Biology subjects

Yoshida, Y.

Publications and source records attributed to Yoshida, Y..

At least 19 recordsLinked to original sources

bioRxiv2026

Structural basis for the intestinal protocadherin-based intermicrovillar adhesion complex

The intestinal brush border (BB), composed of densely packed microvilli on enterocytes, is essential for nutrient absorption and host defense. Its organization relies on the intermicrovillar adhesion complex (IMAC), mediated by protocadherins CDHR2 and CDHR5. Despite their clinical relevance in inflammatory bowel disease and several carcinomas, structural details of IMAC assemblies have remained elusive. Herein, we report the Cryo-EM structure of the adhesive complex at 3.4 [A] resolution, revealing a heterotetrameric ensemble composed of a dimer of CDHR2 and a dimer of CDHR5. This assembly ensures uniform adhesive strength between neighboring microvilli, and facilitates hexagonal packing of microvilli. Biophysical analyses and molecular dynamics simulations revealed a kinked, Ca{superscript 2}-free linker between domains EC3 and EC4 of CDHR5 conferring the necessary flexibility to withstand the shear stress caused during intestinal peristalsis. Collectively, these findings provide a structural framework for understanding BB organization and suggest strategies for therapeutics targeting IMAC in intestinal disorders.

biophysics↗

bioRxiv2026

GTPase-powered progressive contraction of a supramolecular ring driving chloroplast division

The chloroplast division machinery, known as the division ring, is a supramolecular complex composed of bacterial- and host-derived proteins1-5. However, how the division ring generates the force required to sever a chloroplast remains poorly understood. Here, we established an in vitro assay in which chloroplasts isolated from Cyanidioschyzon merolae6,7 undergo GTP hydrolysis-dependent division. Using this assay, we show that Dnm2, rather than FtsZ, acts as the motor driving GTPase-powered contraction of the division ring, thereby physically dividing the chloroplast. We further demonstrate that the division ring is assembled through coiling of a glycosyltransferase-mediated filament and is crosslinked by dimerization of the Dnm2 GTPase domain. Following GTP hydrolysis-dependent force generation, Dnm2 retains its dimeric form in GDP-bound and nucleotide-free states, providing a locking step that suppresses back-slippage of the coiling ring during division. Thus, this mechanical design enables progressive, ratchet-like constriction of the division ring through coiling, overcoming the mechanical load posed by the chloroplast and generating the force required for fission, consistent with quantitative simulations. These findings suggest that a specialized division-ring mechanism, distinct from vesicle fission systems, evolved to mediate endosymbiont fission, allowing host control of endosymbiont proliferation and promoting faithful inheritance of the emerging organelle.

plant biology↗

bioRxiv2025

An orthogonal Cro/OR3 binary system for targeted gene expression

Precise spatiotemporal control of gene expression is essential for dissecting complex biological systems. Here, we introduce a phage-derived Cro/OR3 binary system for transcriptional activation that functions robustly in zebrafish and human cells. A Cro repressor fused to the VP16 activation domain efficiently drives expression of OR3 operator-linked genes, and its activity can be targeted to specific cell types using enhancer-trap and promoter-controlled zebrafish lines. As a proof of concept, we manipulate glutamate transporter Glast/Slc1a3b-expressing cells in the central nervous system by Cro/OR3-mediated expression of a human ataxia-associated, uptake-deficient Glast/Slc1a3b variant, causing spastic, uncoordinated movement and axial deformation. Concurrent Gal4/UAS-mediated visualization reveals substantially disrupted positioning of motor neurons, highlighting their selective sensitivity to extracellular glutamate homeostasis during spinal circuit assembly. Together, these results establish the Cro/OR3 system as an orthogonal platform for targeted gene expression, enabling precise mechanistic dissection of how specific cell types and their interactions underlie diverse biological processes.

bioengineering↗

bioRxiv2025

Discovery of a Selective Inhibitor of ZIP14 with Therapeutic Potential for Cancer-associated Cachexia

ZIP14/SLC39A14, a membrane-bound metal transporter, is essential for systemic metal homeostasis and has been implicated in inflammatory and metabolic disorders, including cancer-associated cachexia. Despite its biological and therapeutic significance, no selective inhibitors have been identified. Here, we identify 1-phenyl-8-(2-phenylethyl)-1,3,8-triazaspiro[4.5]decan-4-one (PPTD) as the first selective small-molecule inhibitor of ZIP14. PPTD efficiently blocks ZIP14-mediated uptake of zinc, iron, manganese, and cadmium, while sparing the closely related transporter ZIP8/SLC39A8. Mechanistically, PPTD binds specifically to a pocket formed at the dimer interface of ZIP14, as revealed by AlphaFold3 structural prediction, ligand-interaction profiling, structure-activity analyses, and site-directed mutagenesis, providing direct evidence for a targeted inhibition mechanism. ZIP14-driven metal influx promotes reactive oxygen species and lipid peroxidation, leading to cytotoxicity, which PPTD effectively reverses. In vivo, PPTD ameliorates major features of cancer cachexia in mice, including weight loss, reduced survival, muscle wasting, impaired locomotor activity, and disease progression. PPTD thus provides both a chemical probe to dissect ZIP14 function and a potential therapeutic candidate for cancer cachexia, establishing a foundation for the development of therapies targeting ZIP14-mediated metal dysregulation.

biochemistry↗

bioRxiv2025

Control of Chloroplast Integrity by the Jasmonate Signaling Pathway is Linked to Growth-Defense Balance

Chloroplasts play a central role in plant responses to environmental stress. Little is known, however, about how chloroplast homeostasis is maintained during stress responses that place high metabolic and bioenergetic demands on the cell. As a chloroplast-derived retrograde signal, jasmonate (JA) promotes broad-spectrum immunity by triggering the degradation of JAZ transcriptional repressors that act in the nucleus to control chloroplast metabolism. Here, we manipulated JAZ abundance to investigate how chloroplast integrity and function is maintained at high levels of defense. A jaz decuple mutant (jazD) lacking 10 of 13 JAZs exhibited strong growth-defense antagonism without loss of photosynthetic efficiency. Treatment of jazD with the JA-receptor agonist coronatine triggered rapid loss of chlorophyll and the turnover of chloroplast proteins and lipids, leading to the collapse of photosynthetic activity and cell death. These findings were supported by global transcript and metabolite profiling over a time course of coronatine treatment. Genetic screens identified MYC2 and the JAZ-destabilizing F-box protein, COI1, as positive regulators of coronatine-induced chloroplast dismantling in jazD plants. These results demonstrate how the progressive loss of JAZ repression drives a continuum of MYC2-dependent growth-defense tradeoffs, including disassembly of the photosynthetic apparatus as a terminal response. In highlighting the critical role for JAZ proteins in maintaining chloroplast integrity at high levels of defense, our results provide insight into the general mechanism by which jasmonate governs chloroplast metabolism to balance growth and stress responses. One sentence summaryThis study shows that the jasmonate-mediated leaf transition from growth- to defense-oriented metabolism culminates in the disassembly of the photosynthetic apparatus.

plant biology↗

bioRxiv2025

Ubiquitination-activated TAB-TAK1-IKK-NF-kB axis modulates gene expression for cell survival in the lysosomal damage response

The lysosomal damage response is important for the maintenance of cellular homeostasis. Although the mechanisms underlying the repair and autophagic elimination of damaged lysosomes have been elucidated, the early signal transduction pathways and genes induced in response to lysosomal damage remain elusive. We performed transcriptome and proteome analyses and found that the TAB-TAK1- IKK-NF-{kappa}B axis is activated by K63-linked ubiquitin chains that accumulate on damaged lysosomes. This activates the expression of various transcription factors and cytokines that promote anti-apoptosis and intercellular signaling. The findings highlight the crucial role of ubiquitin-regulated signal transduction and gene expression in cell survival and cell-cell communication in response to lysosomal damage. The results suggest that the ubiquitin system is not only involved in the removal of damaged lysosomes by lysophagy, but also functions in the activation of cellular signaling for cell survival.

cell biology↗

bioRxiv2025

Activity Protects Spinal Premotor Interneurons from Microglial Phagocytosis and Transneuronal Degeneration After Corticospinal Injury

Spinal premotor circuits play a fundamental role in motor control. The corticospinal tract (CST) provides control signals to premotor circuits in the spinal cord, guiding voluntary skilled movements. Unilateral selective lesion of the CST in the medullary pyramidal tract (PTX) produces transneuronal degeneration, whereby Choline Acetyltransferase-positive (ChAT) premotor interneurons contralesionally undergo non-apoptotic degeneration by microglial phagocytosis. Evidence shows that transneuronal degeneration has an activity dependence: MCX inactivation produces transneuronal degeneration and spinal DC neuromodulation after PTX ameliorates it. This study expands our understanding of transneuronal degeneration mechanisms by examining the activity-dependence of degeneration vulnerability and the implications for motor recovery in a mouse model of a complete CST lesion model (bilateral PTX). We address four key unanswered questions: Are Chx10 (VGlut2) interneurons, the largest spinal interneuron class to receive direct synaptic connections from the CST vulnerable to transneuronal degeneration after CST loss; using DREADD neuromodulation, what are effective sources of presynaptic activation for protecting spinal premotor interneurons after CST loss; how are ameliorating transneuronal interneuron degeneration and reducing microglial activation associated; and does effective rescue of interneuron degeneration rescue grip strength after injury? Transneuronal degeneration is a pervasive pathophysiological change injury; CST lesion produces significant Chx10 interneuron loss. Multiple sources of neuronal DREADD activation--motor cortex, reticular formation, and spinal interneurons--are effective in ameliorating transneuronal degeneration. Interneuron rescue is strongly associated with ameliorating inflammation, showing potential causality between interneuron degeneration and inflammation after CST lesion. Finally, rescuing spinal interneurons was associated with restoring function. Our findings demonstrate the interplay between neuronal activity, microglia actions mediating transneuronal degeneration, and motor recovery following CNS injury.

neuroscience↗

bioRxiv2024

A vestibulospinal pathway for context-dependent motor control of the mouse tail

The tail movement is critical for maintaining balance during locomotion in many animal species, yet its underlying neuro-muscular control remains poorly understood. In this study we investigated what are the neuronal substrates responsible for tail control in mice. Using high-resolution microCT scans and retrograde labeling, we lay out the neuro-muscular organization of the tail and identified distinct pools of motoneurons in the spinal cord that innervate proximal and distal muscles. We further show that the spinal vestibular nucleus (SpVN) in the brainstem sends direct projections to the same spinal cord segments where tail motoneurons are located. The activation of these vestibulospinal neurons using optogenetics results in more accurate tail movements during challenging balance tasks. Our results reveal that the vestibular systems influence on tail control is context-dependent, enhancing balance performance under uncertain sensory conditions. These findings provide novel insights into the neural circuits responsible for maintaining balance, highlighting the role of the vestibulospinal pathway in context-dependent modulation of tail movement to maintain stability during complex locomotor tasks.

neuroscience↗

bioRxiv2024

PISA: versatile microscope for 3D single molecule light sheet imaging

Light sheet microscopy combines high sensitivity and imaging speeds with low photodamage, however a remaining hurdle to its widespread adoption is its requirement for special sample preparation. Our new microscope - planar illumination microscope for single molecule imaging for all purpose (PISA) - significantly relieves these restrictions to enable light sheet imaging of coverslip-mounted samples as commonly analyzed in biological microscopes. We show how this technology now permits high-throughput imaging using multi-well plates. Further, the ability of 3D single molecule imaging in microlitre or mm3 scale volumes over the coverslip improves the sensitivity of biochemical gel-based fluorometric assays to atto/zepto molar levels. PISA will facilitate widespread adoption of light sheet imaging for a variety of biological, biochemical and medical investigations.

biophysics↗

bioRxiv2024

Comparative transcriptome and variant analyses of the pancreatic islets of a rat model of obese type 2 diabetes identifies a frequently distributed nonsense mutation in the lipocalin 2 gene

We have recently established the Zucker fatty diabetes mellitus (ZFDM) rat as a novel model of obese type 2 diabetes (T2D), originating from the obese Zucker fatty (ZF) rat harboring a missense mutation in the leptin receptor gene. Pathogenesis of dysfunction of the pancreatic islets and genetic factors of T2D in ZFDM rats remain unknown. Here, we perform comparative transcriptome and variant analyses of the pancreatic islets between the two strains. Among differentially expressed genes irrespective of obesity and glucose intolerance states, we identify a nonsense mutation, c.409C>T (p.Gln137X), in the lipocalin 2 (Lcn2) gene which encodes a secreted protein called neutrophil gelatinase-associated lipocalin, a well-known biomarker for inflammation. Interestingly, we find that the Lcn2 mutation is distributed widely in rat species, such as commonly used DA and F344 strains. We examine the Lcn2 mutation as a strong candidate gene for T2D in ZFDM rats by using genome editing of ZFDM rats in which the nonsense mutation is replaced with a wild-type nucleotide. We find that the genome editing well works but also observe that there is no significant difference in the development of T2D between genome-edited and original ZFDM rats. Finally, we perform a genetic linkage analysis by using backcross progeny between ZF and ZFDM rats and confirm that the Lcn2 mutation exhibits no significant association with the onset of T2D. Our data indicate that several rat strains would serve as Lcn2 deficient models, contributing to elucidate pathophysiological roles of Lcn2 in a wide variety of phenotypes.

genetics↗

bioRxiv2024

Secretome analysis by proximal labeling in Golgi apparatus for identifying fat body derived humoral factors in Drosophila disc regeneration

Humoral factors act as inter-tissue mediators and regulate various organismal physiologies. Although the importance of humoral factors in tissue repair has been recently recognized, our understanding of how humoral proteins regulate tissue repair remains limited. Glycosylation is an important modification of the conventional secretory pathway, and we have demonstrated that N-glycosylation in the Golgi apparatus of the Drosophila fat body (FB), the major secretory tissue equivalent to the mammalian liver and adipose tissue, remotely contributes to epithelial tissue repair. To identify humoral factors that contribute to repair via the Golgi apparatus, we constructed a Golgi-specific protein biotinylation system and performed hemolymph proteomics. By combining genetic analyses, we found that FB-derived innate immune regulators and iron mediators affect tissue repair. Altogether, our Golgi-specific labeling system has the potential to identify Golgi-mediated secreted factors that regulate inter-organ communication.

developmental biology↗

bioRxiv2024

Anatomical and functional analysis of the corticospinal tract in an FRDA mouse model

Friedreichs ataxia (FRDA) is one of the most common hereditary ataxias. It is caused by a GAA repeat in the first intron of the FXN gene, which encodes an essential mitochondrial protein. Patients suffer from progressive motor dysfunction due to the degeneration of mechanoreceptive and proprioceptive neurons in dorsal root ganglia (DRG) and cerebellar dentate nucleus neurons, especially at early disease stages. Postmortem analyses of FRDA patients also indicate pathological changes in motor cortex including in the projection neurons that give rise to the cortical spinal tract (CST). Yet, it remains poorly understood how early in the disease cortical spinal neurons (CSNs) show these alterations, or whether CSN/CST pathology resembles the abnormalities observed in other tissues affected by FXN loss. To address these questions, we examined CSN driven motor behaviors and pathology in the YG8JR FRDA mouse model. We find that FRDA mice show impaired motor skills, exhibit significant reductions in CSN functional output, and, among other pathological changes, show abnormal mitochondrial distributions in CSN neurons and CST axonal tracts. Moreover, some of these alterations were observed as early as two months of age, suggesting that CSN/CST pathology may be an earlier event in FRDA disease than previously appreciated. These studies warrant a detailed mechanistic understanding of how FXN loss impacts CSN health and functionality.

neuroscience↗

bioRxiv2024

Layer Va neurons, as major presynaptic partners of corticospinal neurons, play critical roles in skilled movements

Corticospinal neurons (CSNs) are located in the cortex and projecting into the spinal cord. The activation of CSNs, which is associated with skilled motor behaviors, induces the activation of interneurons in the spinal cord. Eventually, motor neuron activation is induced by corticospinal circuits to coordinate muscle activation. Therefore, elucidating how the activation of CSNs in the brain is regulated is necessary for understanding the roles of CSNs in skilled motor behaviors. However, the presynaptic partners of CSNs in the brain remain to be identified. Here, we performed transsynaptic rabies virus-mediated brain-wide mapping to identify presynaptic partners of CSNs (pre-CSNs). We found that pre-CSNs are located in all cortical layers, but major pre-CSNs are located in layer Va. A small population of pre-CSNs are also located outside the cortex, such as in the thalamus. Inactivation of layer Va neurons in Tlx3-Cre mice results in deficits in skilled reaching and grasping behaviors, suggesting that, similar to CSNs, layer Va neurons are critical for skilled movements. Finally, we examined whether the connectivity of CSNs is altered after spinal cord injury (SCI). We found that unlike connections between CNSs and postsynaptic neurons, connections between pre-CSNs and CSNs do not change after SCI.

neuroscience↗

bioRxiv2024

Forelimb motor recovery by modulating extrinsic and intrinsic signaling as well as neuronal activity after the cervical spinal cord injury

Singular strategies for promoting axon regeneration and motor recovery after spinal cord injury (SCI) have been attempted with limited success. Here, we propose the combinatorial approach of deleting extrinsic and intrinsic factors paired with neural stimulation, will enhance adaptive axonal growth and motor recovery after SCI. We previously showed the deletion of RhoA and Pten in corticospinal neurons inhibits axon dieback and promotes axon sprouting after lumbar SCI. Here, we examined the effects of RhoA;Pten deletion coupled with neural stimulation after cervical SCI. This combinatorial approach promoted more boutons on injured corticospinal neurons in the spinal cord compared to sole RhoA;Pten deletion. Although RhoA;Pten deletion does not promote motor recovery in the forelimb after SCI, stimulating corticospinal neurons in those mice results in partial motor recovery. These results demonstrate that a combinatorial approach that pairs genetic modifications with neuronal stimulation can promote axon sprouting and motor recovery following SCI.

neuroscience↗

bioRxiv2024

Sexual dimorphism in the tardigrade Paramacrobiotus metropolitanus transcriptome

BackgroundIn gonochoristic animals, the sex determination pathway induces different morphological and behavioral features that can be observed between sexes, a condition known as sexual dimorphism. While many components of this sex differentiation cascade shows high levels of diversity, factors such as the Doublesex-Mab-3-related transcription factor (DMRT) are highly conserved throughout animals. Species of the phylum Tardigrada exhibits remarkable diversity in morphology and behavior between sexes, suggesting a pathway regulating such dimorphism. Despite the wealth of genomic and zoological knowledge accumulated in recent studies, the sexual differences in tardigrades genomes have not been identified. In this study, we focused on the gonochoristic species Paramacrobiotus metropolitanus and employed omics analyses to unravel the molecular basis of sexual dimorphism. ResultsTranscriptome analysis between sex identified numerous differentially expressed genes, of which approximately 2,000 male-biased genes were focused on 29 non-male-specific genomic loci. From these regions, we identified two Macrobiotidae family specific DMRT paralogs, which were significantly upregulated in males and lacked sex specific splicing variants. Furthermore, phylogenetic analysis indicated all tardigrade genomes lacks the doublesex ortholog, suggesting doublesex emerged after the divergence of Tardigrada. In contrast to sex-specific expression, no evidence of genomic difference between the sexes were found. We also identified several anhydrobiosis genes exhibiting sex-biased expression, possibly suggesting a mechanism for protection of sex specific tissues against extreme stress. ConclusionsThis study provides a comprehensive analysis for analyzing the genetic differences between sexes in tardigrades. The existence of male-biased, but not male-specific, genomic loci and identification of the family specific male-biased DMRT subfamily would provide the foundation for understanding the sex determination cascade. In addition, sex-biased expression of several tardigrade-specific genes which are involved their stress tolerance suggests a potential role in protecting sex-specific tissue and gametes.

zoology↗

bioRxiv2024

The Dorsal Column Nuclei Scales Mechanical Allodynia During Neuropathic Pain

Tactile perception relies on reliable transmission and modulation of low-threshold information as it travels from the periphery to the brain. During pathological conditions, tactile stimuli can aberrantly engage nociceptive pathways leading to the perception of touch as pain, known as mechanical allodynia. Two main drivers of peripheral tactile information, low-threshold mechanoreceptors (LTMRs) and postsynaptic dorsal column neurons (PSDCs), terminate in the brainstem dorsal column nuclei (DCN). Activity within the DRG, spinal cord, and DCN have all been implicated in mediating allodynia, yet the DCN remains understudied at the cellular, circuit, and functional levels compared to the other two. Here, we show that the gracile nucleus (Gr) of the DCN mediates tactile sensitivity for low-threshold stimuli and contributes to mechanical allodynia during neuropathic pain in mice. We found that the Gr contains local inhibitory interneurons in addition to thalamus-projecting neurons, which are differentially innervated by primary afferents and spinal inputs. Functional manipulations of these distinct Gr neuronal populations resulted in bidirectional changes to tactile sensitivity, but did not affect noxious mechanical or thermal sensitivity. During neuropathic pain, silencing Gr projection neurons or activating Gr inhibitory neurons was able to reduce tactile hypersensitivity, and enhancing inhibition was able to ameliorate paw withdrawal signatures of neuropathic pain, like shaking. Collectively, these results suggest that the Gr plays a specific role in mediating hypersensitivity to low-threshold, innocuous mechanical stimuli during neuropathic pain, and that Gr activity contributes to affective, pain-associated phenotypes of mechanical allodynia. Therefore, these brainstem circuits work in tandem with traditional spinal circuits underlying allodynia, resulting in enhanced signaling of tactile stimuli in the brain during neuropathic pain.

neuroscience↗

bioRxiv2024

Simple prerequisite of presequence for mitochondrial protein import in the unicellular red alga Cyanidioschyzon merolae

Mitochondrial biogenesis relies on hundreds of proteins which are derived from genes encoded in the nucleus. According to characteristic properties of N-terminal targeting peptides (TP) and multi-step authentication by the protein translocase called the TOM complex, nascent polypeptides satisfying the requirements are imported into mitochondria. However, it has not been investigated whether the eukaryotic cell with a simple proteome and a single mitochondrion in a cell has a similar or simpler complexity of presequence requirements for mitochondrial protein import as other eukaryotes with multiple mitochondria. Based on the amino acid compositions of putative mitochondrial TP sequences in the unicellular red alga Cyanidioschyzon merolae, we designed the synthetic TP (synTP) and confirmed that synTP-fused mVenus were translocated into the mitochondrion in vivo. Through a series of experimental evaluations using modified synTPs, we showed that functional TP must have some basic residues, at least one, and compose the specific amino acid composition, but the physicochemical properties of net charge, hydrophobicity and hydrophobic moment are not strictly determined in C. merolae. Combined with the simple composition of the TOM complex in C. merolae, our results suggest that a regional positive charge in TP would be recognized and verified solely by TOM22 as a single-step authentication for mitochondrial protein import in C. merolae. The simple authentication mechanism indicates that the C. merolae cell, with its simple cell structure and genome, would not need to increase the cryptographic complexity of the lock-and-key for mitochondrial protein import.

cell biology↗

bioRxiv2024

Transcriptome analysis of the tardigrade Hypsibius exemplaris exposed to the DNA-damaging agent bleomycin

Tardigrades are microscopic animals that are renowned for their capabilities of tolerating near-complete desiccation by entering an ametabolic state called anhydrobiosis. However, many species also show high tolerance against radiation in the active state as well, suggesting cross-tolerance via the anhydrobiosis mechanism. Previous studies utilized indirect DNA damaging agents to identify core components of the cross-tolerance machinery; however, it was difficult to distinguish whether transcriptomic changes were the result of DNA damage or residual oxidative stress. To this end, we performed transcriptome analysis on bleomycin-exposed Hypsibius exemplaris. We observed induction of several tardigrade-specific gene families that may be the core components of the cross-tolerance mechanism. We also identified an enrichment of the tryptophan metabolism pathway, which metabolomic analysis suggested the engagement of this pathway in stress tolerance. These results provide several candidates for the core component of the cross-tolerance, as well as possible anhydrobiosis machinery.

molecular biology↗