Biology subjects

Shibata, M.

Publications and source records attributed to Shibata, M..

At least 19 recordsLinked to original sources

bioRxiv2026

Dual role of the RIG-I 2CARD as a signaling module and a brake on dsRNA translocation

RIG-I is an innate immune receptor for viral double-stranded RNA (dsRNA). Upon binding dsRNA, RIG-I nucleates MAVS filaments through its two caspase activation and recruitment domains (2CARD). However, how the 2CARD behaves during activation has remained unclear. Using high-speed atomic force microscopy, we visualized the 2CARD in full-length RIG-I across its apo, dsRNA-bound, translocating, multimeric, and polyubiquitin-bound states. Protrusions consistent with the 2CARD appeared only transiently in the apo state, whereas a mobile 2CARD emerged upon RNA binding. During ATP-driven translocation, wild-type RIG-I moved approximately twofold slower and showed more frequent pauses and multimers than RIG-I {Delta}2CARD. Within multimers, 2CARDs formed a complex that polyubiquitin stabilized into larger, dsRNA-deforming assemblies. These findings reveal the 2CARD as a brake on the RIG-I motor, beyond signaling.

biophysics↗

bioRxiv2026

Conformational dynamics of an intrinsically disordered receptor enable signal transduction

Signal transduction is initiated when activated cell-surface receptors physically engage intracellular signaling proteins. Although structural and biochemical studies have established the molecular architecture of receptor tyrosine kinase (RTK) signaling, the earliest dynamic events that couple receptor activation to intracellular signal propagation have remained inaccessible because existing approaches provide static structural snapshots or ensemble-averaged measurements rather than direct observation of molecular motion. Here, using high-speed atomic force microscopy (HS-AFM), we directly visualize these initial molecular events at the single-molecule level in real time. Using fibroblast growth factor receptor 1 (FGFR1) as a model, we show that receptor autophosphorylation releases its intrinsically disordered juxtamembrane region, enabling extensive conformational dynamics of the kinase domain that permit productive engagement of the signaling adaptor FRS2, a key initiator of downstream RTK signaling. In contrast, a kinase-inactive mutation or the clinically approved FGFR inhibitor futibatinib restricts receptor motion, stabilizes a compact receptor configuration, and markedly reduces adaptor engagement. Molecular dynamics simulations suggest that the disruption of inhibitory interactions between the juxtamembrane region and the kinase domain provides a structural explanation for the observed receptor dynamics. Together, our findings uncover a previously inaccessible physical mechanism underlying the initiation of RTK signaling and establish HS-AFM as a powerful platform for visualizing receptor dynamics, providing a framework for mechanistic studies of signal transduction and for the development and mechanistic evaluation of compounds that modulate receptor signaling.

biophysics↗

bioRxiv2026

Gradient Multinozzle 3D Printing

Direct ink writing is compatible with an expansive materials palette. While enabling diverse applications, this materials versatility brings significant bottlenecks in ink formulation, often requiring the mixing, printing, and testing of dozens to hundreds of ink compositions over the course of a project. To accelerate ink-space exploration, we introduce gradient embedded multinozzle (GEM) printheads that combine the high-throughput parallelized printing of multinozzles with combinatorial ink mixing. These printheads allow simultaneous mixing of two-, three-, and four-input inks which are distributed to printer nozzles to create complex 3D structures with graded compositions of inks. Using a two-way GEM printhead, we vali-date cell compatibility by printing scaffolds containing various concentrations of fibroblasts and observing non-linear compaction behaviours. We next test a three-way GEM multinozzle to print ten compositions of di- and multi-functionalized poly(ethylene-glycol) diacrylate hydrogel tri-leaflet valves, optimizing for stiffness, swelling ratio, and toughness. Our GEM multinozzles are compatible with open-source printers and either pressure- or volume-driven extrusion systems and promise to accelerate iterative ink design and testing.

bioengineering↗

bioRxiv2026

CaMKIIα holoenzymes self-organize into worm-like mesoscale clusters

Ca2+/calmodulin-dependent protein kinase II (CaMKII) is highly enriched in dendritic spines at concentrations comparable to those of cytoskeletal proteins and plays a central role in synaptic plasticity. During long-term potentiation (LTP), CaMKII further accumulates in spines. However, the mechanisms governing its higher-order organization remain poorly understood. Here, we use high-speed atomic force microscopy to visualize inter-holoenzyme interaction of CaMKII at mesoscopic scales (5-500 nm). Under freely diffusible conditions, CaMKII holoenzymes do not form stable clusters. In contrast, when spatially confined, they assemble into worm-like chain clusters mediated by kinase-domain interactions. These clusters expand upon activation, concomitant with the dissociation of the regulatory segment. Notably, the CaMKII P212L mutant associated with neurodevelopmental disorders, forms extensive clusters even in the basal state. Together, our findings demonstrate that CaMKII-CaMKII interactions drive mesoscale cluster formation and that precise regulation of cluster size and activation-dependent growth might be critical for synaptic signaling.

neuroscience↗

bioRxiv2025

A Retinoic Acid Autoregulatory Loop Governing Prefrontal-Motor Arealization

The frontal lobe comprises the prefrontal association cortex (PFC), which supports complex cognition and goal-directed behavior, and the motor cortex (MC), which executes movement. A hallmark of primate brain evolution is PFC expansion accompanied by a posterior displacement of the MC. Retinoic acid (RA) signaling has emerged as a key regulator of PFC specification and expansion. However, the mechanisms that spatially confine RA signaling within the developing PFC, and the downstream RA-responsive gene networks, remain poorly understood. Here we defined an RA-associated gene regulatory network (RA-GRN) in the developing human PFC and identified MEIS2, which encodes a transcription factor linked to intellectual disability and autism spectrum disorder (ASD), as its key hub of this network. Conditional deletion of Meis2 in postmitotic cortical excitatory neurons in mice results in a partial respecification of prospective prefrontal association territories toward motor-like molecular and connectional features, highlighting a critical role of postmitotic neurons in establishing and maintaining cortical areal identities. Concomitant with Meis2 loss, the population of excitatory neurons expressing the RA-synthesizing enzyme ALDH1A3, and consequently RA signaling itself, is markedly reduced in the developing medial prefrontal cortex (mPFC). These findings revealed a conserved autoregulatory loop: RA [->] MEIS2 [->] ALDH1A3 [->] RA that reinforces a PFC-enriched RA gradient and organizes the MC-PFC axis. Together, our findings reveal a postmitotic mechanism by which specific features of neuronal identity reinforce RA signaling to define key features of prefrontal and motor cortical territories, linking a classic morphogen to transcriptional identity, neural circuit formation and function, and potentially to psychiatric disorders.

neuroscience↗

bioRxiv2025

Evolution of HMA-integrated tandem kinases accompanied by expansion of target pathogens

Tandem kinase proteins (TKPs) are an emerging family of plant intracellular immune receptors that offer potential for developing novel disease resistance. We cloned Rmo2 and Rwt7, genes for resistance to the blast fungus, from barley and wheat, respectively, and discovered that they are orthologs encoding TKPs with an integrated N-terminal heavy metal-associated (HMA) domain. Rmo2 was collocated with Rpg1, a TKP gene for resistance to stem rust, while Rwt7 was collocated with WTK4, a TKP gene for resistance to powdery mildew. Domain swapping suggested that the HMA domain determines target effector specificity of these genes. We propose a model illustrating an evolutionary process in which a TKP gene has differentiated into paralogs and orthologs that recognize various effectors through diversification of their HMA domains.

plant biology↗

bioRxiv2025

Competing Programs Shape Cortical Sensorimotor-Association Axis Development

The neocortex is organized along a dominant sensorimotor-to-association (S-A) axis, anchored by modality-specific primary sensorimotor areas at one end and transmodal association areas that form distributed networks supporting abstract cognition at the other. The developmental mechanisms shaping this axis remain elusive. Here, we present converging multispecies evidence supporting the Multinodal Induction-Exclusion in Network Development (MIND) model, in which S-A patterning is governed by competing processes of induction and exclusion, driven by opposing transcriptomically-defined identity programs emerging from different nodes. Key molecular and connectional features of association cortices arise through pericentral programs, originating around fronto-temporal poles and partially regulated by retinoic acid. They progress inward toward central territories of the naive neocortex along fronto-temporally polarized trajectories. Central programs are induced through interactions between topographically separated first-order sensorimotor thalamocortical inputs and the neocortex, promoting the formation of primary areas while excluding pericentral programs. Influenced by SATB2 and ZBTB18, these evolutionarily conserved programs compete for the same territory and create spatial compartmentalization of axon guidance, cell-cell adhesion, retinoic acid signaling, synaptogenesis, Wnt signaling, and autism risk genes. Notably, PLXNC1 and SEMA7A exhibit anti-correlated expression and repulsive functions in shaping cortico-cortical connectivity along the S-A axis. These processes of induction and exclusion establish an S-A equilibrium and topography in which primary sensorimotor areas emerge as focal islands within the broader ocean of distributed associative networks. The MIND model provides a unifying framework for understanding experimental, evolutionary, and clinical phenomena, revealing induction and exclusion as antagonistic complementary principles shaping the S-A axis and processing hierarchies.

neuroscience↗

bioRxiv2025

Adaptive Evolution of Gene Regulatory Networks in Mammalian Neocortical Neurons

Mammals have evolved a plethora of adaptations that have enabled them to thrive in diverse environments. Among the most significant is the emergence of a more complex brain, exemplified by the dramatic transformation of the dorsal cortex from a single layer of excitatory projection neurons (ExNs) in ancestors to a multilayered cerebral neocortex enriched with diverse intratelencephalic (IT) and extratelencephalic (ET) ExN subtypes. These ExNs established specialized projection systems, such as the corticospinal tract and corpus callosum, enhancing brain connectivity and functionality. However, the evolutionary mechanisms underlying these mammalian-specific adaptations remain elusive. By comparing the landscape of gene expression and cis-regulatory elements (CREs) in mouse ExN subtypes and by cross-species examination of mammalian and non-mammalian CREs, we identified mammalian-specific CREs and expression patterns. The mammalian-specific CREs include a subset bound by ZBTB18 that are associated with genes defining IT and ET subtypes and connectivity. Both ZBTB18 and these target genes have previously been implicated in intellectual disability and autism. Deletion of Zbtb18 in mouse ExNs dysregulated target gene expression, reduced molecular diversity, diminished corticospinal and callosal projections, and increased intrahemispheric cortico-cortical association projections to the prefrontal cortex, resembling features of non-mammalian dorsal pallium. Interestingly, ZBTB18 binding motifs are highly enriched in callosally projecting IT-biased CREs, where they show higher conservation specifically in mammals. This study uncovers critical components and mammalian-specific evolutionary adaptations within a regulatory node essential for neocortical ExN identity and connectivity, with implications for neurodevelopmental and neuropsychiatric disorders.

neuroscience↗

bioRxiv2025

Structural dynamics of mixed-subunit CaMKIIα/β heterododecamers filmed by high-speed AFM

CaMKII predominantly assembles into a 12-meric ring assembly, primarily consisting of CaMKII and CaMKII{beta} variants in the brain. Previous biochemical studies have reported varying ratios of these CaMKII variants across different brain regions and developmental stages. However, direct evidence for the formation of CaMKII/{beta} heterooligomers within a 12-meric ring assembly has been lacking at the single-molecule level. Here, we employed high-speed atomic force microscopy to visualize the conformational dynamics of forebrain-mimicked CaMKII/{beta} at a 3:1 ratio. Our findings revealed that the CaMKII and CaMKII{beta} subunits are intermixed within the 12-meric ring assembly, with more than 83% probability that CaMKII{beta} subunits adjacent to one another. Furthermore, in the activated state, CaMKII/{beta} heterooligomers form a stable kinase domain complex via interactions between adjacent CaMKII{beta} subunits, resulting in a long-lasting structure with an exposed target binding site. Collectively, our observations provide insights into the structural role of CaMKII{beta} subunits within the CaMKII/{beta} heterododecamer.

neuroscience↗

bioRxiv2025

Structural insights into heterohexameric assembly of epilepsy-related ligand-receptor complex LGI1-ADAM22

Leucine-rich glioma-inactivated 1 protein (LGI1) is a secreted neuronal protein consisting of the N-terminal leucine-rich repeat (LRR) and C-terminal epitempin repeat (EPTP) domains. LGI1 is linked to epilepsy, a neurological disorder that can be caused by genetic mutations of genes regulating neuronal excitability (e.g., voltage- or ligand-gated ion channels). ADAM22 is a membrane receptor that binds to LGI1 extracellularly and interacts with AMPA-type glutamate receptors via PSD-95 intracellularly to maintain normal synaptic signal transmission. Structural analysis of the LGI1-ADAM22 complex is important for understanding the molecular mechanism of epileptogenesis and developing new therapies against epilepsy. We previously reported the crystal structure of a 2:2 complex consisting of two molecules of LGI1 and two molecules of the ADAM22 ectodomain (ECD), which is suggested to bridge neurons across the synaptic cleft. On the other hand, multiangle light scattering, small-angle X-ray scattering, and cryo-EM analyses have suggested the existence of a 3:3 complex consisting of three molecules of LGI1 and three molecules of ADAM22. In the previous cryo-EM analysis, many observed particles were in a dissociated state, making it difficult to determine the three-dimensional (3D) structure of the 3:3 complex. In this study, we stabilized the 3:3 LGI1-ADAM22ECD complex using chemical crosslinking and determined the cryo-EM structures of the LGI1LRR-LGI1EPTP- ADAM22ECD and 3:3 LGI1-ADAM22ECD complexes at 2.78 [A] and 3.79 [A] resolutions, respectively. Furthermore, high-speed atomic force microscopy (HS-AFM) visualized the structural features and flexibility of the 3:3 LGI1-ADAM22ECD complex in solution. We discuss new insights into the interaction modes of the LGI1-ADAM22 higher-order complex and the structural properties of the 3:3 LGI1-ADAM22 complex. SignificanceThe neuronal secretory protein Leucine-rich glioma-inactivated 1 (LGI1) and its receptor protein ADAM22 play a critical role in maintaining normal synaptic signal transmission. Genetic mutations in LGI1 are linked to epilepsy. Structural analysis of the LGI1-ADAM22 complex is crucial for understanding the molecular mechanisms of epileptogenesis and for developing targeted treatments. In this study, we determined the cryo-EM structure of the heterohexameric LGI1-ADAM22 complex and visualized the dynamics of this complex by high-speed atomic force microscopy.

biochemistry↗

bioRxiv2024

A low-cost, open-source 3D printer for multimaterial and high-throughput direct ink writing of soft and living materials

Direct ink writing is a 3D printing method that is compatible with a wide range of structural, elastomeric, electronic, and living materials, and it continues to expand its uses into physics, engineering, and biology laboratories. However, the large footprint, closed hardware and software ecosystems, and expense of commercial systems often hamper widespread adoption. Here, we present a compact, simple-to-build, low-cost, multimaterial, and high-throughput direct ink writing 3D printer platform with detailed assembly files and instructions provided freely online. In contrast to existing low-cost 3D printers and bioprinters, which typically modify off-the-shelf plastic 3D printers, this system is built from scratch, offering a lower cost and full customizability. Despite its low cost, we demonstrate advanced active mixing and multimaterial multinozzle 3D (MM3D) printing methods, which previously have relied on expensive and custom motion control platforms. We finally introduce embedded multinozzle and 3D gradient nozzle designs that offer high throughput and graded 3D parts. This powerful, easy-to-build, and customizable printing platform can help stimulate a vibrant biomaker community of engineers, biologists, and educators.

bioengineering↗

bioRxiv2023

High-speed AFM reveals fluctuations and dimer splitting of the N-terminal domain of GluA2-γ2

AMPA glutamate receptors (AMPARs) enable rapid excitatory synaptic transmission by localizing to the postsynaptic density of glutamatergic spines. AMPARs possess large extracellular N-terminal domains (NTDs), which participate in AMPAR clustering at synapses. Nevertheless, the dynamics of NTDs and the molecular mechanism governing their synaptic clustering remain elusive. Here, we employed high-speed atomic force microscopy (HS-AFM) to directly visualize the conformational dynamics of NTDs in the GluA2 subunit with TARP {gamma}2 in lipid environments. HS-AFM videos of GluA2-{gamma}2 in the resting and open states revealed fluctuations in NTD dimers. Conversely, in the desensitized state, the two NTD dimers adopted a separated conformation with less fluctuation. Notably, we visualized individual NTD dimers transitioning into monomers. Furthermore, this NTD-dimer splitting resulted in intersubunit exchange between NTD dimers and an increased number of binding sites with the synaptic protein neuronal pentraxin 1. Therefore, our findings illuminate the significance of NTD dynamics in the synaptic clustering of AMPARs.

neuroscience↗

bioRxiv2023

Evolution of wheat blast resistance gene Rmg8 accompanied by differentiation of variants recognizing the powdery mildew fungus

Wheat blast, a devastating disease having spread recently from South America to Asia and Africa, is caused by Pyricularia oryzae pathotype Triticum which emerged in 1985. Rmg8 and Rmg7, genes for resistance to wheat blast found in common wheat and tetraploid wheat, respectively, recognize the same avirulence gene, AVR-Rmg8. Here, we show an evolutionary process in which resistance gene(s), which had obtained an ability to recognize AVR-Rmg8 before the differentiation of Triticum and Aegilops, has expanded its target pathogens. Molecular cloning revealed that Rmg7 was one of alleles of Pm4 (Pm4a), a gene for resistance to wheat powdery mildew on 2AL, whereas Rmg8 was its homoeolog on 2BL ineffective against wheat powdery mildew. Rmg8 variants with the ability to recognize AVR-Rmg8 were distributed not only in Triticum spp. but also in Aegilops speltoides, Ae. umbellulata, and Ae. comosa. This result suggests that the origin of resistance gene(s) recognizing AVR-Rmg8 dates back to the time before differentiation of A, B, S, U, and M genomes, that is, [~]5 million years before the emergence of its current target, the wheat blast fungus. Phylogenetic analyses suggested that, in the evolutionary process thereafter, some of their variants gained the ability to recognize the wheat powdery mildew fungus and evolved into genes for resistance to wheat powdery mildew.

plant biology↗

bioRxiv2023

Breeding of a near-isogenic wheat line resistant to wheat blast at both seedling and heading stages through incorporation of Rmg8

Wheat blast caused by Pyricularia oryzae Triticum pathotype (MoT) has been transmitted from South America to Bangladesh and Zambia and is now spreading in these countries. To prepare against its further spread to Asian countries, we introduced Rmg8, a gene for resistance to wheat blast, into a Japanese elite cultivar, Chikugoizumi (ChI), through recurrent backcrosses, and established ChI near-isogenic lines, #2-1-10 with the Rmg8/Rmg8 genotype and #4-2-10 with the rmg8/rmg8 genotype. A molecular analysis suggested that at least 96.6% of the #2-1-10 genome was derived from the recurrent parent ChI. The #2-1-10 line was resistant to MoT not only in primary leaves at the seedling stage but also in spikes and flag leaves at the heading stage. The strength of the resistance in spikes of this Rmg8 carrier was comparable to that of a carrier of the 2NS segment which has been the only one genetic resource released to farmers field for wheat blast resistance. On the other hand, the 2NS resistance was not expressed on leaves at the seedling stage nor flag leaves at the heading stage. Considering that leaf blast has been increasingly reported and regarded as an important inoculum source for spike blast, Rmg8 expressed at both the seedling and heading stages, or more strictly in both leaves and spikes, is suggested to be useful to prevent the spread of MoT in Asia and Africa.

plant biology↗

bioRxiv2023

Antithetic effects of agonists and antagonists on the structural fluctuations of TRPV1 channel

Transient receptor potential vanilloid member 1 (TRPV1) is a heat and capsaicin receptor that allows cations to permeate and cause pain. As the molecular basis for temperature sensing, the heat capacity ({Delta}Cp) model (D. E. Clapham, C. Miller, Proc. Natl. Acad. Sci. U. S. A. 108, 19492-19497 (2011).) has been proposed and experimentally supported. Theoretically, heat capacity is proportional to a variance in enthalpy, presumably related to structural fluctuation; however, the fluctuation of TRPV1 has not been directly visualized. In this study, we directly visualized single-molecule structural fluctuations of the TRPV1 channels in a lipid bilayer with the ligands resiniferatoxin (RTX: agonist, 1000 times hotter than capsaicin) and capsazepine (CPZ: antagonist) by high-speed atomic force microscopy (HS-AFM). We observed the structural fluctuations of TRPV1 in an apo state and found that RTX binding enhances structural fluctuations, while CPZ binding suppresses fluctuations. These ligand-dependent differences in structural fluctuation would play a key role in the gating of TRPV1.

physiology↗

bioRxiv2023

Evolutionarily acquired activity-dependent transformation of the CaMKII holoenzyme

Ca2+/calmodulin-dependent protein kinase II (CaMKII) has long been central in synaptic plasticity research. CaMKII is a dodecameric serine/threonine kinase that has been essentially conserved across metazoans for over a million years. While the mechanisms of CaMKII activation are well studied, its "behavior" at the molecular level has remained unobserved. Here, high-speed atomic force microscopy was used to visualize the activity-dependent structural dynamics of rat/hydra/C. elegans CaMKII in various states at nanometer resolution. Among the species, rat CaMKII underwent internal kinase domain aggregation in an activity-dependent manner and showed a higher tolerance to dephosphorylation by phosphatase. Our findings suggest that mammalian CaMKII has evolutionarily acquired a new structural form and a tolerance to phosphatase to maintain robust CaMKII activity for proper neuronal function. One-Sentence SummaryHigh-speed atomic force microscopy reveals the activity-dependent structural dynamics of rat/hydra/C. elegans CaMKII

neuroscience↗

bioRxiv2022

Large-area electrical imaging having single neuron resolution using 236,880 electrodes CMOS-MEA technology

The electrophysiological technology having a high spatio-temporal resolution at the single-cell level, and noninvasive measurements of large areas provides insights on underlying neuronal function. Here, we used a complementary metal-oxide semiconductor (CMOS)-microelectrode array (MEA) that uses 236,880 electrodes each with an electrode size of 11.22 x 11.22 {micro}m and 236,880 covering a wide area of 5.5 x 5.7 mm in presenting a detailed and single-cell-level neural activity analysis platform for brain slices, human iPS cell-derived cortical networks, peripheral neurons, and human brain organoids. Propagation pattern characteristics between brain regions changes the synaptic strength into compounds based on single-cell time-series patterns, classification based on single DRG neuron firing patterns and compound responses, axonal conduction characteristics and changes to anticancer drugs, and network activities and transition to compounds in brain organoids were extracted. This detailed analysis of neural activity at the single-cell level using our CMOS-MEA provides a new understanding the basic mechanisms of brain circuits in vitro and ex vivo, on human neurological diseases for drug discovery, and compound toxicity assessment.

neuroscience↗

bioRxiv2022

The OncoLoop network-based precision oncology framework

At present, prioritizing cancer treatments at the individual patient level remains challenging, and performing co-clinical studies using patient-derived models in real-time is often not feasible. To circumvent these challenges, we introduce OncoLoop, a precision medicine framework to predict and validate drug sensitivity in human tumors and their pre-existing high-fidelity (cognate) model(s) by leveraging perturbational profiles of clinically-relevant oncology drugs. As proof-of-concept, we applied OncoLoop to prostate cancer (PCa) using a series of genetically-engineered mouse models (GEMMs) that recapitulate a broad spectrum of disease states, including castration-resistant, metastatic, and neuroendocrine prostate cancer. Interrogation of published cohorts using Master Regulator (MR) conservation analysis revealed that most patients were represented by at least one cognate GEMM-derived tumor (GEMM-DT). Drugs predicted to invert MR activity in patients and their cognate GEMM-DTs were successfully validated, including in two cognate allografts and one cognate patient-derived xenograft (PDX). OncoLoop is a highly generalizable framework that can be extended to other cancers and potentially other diseases. Significance StatementOncoLoop is a transcriptomic-based experimental and computational framework that can support rapid-turnaround co-clinical studies to identify and validate drugs for individual patients, which can then be readily adapted to clinical practice. This framework should be applicable in many cancer contexts for which appropriate models and drug perturbation data are available.

cancer biology↗