Biology subjects

Miyata, T.

Publications and source records attributed to Miyata, T..

At least 19 recordsLinked to original sources

bioRxiv2026

Embryonic macrophages chronologically change their brain-entry routes and competence for neural progenitor-induced microglial differentiation

Microglia arise from macrophage precursors that enter the developing brain and acquire microglial identity, but how these processes are coordinated remains unclear. We show that despite prominent entry of macrophages from the ventricles into the mouse telencephalon at embryonic day (E) 12.5, they enter from the meninges at E11.5, preferentially in the rostral pallium. Consistent with this regional pattern, whole-mount en face imaging revealed that focal holes in the pial basement membrane were most frequently observed in the rostral pallium. We found that neural progenitors, but not endothelial cells, promote acquisition of microglial properties through a process that requires close cellular proximity and partly depends on TGF-{beta} receptor signaling. This responsiveness is substantially stronger in E13.5 than in E17.5 meningeal macrophages, indicating stage-dependent competence to acquire microglial properties. These findings suggest that microglial establishment is developmentally regulated by macrophage access to the telencephalon and responsiveness to local neural cues.

developmental biology↗

bioRxiv2026

Reconstitution in a minimal synthetic bacterium reveals distinct Mollicute MreB-based motility systems driven by orthogonal force directions

MreB, a bacterial actin homolog, is widely conserved in bacteria where it functions as a scaffold for a peptidoglycan synthesis. Intriguingly, members of the cell wall-lacking Mollicutes retain multiple mreB genes despite the absence of peptidoglycan. Previous work demonstrated that two MreB isoforms from Spiroplasma can reconstitute swimming motility in the minimal synthetic bacterium syn3B, indicating that MreB was repurposed for motility. Here, we expressed seven MreBs from Haloplasma contractile, an early-diverging Mollicute, in syn3B. Approximately 50% of cells elongated, and 15% exhibited curving and coiling movements that resembled Haloplasma but differed from Spiroplasma motility. Systematic analysis of isoform combinations revealed that movement requires specific MreB pairs, and that additional isoforms enhance motility efficiency. Cryo-electron tomography showed membrane-associated ribbons composed of MreB filaments. Mathematical modeling demonstrated that Haloplasma- and Spiroplasma-type movements can be reproduced by altering only the direction of force generation. Thus, diversification of force orientation within an actin homolog enabled independent evolution of distinct motility systems in Mollicutes.

Synthetic Biology↗

bioRxiv2026

Replication protein A prevents unregulated priming and Rad51 loading on single-stranded DNA in nuclear extracts of Xenopus eggs

In eukaryotes, single-stranded DNA (ssDNA) generated during DNA replication, recombination, and repair is rapidly bound and protected by the major single-stranded DNA-binding protein replication protein A (RPA). RPA not only stabilizes ssDNA but also acts as a central platform that coordinates diverse DNA transactions. Exhaustion of RPA due to unregulated ssDNA production leads to replication fork breakage and replication catastrophe, underscoring its critical role in genome stability. However, the direct consequences of RPA limitation remain incompletely understood. Using Xenopus egg extracts, we show that excess ssDNA induces spontaneous priming, a reaction that is otherwise prevented in a physiological nuclear environment. We provide evidence that priming suppression is mediated by stoichiometric binding of RPA to ssDNA. Analysis of the ssDNA-binding proteome reveals that RPA promotes the association of ATR checkpoint factors, Pol-primase, and the RFWD3 ubiquitin ligase with ssDNA. In contrast, RPA depletion induces the recruitment of Rad51, Rad51 paralogs, and Fbh1, a DNA helicase that interacts with both RPA and Rad51 and promotes fork breakage under replication stress. Collectively, our findings suggest that RPA contributes to genome stability by protecting ssDNA from inappropriate DNA synthesis and unscheduled recruitment of recombination and fork-processing factors.

biochemistry↗

bioRxiv2026

Tractometry reproducibility and generalizability across scanners, scanner models, and acquisition protocols

Diffusion-weighted magnetic resonance imaging (dMRI)-based tractometry enables the quantification of white matter tissue properties in living humans while preserving anatomical specificity. Although tractometry is highly reproducible when the same scanner and acquisition protocol are used, its generalizability across scanners and protocols remains unclear. To address this gap, we performed a traveling-head experiment involving five subjects to evaluate tractometry across progressively different acquisition conditions, including multiple scanners, different scanner models, and two distinct protocols. Tractometry was performed for 20 major white matter tracts using diffusion tensor imaging metrics, neurite orientation dispersion and density imaging (NODDI) metrics, and a semi-quantitative ratio metric (T1w/b0). Generalizability across dataset pairs was quantified using the intraclass correlation coefficient (ICC). Tractometry showed consistently high ICCs when the scanner and protocol were identical; however, ICCs declined as differences in scanner model and acquisition protocol increased. Fractional anisotropy and orientation dispersion index retained relatively high ICCs across these comparisons, whereas other metrics showed marked declines when scanners or protocols differed. ComBat harmonization partially mitigated these declines, but ICCs did not reach the levels observed for datasets acquired using identical scanners and protocols. Finally, the minimum detectable change (MDC) for tractometry in datasets pooled across scanners and protocols varied by tract; for example, the optic radiation showed a lower MDC than the cingulum hippocampus. These findings highlight both the strengths and limitations of tractometry in multisite studies and highlight the importance of quantifying scanner- and protocol-dependent effects for specific metrics and tracts when interpreting measurements from heterogeneous datasets.

neuroscience↗

bioRxiv2026

Inward thickening of embryonic neuron-packing brain walls: Mechanical integrity via curvature-associated inner-surface contractility

Despite recent explorations of tissue-level epithelial morphogenesis, how the embryonic brain wall, an epithelial derivative unique in its extensive cellular stratification coupled with epithelial-cell heightening to [~]0.5 mm, achieves thickening is unknown. Furthermore, the role of the inner curvature of the wall in this process remains unclear. Thus, whether the apically concave dorsal cerebral (pallial) wall thickens inward, not only outward as previously thought, was examined in culture and in vivo. The pallial wall in the midembryonic period, but not the earlier pallial wall, thickened inward at a pace equivalent to that of neuronal accumulation, which was found via stress-release tests to proceed in a compressive manner. The inhibition of actomyosin-mediated contraction of the inner/apical surface prevented the pallium from thickening, while inducing apical-surface buckling, suggesting the necessity of this inward thickening to actively avoid overcrowding. In contrast, inward bulging of the apically convex ganglionic eminence occurs more passively via pushing of the low-actomyosin apical surface by the constituent cells.

developmental biology↗

bioRxiv2025

Catalytic regulation of CoA transferase by an NAD+-sensing accessory protein and protein acetylation

CoA transferases play essential roles in short-chain fatty acid metabolism by catalyzing the reversible transfer of CoA groups between acyl substrates. However, how their activities are regulated in response to cellular metabolic states remains unclear. Here we identify a dual regulatory mechanism of a CoA transferase from Thermus thermophilus, which is controlled by both interaction with an NAD+-sensing accessory protein and protein acetylation. The enzyme associates with an alanine dehydrogenase-like protein that lacks catalytic activity but binds NAD+ and NADH. Biochemical analyses revealed that inhibition occurs specifically upon NAD+ binding, but not NADH, indicating that the alanine dehydrogenase-like protein senses the intracellular NAD+/NADH ratio. Cryo-electron microscopy structures of the complex of CoA transferase and alanine dehydrogenase-like protein reveal the structural basis of this redox-dependent inhibition. Furthermore, acetylation of the CoA transferase alleviates the inhibition. Since the NAD+/NADH ratio and acetyl-CoA levels reflect the cellular energy and metabolic states, these findings uncover a previously unrecognized regulatory link through which redox sensing and acetylation cooperate to fine-tune the {beta}-oxidation flux.

biochemistry↗

bioRxiv2025

Machine Learning Matches Human Performance at Segmenting the Human Visual Cortex

A major problem in human visual neuroscience research is the localization of visual areas on the cortical surface. Currently available methods are capable of making detailed predictions about many areas, but human raters do not agree as well with these methods as they do with each other. Although highly accurate, human raters require substantial time and expertise that many researchers do not have. Additionally, human raters require functional data for drawing visual area boundaries that requires additional scan time, budget, and expertise to collect. Here, we train convolutional neural network (CNN) models to predict the boundaries and iso-eccentric regions of V1, V2, and V3 in both the Human Connectome Project dataset and the NYU Retinotopy dataset. CNNs trained to use the same functional data available to human raters predicted these boundaries with an accuracy similar to humans, while CNNs trained to use only anatomical data had a lower accuracy that was nonetheless higher than that of any currently available method. A comparison of the model accuracies when predicting eccentricity-based boundaries and polar angle-based boundaries suggests that eccentricity is substantially less closely tied to anatomical structure than polar angle and that the cortical magnification function, at least in terms of eccentricity, varies substantially between subjects. We further find that the fraction of V1, V2, and V3 that can be accurately parcellated into function regions using gray matter structural data alone is [~]75% ([~]80% of the inter-rater reliability of human experts), implying a much tighter coupling between structure and function in these areas than previously estimated. We conclude that machine learning techniques such as CNNs provide a powerful tool for mapping the brain with human accuracy and predict that such tools will become integral to neuroscience research going forward.

neuroscience↗

bioRxiv2025

Perturbing postural stability during treadmill walking with dysfunctional electrical stimulation

Functional electrical stimulation is commonly used to enhance human movement through low-level electrical activation of muscles. More recently, dysfunctional electrical stimulation (DFES) has been proposed as a method to perturb gait by artificially inducing discomfort and mimicking inadequate muscle activity. Here we investigated strategies to induce internal perturbations during treadmill walking using DFES by systematically varying the timing and target muscle. Eleven healthy participants walked at three different speeds while DFES was applied to the tibialis anterior (TA), soleus (SOL), rectus femoris (RF), and biceps femoris (BF) muscles at 25%, 50%, 75%, and 100% of the gait cycle, each for a duration of 0.2 seconds. The gait cycle was time-locked to heel contact (0%). Results showed a significant reduction in the anterior-posterior margin of stability compared to baseline, particularly when DFES was applied to the SOL at 75%, the RF at 50%, and the BF at 75% of the gait cycle. Under these conditions, increased knee flexion and shorter stride intervals were observed relative to baseline. In conclusion, we identified effective DFES conditions to induce postural instability during walking. By mimicking inadequate muscle activity, DFES provides a promising method to study dynamic balance control and mechanisms underlying falls in neurological populations.

bioengineering↗

bioRxiv2025

Conversion of an agonistic anti-TNFR2 biparatopic antibody into an antagonist by insertion of peptide linkers into the hinge region

Biparatopic antibodies (BpAbs) bind two different antigen epitopes to form characteristic immunocomplexes. Many BpAbs have been developed for enhanced cross-linking to induce signal transduction or cell internalization, whereas few were reported with smaller immunocomplexes to suppress unwanted signaling. Here, we developed a strategy to induce 1:1 immunocomplex formation to maximize antagonistic function. Various peptide linkers were introduced into the hinge regions of IgG-like agonist BpAbs against tumor necrosis factor receptor 2. Loss of crosslinking activity was observed for one BpAb, allowing the conversion of its function from an agonist to an antagonist. However, cross-linking activity was retained for another agonist BpAb, which binds to a different epitope pair. In a combined analysis of cryo-electron microscopy and coarse-grained molecular dynamics simulations, effect of epitope combination on the stability of 1:1 complexes was observed. These results lead to an understanding of the mechanism and design of BpAbs to adopt a 1:1-binding mode.

biochemistry↗

bioRxiv2025

Structural basis of flagellar rod assembly on the FliPQR protein-export channel

The FliPQR complex constitutes a channel for export of the bacterial flagellar proteins involved in axial structure assembly. It also serves as a template for flagellar rod assembly. A periplasmic gate, formed by the N-terminal -helices of FliP and FliR, remains closed until FliE assembles onto FliP and FliR. The mechanism by which FliE opens the gate and assembles has remained unclear. Here, we present a cryoEM structure of the FliPQR complex in closed form at 3.0 [A] resolution. A {beta}-cap formed by the N-terminal {beta}-strands of FliP and FliR creates a tight seal in the closed gate. Interaction of FliE with FliP and FliR induces a conformational change in FliP and FliR, with their N-terminal -helices move up and outward. Consequently, the N-terminal {beta}-strands of FliP and FliR start opening the periplasmic gate one after another and form a docking site for FliE to initiate rod assembly.

microbiology↗

bioRxiv2025

Chloroplast genome editing of Rubisco boosts photosynthesis and plant growth

Photosynthetic inefficiencies limit the productivity and sustainability of crop production and the resilience of agriculture to future societal and environmental challenges. Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) has inherently low catalytic efficiency, making it a key target for photosynthesis and crop improvement. However, introducing mutations to the chloroplast-encoded Rubisco large subunit (rbcL), which contains the enzymes catalytic sites, is technically challenging. In this study, we successfully generated a range of chloroplast-genome-edited Arabidopsis thaliana plants targeting rbcL by a targeted base editor, ptpTALECD. The M309I and D397N substitutions in rbcL resulted in an increased Rubisco catalytic rate (kcat) without any reductions of Rubisco content, thereby enhancing photosynthetic rates and plant growth under both current atmospheric CO2 concentrations (i.e., 381 mol mol-1) and projected future concentrations (i.e., 549 mol mol-1). Cryo-electron microscopy (cryo-EM) structural analysis showed that the M309I and D397N substitutions, although located far from the catalytic site, induce structural alterations in the catalytic (60s) loops. Our findings highlight the potential of Rubisco engineering to improve plant photosynthesis and growth, and underscore the unique opportunities that chloroplast genome editing offers for enhancing photosynthesis and crop productivity and reducing atmospheric CO2 levels in a non-GMO context.

plant biology↗

bioRxiv2024

Quantitative in toto live imaging analysis of apical nuclear migration in the mouse telencephalic neuroepithelium

In the embryonic neuroepithelium (NE), neural progenitor cells undergo cell cycle-dependent interkinetic nuclear migration (IKNM) along the apicobasal axis. Extensive IKNM supports increasing cell production rates per unit apical surface, as typically observed in the mammalian telencephalic NE. Apical nucleokinesis during the G2 phase is an essential premitotic event, but its occurrence has not yet been quantitatively analyzed at a large 3D scale with sufficient spatiotemporal resolution. Here, we comprehensively analyzed apically migrating nuclei/somata in reference to their surroundings from embryonic day (E)11 to E13 in the mouse telencephalon. The velocity of apical nucleokinesis decreased, with more frequent nuclear pausing occurring at E12 and E13, whereas the nuclear density in the middle NE zone (20-40 m deep) increased. This result, together with the results of Shh-mediated overproliferation experiments in which the nuclear density was increased in vivo at E11, suggests that apical nucleokinesis is physically influenced by the surrounding nuclei. Mean square displacement analysis for nuclei being passed by the apically migrating nuclei via horizontal sectioning in toto-recorded movies revealed that the "tissue fluidity" or physical permissiveness of the NE to apical nucleokinesis gradually decreased (E11 > E12 > E13). To further investigate the spatial relationship between preexisting mitoses and subsequent premitotic apical nucleokinesis, the horizontal distribution of mitoses was cumulatively ([~]3 hours) analyzed under in toto monitoring. The four-dimensional cumulative apical mitoses presented a "random", not "clustered" or "regular", distribution pattern throughout the period examined. These methodologies provide a basis for future comparative studies of interspecies differences.

developmental biology↗

bioRxiv2024

From Monomers to Oligomers: Structural Mechanism of Receptor-Triggered MyD88 Assembly in Innate Immune Signaling

MyD88 plays a pivotal role in Toll-like receptor (TLR) and interleukin-1 family signaling through its oligomerization upon receptor activation, leading to downstream protein recruitment. The Toll/interleukin-1 receptor domain of MyD88 (TIRMyD88) is responsible for this receptor-mediated oligomerization, but the detailed mechanism involved remains elusive. We investigated the structure of TIRMyD88 oligomers and their interactions with TLRs. Cryoelectron microscopy revealed that tandemly arrayed TIRMyD88 subunits formed an antiparallel double-stranded filament that could further form rings and cylindrical filaments. Moreover, the self-assembly of TIRMyD88 in vitro was markedly accelerated by dimeric rather than monomeric receptor TIRs, possibly reflecting the signal initiation step in vivo. High-speed atomic force microscopy further captured the dynamic processes of oligomerization of TIRMyD88, in addition to its direct interaction with the receptor TIRs. Based on these results, a novel regulatory mechanism of TIRMyD88 oligomerization underlying the signal initiation step was revealed.

biophysics↗

bioRxiv2024

CSF pressure in fetal mice in utero: External factors pressurize the intraventricular space

Previous experiments inducing leakage of embryonic cerebrospinal fluid (CSF) suggest the necessity of intraventricular CSF pressure (PCSF) for brain morphogenesis. Nevertheless, how embryonic PCSF occurs is unclear, especially in utero. Using a Landis water manometer, we measured PCSF in fetal mice isolated from the amniotic cavity (PCSF-ISO). At embryonic day (E) 13, PCSF-ISO was 82.7 Pa. Intraventricular injections of [≥]2 l of saline elevated PCSF-ISO by [~]30%. Intraventricularly injecting inhibitors of CSF secretion decreased PCSF-ISO by [~]30%. Shh-mediated cerebral-wall expansion and the resulting ventricular narrowing did not significantly increase PCSF-ISO. Removal of the brain-surrounding contractile tissues decreased PCSF-ISO by 80-90%. The intraamniotic pressure measured in utero (PAF-IU) was 1030.7 Pa. Our direct measurement revealed that the PCSF in utero (PCSF-IU) was 1076.4 Pa, confirming the susceptibility of PCSF to external factors. Subsequent PCSF measurements under hydrostatic pressure loading suggested that PCSF-IU = PCSF-ISO + PAF-IU, a relationship further used to estimate PCSF-IU at other ages when direct measurement was not possible. The estimated PCSF-IU decreased almost constantly from E10 to E16 (2000 500 Pa).

developmental biology↗

bioRxiv2024

Structural insights into the assembly mechanism of the flagellar MS-ring with three different symmetries

The flagellar basal body MS-ring, formed by 34 FliF subunits, is the core of the flagellar motor as well as the base for flagellar assembly. The MS-ring is also a housing for the flagellar protein export gate complex that is required for construction of the flagellum on the cell surface. A large periplasmic region of FliF contains three ring-building motifs named RBM1, RBM2, and RBM3. RBM3 forms the S-ring and {beta}-collar with C34 symmetry. RBM2 forms the inner core ring of the M-ring with C23 symmetry surrounded by 11 cog-like structures formed by RBM1 and RBM2. However, it remains unknown how FliF assembles to generate these three different symmetries within the MS-ring. Here, we report the two cryoEM structures of the MS-ring formed by FliF co-expressed with FliG and transmembrane export gate proteins. Structural comparison of 33-mer and 34-mer MS-rings revealed that a subtle change in the conformation of RBM3 produces the different rotational symmetries. Combination of cryoEM structural and mutational analyses of the MS-ring with C33 symmetry provides evidence that the well-conserved DQxGxxL motif within a flexible loop connecting RBM2 and RBM3 allows RBM2 to take two different orientations relative to RBM3 to form not only 11 cog-like structures just outside the inner core ring with C22 symmetry but also an appropriately sized central pore in the inner core ring to accommodate the export gate complex. IMPORTANCEThe flagellar MS-ring is the core of the flagellar motor and serves not only as an initial template for flagellar assembly but also as a base to accommodate the flagellar protein export complex. The MS-ring is formed by 34 subunits of FliF with two transmembrane helices and a large periplasmic region containing ring-building motifs, RBM1, RBM2, and RBM3. FliF adopts two different conformations in the MS-ring to generate three different rotational symmetries, C34, C23, and C11. However, how FliF assembles to produce these three symmetries remains a mystery. Combination of cryoEM structural and mutational analyses has provided evidence that the well-conserved DQxGxxL motif within a hinge loop connecting RBM2 and RBM3 allows RBM2 to take two different orientations relative to RBM3, allowing 23 RBM2 domains of 34 subunits to form the inner core ring with a properly sized central pore to accommodate the flagellar protein export complex.

microbiology↗

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↗

bioRxiv2023

Antibodies halting motility in Mycoplasma pneumoniae reveal the dynamic nature of the adhesion complex

Mycoplasma pneumoniae is a bacterial wall-less human pathogen and the etiological agent of atypical pneumonia and tracheobronchitis in both adults and children. M. pneumoniae infectivity, gliding motility and adherence to host target respiratory epithelial cells are mediated by adhesin proteins P1 and P40/P90 forming a transmembrane complex that binds to sialylated oligosaccharides human cell ligands. Here we report the cryo-EM structure of P1 bound to the Fab fragment of monoclonal antibody P1/MCA4, which stops gliding and induces detachment of motile M. pneumoniae cells. On the contrary, polyclonal antibodies generated against the N-domain of P1 or against the whole ectodomain of P40/P90 have little or no effects on adhesion or motility. The epitope of P1/MCA4, centred on loop Thr1426-Asp1438 in the small C-terminal domain of P1, is inaccessible to antibodies in the "open" conformation of the adhesion complex, when ready for attachment to sialylated oligosaccharides. Mutations in the highly conserved Engelman motifs found in the transmembrane helix of P40/P90 also alter adhesion and motility. During the attachment/detachment cycle of the adhesion complex, the C-terminal domain of P1 experiences large conformational rearrangements that are hindered by the antibodies against the domain. Interfering with the gliding of mycoplasma cells suggests new ways to confront M. pneumoniae infections.

molecular biology↗

bioRxiv2023

Cryo-EM of cytoskeletal 'fibril' protein involved in Spiroplasma swimming

Spiroplasma, parasitic or commensal bacteria, can swim by switching the handedness of its helical cell body. A helical cell body is formed by an internal ribbon of MreB, an actin superfamily, and Spiroplasma-specific fibril proteins. Here we have solved the structure of the fibril filament by single-particle cryo-electron microscopy at 3.6 [A] resolution and built its atomic structure. The structure is composed of repeated rings and cylinders. The N-terminal cylinder of the fibril protein shows a structure similar to that of S-adenosylhomocysteine nucleosidase, while the C-terminal ring does not show similarity to other proteins. The filament is nonpolar and flexible, possessing a helical pitch of 700 nm, consistent with cell helicity. Cryo-electron tomography revealed aligned several MreB filaments in the center of the ribbon, flanked by membrane-binding fibril filaments through electrostatic interactions. This study discusses the evolution and roles of the fibril filament.

microbiology↗