Biology subjects

Kawakami, K.

Publications and source records attributed to Kawakami, K..

At least 19 recordsLinked to original sources

bioRxiv2026

Cellular hydraulics ensures robust endothelial-to-haematopoietic transition

Haematopoietic stem and progenitor cells (HSPCs) emerge from specialised haemogenic endothelial cells (HECs) in the ventral wall of the dorsal aorta (VDA) through a process known as endothelial-to-haematopoietic transition (EHT). During EHT, elongated HECs undergo actomyosin-driven rounding and extrusion, a mechanically demanding transformation that requires cell integrity to be maintained. Here, we identify an osmo-hydraulic volume-regulation mechanism that enables HECs to adapt to and withstand this mechanical challenge. Haemodynamic forces increase Piezo-dependent calcium ion activity in ventral endothelial cells, while Piezo activation promotes HEC swelling. Combined genetic and pharmacological analyses support a pathway in which Piezo-mediated swelling triggers VRAC-dependent osmolyte efflux and Aqp1a.1-mediated water efflux to reduce HEC volume. Disrupting either osmolyte or water efflux causes excessive HEC swelling, compromises HEC integrity and reduces HSPC production. Together, our findings establish osmo-hydraulic volume regulation as a mechanism that preserves cellular robustness during EHT and support a model in which aquaporins act as pressure-relief valves, dissipating intracellular hydrostatic pressure to sustain HEC survival and definitive haematopoiesis.

cell biology↗

bioRxiv2026

A Rapid and Universal Pipeline for High-Resolution GPCR Structure Determination through In Silico Construct Optimization and de novo Protein Design

G protein-coupled receptors (GPCRs) are critical regulators of human physiology and major drug targets. Although structural studies have provided valuable insights, determining GPCR structures remains challenging, especially for inactive state receptors. Recent advances in cryo-electron microscopy (cryo-EM) have enabled structural determination of small GPCRs by using fusion partner proteins and binders to increase molecular weight. However, current methods require extensive experimental screening of fusion constructs. Widely adopted strategies, such as BRIL-Fab complexes, also face limitations due to inherent flexibility. Here, we introduce a streamlined and universal pipeline that integrates an in silico fusion construct screening program, NOAH (NOAH: NOn-experimental, AI-assisted High-throughput construct screening), with a de novo designed fusion protein called ARK1 (ARtificially-designed fiducial marKer). We validate the efficacy of NOAH by determining the structures of the vasopressin V2 receptor (V2R) bound to the clinical antagonist tolvaptan and the partial agonist OPC51803, as well as the bradykinin B2 receptor (B2R) bound to the clinical antagonist icatibant, thereby elucidating their activation and deactivation mechanisms. Furthermore, we demonstrate the capability of NOAH-ARK1 by solving the tolvaptan-bound V2R structure at higher resolution and showcase the methods versatility by determining the structure of lysophosphatidic acid receptor 2 (LPA2) bound to the antagonist Ki16425. This approach eliminates the need for time-consuming and labor-intensive construct optimization, providing a rapid and widely applicable solution for high-resolution GPCR structure determination and drug discovery.

biophysics↗

bioRxiv2026

The not-so-great speciator: Systematics and species limits in a rapid radiation, the Asiatic white-eye complex (Zosterops spp.)

Abstract/SummaryHere we untangle the systematics of the Asiatic white-eye complex (Zosterops spp.) to better understand the early stages of a recent island radiation. We adopt an integrative approach involving allelic data, genome-scale single nucleotide polymorphisms (SNPs), and museum-based morphometrics coupled with a comprehensive sampling to provide the most holistic understanding of the group to date. The island lineages of Asiatic white-eyes across Indonesia, the Philippines, East Asia, the adjacent oceanic islands of the Western Pacific underwent a deep split separating Zosterops everetti and Z. nigrorum in the Phillippines from a very rapid radiation including Z. japonicus, Z. meyeni, and Z. montanus in the Philippines, Japan, and Indonesia. Z. nigrorum catarmanensis on Camiguin South in the Philippines was found to be nested within Z. montanus and a species limit between Z. nigrorum populations on Panay and Luzon was strongly supported. Phylogenetic splits and population structure were detected within the clade containing Z. japonicus, Z. meyeni, and Z. montanus. A well-supported split separates a northern group including Northern Philippines Z. montanus subspecies, Z. meyeni, and Z. japonicus from the southerly Z. montanus taxa. This creates a paraphyletic Z. montanus. However, based on speciation rates within the broader Asiatic white-eye clade this break likely does not yet represent evolutionarily independent species lineages. Morphological evolution is taking place within the Asiatic white-eyes especially within the robust, large-billed subspecies of Z. japonicus on the oceanic islands of Japan and in the newly identified yellow-morph of Z. montanus on Camiguin South.

evolutionary biology↗

bioRxiv2025

Hierarchical processing of sensory information across topographically organized thalamocortical-like circuits in the zebrafish brain

Thalamocortical projections contribute to the spatial organization and functional hierarchies of the mammalian cortex. Primary sensory cortices receive topographically segregated information from first-order thalamic nuclei, which process distinct sensory modalities. In contrast, higher-order cortical regions integrate information from multiple information channels. While such hierarchical processing and integration of information is the foundation for neural computations in the mammalian cortex, the fundamental principles of thalamocortical computations in non-mammalian vertebrates remains unexplored. The zebrafish pallium, located in the dorsal telencephalon, is regarded as the homolog of the mammalian cortex. However, it remains unclear how the zebrafish pallium receives and processes sensory information, and how the architecture and function of these processes compare to the thalamocortical circuits in other vertebrates. Using anatomical tracing, electrophysiological circuit mapping, and in vivo Ca2+ imaging, we revealed a thalamocortical-like pathway in the zebrafish brain. We found that the preglomerular nucleus (PG) is the primary source of visual and mechano-vibrational information to the zebrafish pallium. We demonstrated that PG neurons and their pallial projections exhibit sensory-specific and topographically organized responses. In contrast, the sensory responses of pallial neurons display multiple layers of topographically organized hierarchies, ranging from simple sensory-specific responses to multimodal and coincidence-detecting nonlinear responses. Notably, we observed a progressive increase in the complexity of sensory computations, which is organized topographically along the posterior-anterior axis of the zebrafish pallium. Collectively, our results suggest that hierarchical sensory processing across topographically organized pallial regions is a conserved functional feature of the vertebrate pallium.

neuroscience↗

bioRxiv2025

Organ-specific immune responses are strain-dependent in a mouse model of Cryptococcus neoformans brain infection

Cryptococcal meningitis remains the top cause of death from an invasive fungal infection in humans, responsible for [~]100,000 deaths annually of vulnerable patients with underlying immune deficiencies. Animal models of cryptococcal meningitis are important for understanding the immune parameters that correlate with protection. However, modelling this infection in mice is challenging. There is wide variability in infection routes, doses and mouse background used in the field, which makes understanding phenotypes of mutants and immune interventions difficult to broadly apply. Our intention was to create a resource for the field on how Cryptococcus neoformans strain influences analysis of organ-specific immune responses in an intravenous mouse model of cryptococcal meningitis, focusing on impact of the fungal strain while keeping mouse genetic background (C57BL/6J) and infection route constant. We quantified myeloid and lymphoid cell recruitment and fungal-specific CD4 T-cell activation, correlating these results with fungal burdens in mice infected with the commonly used reference strain H99 or with two recently isolated clinical strains that were the same molecular type (VNI) or an unrelated type (VNB). We also analysed how dose used in murine infection models affected brain immune responses during C. neoformans infection. Our work reveals intriguing patterns of organ-specific immunity that are dependent on C. neoformans strain but not always explained by virulence potential, raising important questions for the field regarding the impact of C. neoformans strain on cellular immune responses in experimental animal models.

immunology↗

bioRxiv2025

Molecular and functional dissection using CaMPARI-seq reveals the neuronal organization for dissociating optic flow-dependent behaviors

Optic flow processing is critical for the visual control of body and eye movements in many animals. Rotational and translational binocular optic flow patterns need to be clearly distinguished to induce different behavior outputs. However, the specific neuron types and their connectivity involved in this computation remain unclear. Here, we developed a method to link the functional labeling using a photoconvertible calcium indicator called CaMPARI2 and single-cell RNA-sequencing (CaMPARI-seq) to investigate the transcriptional profile of the pretectum, a center for processing optic flow in larval zebrafish. Using this technique, we identified a pretectal cluster expressing tcf7l2, which can be further classified into molecularly distinct subclusters. In vivo calcium imaging and cell ablation revealed that nkx1.2lb-positive pretectal neurons are commissural inhibitory neurons required for the optomotor response but not for the optokinetic response. Our genetic and functional dissection using CaMPARI-seq uncovered the neuronal organization essential for dissociating different optic flow-dependent behaviors.

neuroscience↗

bioRxiv2025

Intrinsic and TDP-43 dysfunction-induced catabolic stress elicit neuroprotective cellular degradation in ALS-vulnerable motor neurons

Selective neuronal vulnerability is a defining feature of neurodegenerative disorders, exemplified by motor neuron degeneration in amyotrophic lateral sclerosis (ALS). The nature of motor neurons underlying this selectivity remains unresolved. Here, by monitoring autophagy at single-cell resolution across the translucent zebrafish spinal cord, we identify motor neurons as the cell population with the highest autophagic flux. Large spinal motor neurons (SMNs), most susceptible to ALS, exhibit higher flux compared to smaller SMNs and ALS-resistant ocular motor neurons. Notably, large SMNs accelerates both autophagy and proteasome-mediated degradation, which are further augmented by TDP-43 loss. Additionally, acceleration of multiple unfolded protein response pathways indicates their innate tendency to accumulate misfolded proteins. Enhanced cellular degradation in large SMNs is neuroprotective as its inhibition halts axon outgrowth. These findings propose that cell size-associated degradation load underlies selective neuronal vulnerability in ALS, highlighting the alleviation of catabolic stress as a target of therapy and prevention.

neuroscience↗

bioRxiv2025

Analysis of Treacher Collins syndrome 4-associated mutations in Schizosaccharomyces pombe

Treacher Collins syndrome (TCS) is a rare congenital disorder characterized by craniofacial deformities. Although mutations in several genes involved in ribosome biogenesis have been identified in patients with TCS, the molecular mechanisms by which these mutations exert their effects remain poorly understood. In this study, we established Schizosaccharomyces pombe models for TCS type 4 (TCS4) by introducing specific mutations in the rpa2 gene, which encodes Rpa2, the second largest subunit of RNA polymerase I (Pol I). TCS4-associated rpa2R1022C and rpa2R1022S mutations impaired cell growth without altering Rpa2 protein levels. These mutants were defective in 35S pre-rRNA biogenesis and were sensitive to the Pol I inhibitor BMH-21. These findings underscore the essential role of Rpa2 residues associated with TCS4 in rRNA transcription and cell growth.

molecular biology↗

bioRxiv2025

Immune-induced TCR-like antibodies regulate specific T cell response

Antigen-specific regulation of T cell response is crucial for limiting hyperimmune response. In this study, we discovered that antibodies specific to the antigen peptide-MHC class II complex are produced during helper T cell responses to various antigens. These antibodies specifically inhibited T cell receptor (TCR) recognition of MHC class II molecules presenting specific antigen peptide. We have termed these antibodies immuneinduced TCR-like antibodies (iTabs). Immunization with peptides containing flanking residues induced iTabs, whereas immunization with peptides lacking flanking residues did not. Furthermore, immunization of iTab-inducible peptide as well as iTab treatment suppressed autoimmune disease development. Our findings provide a strategy for suppressing antigen-specific helper T cell responses using specific peptides, potentially controlling autoimmune diseases.

immunology↗

bioRxiv2024

Gene identification for ocular congenital cranial motor neuron disorders using human sequencing, zebrafish screening, and protein binding microarrays

PurposeTo functionally evaluate novel human sequence-derived candidate genes and variants for unsolved ocular congenital cranial dysinnervation disorders (oCCDDs). MethodsThrough exome and genome sequencing of a genetically unsolved human oCCDD cohort, we previously identified variants in 80 strong candidate genes. Here, we further prioritized a subset of these (43 human genes, 57 zebrafish genes) using a G0 CRISPR/Cas9-based knockout assay in zebrafish and generated F2 germline mutants for seventeen. We tested the functionality of variants of uncertain significance in known and novel candidate transcription factor-encoding genes through protein binding microarrays. ResultsWe first demonstrated the feasibility of the G0 screen by targeting known oCCDD genes phox2a and mafba. 70-90% of gene-targeted G0 zebrafish embryos recapitulated germline homozygous null-equivalent phenotypes. Using this approach, we then identified three novel candidate oCCDD genes (SEMA3F, OLIG2, and FRMD4B) with putative contributions to human and zebrafish cranial motor development. In addition, protein binding microarrays demonstrated reduced or abolished DNA binding of human variants of uncertain significance in known and novel sequence-derived transcription factors PHOX2A (p.(Trp137Cys)), MAFB (p.(Glu223Lys)), and OLIG2 (p.(Arg156Leu)). ConclusionsThis study nominates three strong novel candidate oCCDD genes (SEMA3F, OLIG2, and FRMD4B) and supports the functionality and putative pathogenicity of transcription factor candidate variants PHOX2A p.(Trp137Cys), MAFB p.(Glu223Lys), and OLIG2 p.(Arg156Leu). Our findings support that G0 loss-of-function screening in zebrafish can be coupled with human sequence analysis and protein binding microarrays to aid in prioritizing oCCDD candidate genes/variants.

neuroscience↗

bioRxiv2024

Dimeric assembly of F1-like ATPase for Mycoplasma gliding motility

Rotary ATPases, including F1Fo- and V/A-ATPases, are molecular motors that exhibit rotational movements for energy conversion1. In the gliding bacterium, Mycoplasma mobile, a dimeric F1-like ATPase forms a chain structure with the glycolytic enzyme, phosphoglycerate kinase (PGK), within the cell2, which is proposed to drive the bacterial gliding motility2-4. However, the mechanisms of force generation and transmission remain unclear. Here, we present a 3.2 [A] resolution structure of the dimeric ATPase complex obtained using electron cryomicroscopy (cryo-EM). Notably, the structure revealed an assembly distinct from that of known dimeric forms of F1Fo-ATPase5, despite containing conserved F1-ATPase structures. The two ATPase units were interconnected by GliD dimers, previously identified as MMOB16202,6. Gli{beta}, a homologue of the F1-ATPase catalytic subunit6, exhibited a specific N-terminal region that incorporates PGK into the complex. Structural conformations of the catalytic subunits, catalytically important residues, and nucleotide-binding pattern of the catalytic sites of the ATPase displayed strong similarities to F1-ATPase, suggesting a rotation based on the rotary catalytic mechanism conserved in rotary ATPases1,7-10. Overall, the cryo-EM structure underscores an evolutionary connection in the rotary ATPases and provides insights into the mechanism through which F1-like ATPase drives bacterial gliding motility.

microbiology↗

bioRxiv2024

Induction of NASH and vacuolar structures in the Nwd1-/- mouse liver via SERCA2-dependent endoplasmic reticulum stress

The endoplasmic reticulum (ER) stores Ca2+ and plays crucial roles in protein folding, lipid transfer, and its perturbations trigger an ER stress. In the liver, chronic ER stress is involved in the pathogenesis of nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH). Previous studies revealed that dysfunction of sarco/endoplasmic reticulum calcium ATPase (SERCA2), a key regulator of Ca2+ transport from the cytosol to the ER, is associated with the induction of ER stress and lipid droplet formation. We previously identified NACHT and WD repeat domain-containing protein 1 (Nwd1), which is localized in the ER and mitochondria. However, the physiological significance of Nwd1 outside the central nervous system remains unclear. In this study, we revealed that Nwd1 knockout mice exhibited pathological manifestations comparable to NASH. Nwd1 interacts with SERCA2 near ER membranes. Nwd1-/- livers exhibited reduced SERCA2 ATPase activity and a smaller Ca2+ pool in the ER, leading to an exacerbated state of ER stress. These findings highlight the importance of SERCA2 activity mediated by Nwd1 in the pathogenesis of NASH. HighlightsO_LINwd1-/- mice exhibited NASH-like liver steatosis. C_LIO_LIElevated ER stress, fibrosis, and pyroptosis were observed in Nwd1-/- livers. C_LIO_LINwd1 interacts with SERCA2, an ER membrane Ca2+ pump. C_LIO_LINwd1-/- livers exhibited reduced SERCA2 activity and smaller Ca2+ pools in the ER. C_LI

molecular biology↗

bioRxiv2023

Structure of PSI-LHCI from Cyanidium caldarium provides evolutionary insights into conservation and diversity of red-lineage LHCs

Light-harvesting complexes (LHCs) are diversified among photosynthetic organisms, and their structural variety in photosystem I-LHC (PSI-LHCI) supercomplexes has been shown. However, structural and evolutionary correlations of red-lineage LHCs are unknown. Here we determined a 1.92-[A] resolution cryo-electron microscopic structure of a PSI-LHCI supercomplex isolated from the red alga Cyanidium caldarium RK-1 (NIES-2137) which is an important taxon in the Cyanidiophyceae, and subsequently investigated these correlations through structural comparisons and phylogenetic analysis. The PSI-LHCI structure shows five LHCI subunits together with a PSI-monomer core. The five LHCIs are composed of two Lhcr1s, two Lhcr2s, and one Lhcr3. Phylogenetic analysis of LHCs bound to PSI in red-lineage algae showed clear orthology of LHCs between C. caldarium and Cyanidioschyzon merolae, whereas no orthologous relationships were found between C. caldarium Lhcr1-3 and LHCs in other red-lineage PSI-LHCI structures. These findings provide evolutionary insights into conservation and diversity of red-lineage LHCs associated with PSI.

plant biology↗

bioRxiv2023

Dysregulated TDP-43 proteostasis perturbs excitability of spinal motor neurons during brainstem-mediated fictive locomotion in zebrafish

Spinal motor neurons (SMNs) are the primary target of degeneration in amyotrophic lateral sclerosis (ALS). Degenerating motor neurons accumulate cytoplasmic TAR DNA-binding protein 43 (TDP-43) aggregates in most ALS cases. This SMN pathology can occur without mutation in the coding sequence of the TDP-43-encoding gene, TARDBP. Whether and how wild-type TDP-43 drives pathological changes in SMNs in vivo remain largely unexplored. In this study, we develop a two-photon calcium imaging setup in which tactile-evoked neural responses of motor neurons in the brainstem and spinal cord can be monitored using the calcium indicator GCaMP. We devise a piezo-assisted tactile stimulator that reproducibly evokes a brainstem descending neuron upon tactile stimulation of the head. A direct comparison between caudal primary motor neurons (CaPs) with or without TDP-43 overexpression in contiguous spinal segments demonstrates that CaPs overexpressing TDP-43 display attenuated Ca2+ transients during fictive escape locomotion evoked by the tactile stimulation. These results show that excessive amounts of TDP-43 protein reduce the neuronal excitability of SMNs and potentially contribute to asymptomatic pathological lesions of SMNs and movement disorders in patients with ALS.

neuroscience↗

bioRxiv2023

Mrc1Claspin is essential for heterochromatin maintenance in Schizosaccharomyces pombe

Heterochromatin is a highly condensed chromatin structure that represses gene expression. In eukaryotes, maintenance of the heterochromatin structure during cell proliferation is essential for guaranteeing cell identity. However, how heterochromatin is maintained and transmitted to the daughter cells remains largely unknown. In this study, we constructed a reporter system to study the maintenance of heterochromatin in the subtelomeric region of the fission yeast, Schizosaccharomyces pombe. We demonstrated that once subtelomeric heterochromatin was established, it tended to be maintained as a metastable structure through cell proliferation. Using this system, we screened an S. pombe genome-wide gene deletion library and identified 57 factors required for the maintenance of subtelomeric heterochromatin. We focused on Mrc1Claspin, a mediator of DNA replication checkpoint. We found that Mrc1 maintains heterochromatin structure not only at the subtelomeres but also at other heterochromatic loci, such as the pericentromeres and mating-type regions. Furthermore, we showed that Mrc1 facilitates the hypoacetylation state of histone H3K14 by recruiting the Snf2/Hdac-containing Repressor Complex (SHREC), via physical interaction. In addition, depletion of Mst2, an H3K14 acetyltransferase, restored heterochromatin integrity in mrc1 mutants. This is the first report to show a link between DNA replication factors and H3K14 deacetylation in heterochromatin.

molecular biology↗

bioRxiv2023

Phosphorylation barcodes direct biased chemokine signaling at CXCR3

G protein-coupled receptor (GPCR) biased agonism, the activation of some signaling pathways over others, is thought to largely be due to differential receptor phosphorylation, or "phosphorylation barcodes." At chemokine receptors, ligands act as "biased agonists" with complex signaling profiles, which contributes to the limited success in pharmacologically targeting these receptors. Here, mass spectrometry-based global phosphoproteomics revealed that CXCR3 chemokines generate different phosphorylation barcodes associated with differential transducer activation. Chemokine stimulation resulted in distinct changes throughout the kinome in global phosphoproteomic studies. Mutation of CXCR3 phosphosites altered {beta}-arrestin conformation in cellular assays and was confirmed by molecular dynamics simulations. T cells expressing phosphorylation-deficient CXCR3 mutants resulted in agonist- and receptor-specific chemotactic profiles. Our results demonstrate that CXCR3 chemokines are non-redundant and act as biased agonists through differential encoding of phosphorylation barcodes and lead to distinct physiological processes.

biochemistry↗

bioRxiv2023

A glia cell dependent mechanism at a peripheral nerve plexus critical for target-selective axon regeneration

A critical step for functional recovery from peripheral nerve injury is for regenerating axons to connect with their pre-injury targets. Reestablishing pre-injury target specificity is particularly challenging for limb-innervating axons as they encounter a plexus, a network where peripheral nerves converge, axons from different nerves intermingle, and then re-sort into target-specific bundles. Here, we examine this process at a plexus located at the base of the zebrafish pectoral fin, equivalent to tetrapod forelimbs. Using live cell imaging and sparse axon labeling, we find that regenerating motor axons from three nerves coalesce into the plexus. There, they intermingle and sort into distinct branches, and then navigate to their original muscle domains with high fidelity that restores functionality. We demonstrate that this regeneration process includes selective retraction of mistargeted axons, suggesting active correction mechanisms. Moreover, we find that Schwann cells are enriched and associate with axons at the plexus, and that Schwann cell ablation during regeneration causes profound axonal mistargeting. Our data provide the first real time account of regenerating vertebrate motor axons navigating a nerve plexus and reveal a previously unappreciated role for Schwann cells to promote axon sorting at a plexus during regeneration.

neuroscience↗

bioRxiv2022

Determinants of Motor Neuron Functional Subtypes Important for Locomotor Speed

Locomotion requires precise control of the strength and speed of muscle contraction and is achieved by recruiting functionally-distinct subtypes of motor neurons (MNs). MNs are essential to movement and differentially susceptible in disease, but little is known about how MNs acquire functional subtype-specific features during development. Using single-cell RNA profiling in embryonic and larval zebrafish, we identify novel and conserved molecular signatures for MN functional subtypes, and identify genes expressed in both early post-mitotic and mature MNs. Assessing MN development in genetic mutants, we define a molecular program essential for MN functional subtype specification. Two evolutionarily-conserved transcription factors, Prdm16 and Mecom, are both functional subtype-specific determinants integral for fast MN development. Loss of prdm16 or mecom causes fast MNs to develop transcriptional profiles and innervation similar to slow MNs. These results reveal the molecular diversity of vertebrate axial MNs and demonstrate that functional subtypes are specified through intrinsic transcriptional codes.

neuroscience↗