidance. A majority of axons (~92%) experiences guided outgrowth with positive angular changes towards netrin-1 gradients. These results demonstrate the capability of the precise and real-time manipulation system based on a fs laser and a microfluidic device to control the growth of neurons.

bioengineering↗

bioRxiv2021

A Ca2+ wave generates a force during cell extrusion in zebrafish

When oncogenic transformed or damaged cells appear within an epithelial sheet, they are apically extruded by surrounding cells. Recently, using cultured mammalian epithelial cells and zebrafish embryonic epithelial cells, we found that a calcium (Ca2+) wave propagates from RasV12-transformed cells and laser-irradiated damaged cells to surrounding cells and promotes apical extrusion by inducing polarized movements of the surrounding cells. In mammalian cell cultures, we reported that the inositol trisphosphate (IP3) receptor, gap junctions, and the mechanosensitive Ca2+ channel TRPC1 are involved in Ca2+ wave-mediated polarized movements. However, which molecules regulate Ca2+ wave-mediated polarized movements in zebrafish and whether the Ca2+ wave can generate a force remain unknown. In this study, we aimed to answer these questions. By performing pharmacological and gene knockout experiments, we showed that a Ca2+ wave induced by the IP3 receptor and trpc1 led to formation of cryptic-lamellipodia and polarized movements of surrounding cells toward extruding cells in zebrafish. By using an in vivo force measurement method, we found that the Ca2+ wave generated approximately 1 kPa of force toward extruding cells. Our results reveal a previously unidentified molecular mechanism underlying the Ca2+ wave in zebrafish and demonstrate that the Ca2+ wave generates a force during cell extrusion.

cell biology↗

bioRxiv2021

GTL1 is required for a robust root hair growth response to avoid nutrient overloading

O_LIRoot hair growth is tuned in response to the environment surrounding plants. While most of previous studies focused on the enhancement of root hair growth during nutrient starvation, few studies investigated the root hair response in the presence of excess nutrients. C_LIO_LIWe report that the post-embryonic growth of wild-type Arabidopsis plants is strongly suppressed with increasing nutrient availability, particularly in the case of root hair growth. We further used gene expression profiling to analyze how excess nutrient availability affects root hair growth, and found that RHD6 subfamily genes, which are positive regulators of root hair growth, are down-regulated in this condition. C_LIO_LIOn the other hand, defects in GTL1 and DF1, which are negative regulators of root hair growth, cause frail and swollen root hairs to form when excess nutrients are supplied. Additionally, we observed that the RHD6 subfamily genes are mis-expressed in gtl1-1 df1-1. Furthermore, overexpression of RSL4, an RHD6 subfamily gene, induces swollen root hairs in the face of a nutrient overload, while mutation of RSL4 in gtl1-1 df1-1 restore root hair swelling phenotype. C_LIO_LIIn conclusion, our data suggest that GTL1 and DF1 prevent unnecessary root hair formation by repressing RSL4 under excess nutrient conditions. C_LI

plant biology↗

bioRxiv2019

Tandem paired nicking promotes precise genome editing with scarce interference by p53

Targeted knock-in mediated by double-stranded DNA cleavage is accompanied by unwanted insertions and deletions (indels) at on-target and off-target sites. A nick-mediated approach scarcely generates indels but exhibits reduced efficiency of targeted knock-in. Here, we demonstrate that tandem paired nicking, a method for targeted knock-in involving two Cas9 nickases that create nicks at the homologous regions of the donor DNA and the genome in the same strand, scarcely creates indels at the edited genomic loci, while permitting the efficiency of targeted knock-in largely equivalent to that of the Cas9 nuclease-based approach. Tandem paired nicking seems to accomplish targeted knock-in via DNA recombination analogous to Hollidays model, and creates intended genetic changes in the genome without introducing additional nucleotide changes such as silent mutations. Targeted knock-in through tandem paired nicking neither triggers significant p53 activation nor occurs preferentially in p53-suppressed cells. These properties of tandem paired nicking demonstrate its utility in precision genome engineering.

molecular biology↗