Biology subjects

Otsuka, S.

Publications and source records attributed to Otsuka, S..

15 recordsLinked to original sources

bioRxiv2026

Structural mechanism of nuclear membrane sealing by LEM2-ESCRT-III

In open mitosis, re-establishing nucleocytoplasmic compartmentalization requires the LEM2-ESCRT machinery to coordinate spindle clearance with sealing of the remaining nuclear envelope pores. The structural basis of this topologically unique and fundamental membrane-remodeling process is poorly understood. Here, we combine biochemical reconstitution, cryo-electron tomography, subtomogram averaging and large-scale molecular dynamics simulations to define the structural mechanism of nuclear membrane sealing. We structurally resolve that LEM2s winged-helix domain (WH) co-polymerizes with the ESCRT-II/III protein CHMP7 to form a membrane-bound scaffold whose geometry is progressively remodeled by downstream ESCRT-III proteins as it transitions from the flat membrane surrounding the pore towards the negatively curved membrane neck. In parallel, LEM2 positions its intrinsically disordered low-complexity domain within the pore, where condensation around spindle microtubules mechanically couples the membrane-ESCRT-LEM2 scaffold to the spindle and narrows the remaining diffusion path, restoring compartmentalization before membrane closure is complete. Remarkably, the LEM2-WH domain alone forms tightly constricted membrane tubes, coating the negatively curved inner surface, revealing an intrinsic membrane-remodeling activity of the receptor itself. Together, our work establishes a structural framework for how receptor-ESCRT co-polymerization, low complexity domain-mediated sealing and receptor-driven membrane remodeling guide nuclear-envelope pores from spindle-containing openings to terminal constriction and fusion.

molecular biology↗

bioRxiv2026

Autism associated mutation in Cacna1d causes perseverative behavioral phenotype in mice

Behavioral inflexibility and perseveration are core features of autism spectrum disorder and are frequently modeled in mice using reversal learning and repetitive behavior assays. Mutations in CACNA1D, which encodes the L-type calcium channel Cav1.3, have been linked to autism, yet their behavioral consequences remain incompletely characterized. We examined mice carrying the autism-associated Cacna1dG407R gain-of-function mutation across a battery of assays assessing learning, flexibility, and repetitive behavior. Mutant mice learned spatial discriminations and instrumental contingencies at rates comparable to wild-type controls but exhibited deficits during reversal learning following intermediate and extended overtraining, as well as under probabilistic reinforcement. Across ethological assays, mutant mice showed increased grooming, marble burying, and nestlet shredding, consistent with enhanced perseverative behavior. Anxiety-related measures and general locomotion were largely unaffected. These results identify Cav1.3 gain-of-function as a selective regulator of behavioral flexibility and support a role for calcium-dependent corticostriatal plasticity in autism-associated perseveration.

neuroscience↗

bioRxiv2025

Operant House: A Versatile Open-Source Platform for Automated Operant Conditioning Testing of Mice in Home Cages

Operant conditioning is a valuable method for studying cognitive functions, yet its adoption is limited by low throughput, labor intensity, and high costs. Here, we developed "Operant House," a low-cost, programmable device featuring a touchscreen, retractable levers, and a water reward port, designed for flexible, automated operant conditioning tasks. To validate its utility, we implemented two protocols to assess working memory in mice: a delayed non-match-to-position test and a two-choice spatial discrimination test. Using these protocols, we examined a mouse model of Alzheimers disease carrying familial Alzheimers disease-associated amyloid precursor protein mutations. Results revealed significant working memory deficits as early as 5 months of age. These findings highlight the Operant House as a cost-effective, high-throughput platform for evaluating higher cognitive functions in mice, offering an accessible tool for investigating models of neurological and neuropsychiatric disorders.

neuroscience↗

bioRxiv2024

Subregional activity in the dentate gyrus is amplified during elevated cognitive demands

Neural activity in the dentate gyrus (DG) supports the detection and discrimination of novelty, context, and patterns. Granule cell activation differs between the supra- and infrapyramidal blades across hippocampal-dependent tasks, yet how excitatory dynamics shape this blade-specific bias under varying cognitive demands remains unclear. Here, we combined an automated touchscreen pattern separation task in mice with temporally controlled tagging of active neurons to determine how increasing cognitive demand influences spatial activity patterns in the DG. As task difficulty increased, activation became progressively biased toward the suprapyramidal blade and was accompanied by structured distributions of active mature granule cells (mGCs) along both the radial and transverse axes. Selective inhibition of mGCs did not alter these spatial patterns but profoundly impaired performance, as mice were no longer able to discriminate between closely spaced locations. In contrast, chemogenetic inhibition of adult-born granule cells (abDGCs) beyond a critical maturation window impaired performance under high-demand conditions, increased overall mGC activity, and disrupted blade-specific organization even in animals that successfully completed the task. These findings demonstrate that high cognitive demand recruits spatially organized mGC activity and support a modulatory role for abDGCs in shaping dentate circuit dynamics.

neuroscience↗

bioRxiv2024

Integrative spatiotemporal modeling of biomolecular processes: application to the assembly of the Nuclear Pore Complex

Dynamic processes involving biomolecules are essential for the function of the cell. Here, we introduce an integrative method for computing models of these processes based on multiple heterogeneous sources of information, including time-resolved experimental data and physical models of dynamic processes. We first compute integrative structure models at fixed time points and then optimally select and connect these snapshots into a series of trajectories that optimize the likelihood of both the snapshots and transitions between them. The method is demonstrated by application to the assembly process of the human Nuclear Pore Complex in the context of the reforming nuclear envelope during mitotic cell division, based on live-cell correlated electron tomography, bulk fluorescence correlation spectroscopy-calibrated quantitative live imaging, and a structural model of the fully-assembled Nuclear Pore Complex. Modeling of the assembly process improves the model precision over static integrative structure modeling alone. The method is applicable to a wide range of time-dependent systems in cell biology, and is available to the broader scientific community through an implementation in the open source Integrative Modeling Platform software.

biophysics↗

bioRxiv2024

Ca2+-dependent phosphodiesterase 1 regulates the plasticity of striatal spiny projection neuron glutamatergic synapses

Long-term synaptic plasticity at glutamatergic synapses on striatal spiny projection neurons (SPNs) is central to learning goal-directed behaviors and habits. Although considerable attention has been paid to the mechanisms underlying synaptic strengthening and new learning, little scrutiny has been given to those involved in the attenuation of synaptic strength that attends suppression of a previously learned association. Our studies revealed a novel, non-Hebbian, long-term, postsynaptic depression of glutamatergic SPN synapses induced by interneuronal nitric oxide (NO) signaling (NO-LTD) that was preferentially engaged at quiescent synapses. This form of plasticity was gated by local Ca2+ influx through CaV1.3 Ca2+ channels and stimulation of phosphodiesterase 1 (PDE1), which degraded cyclic guanosine monophosphate (cGMP) and blunted NO signaling. Consistent with this model, mice harboring a gain-of-function mutation in the gene coding for the pore-forming subunit of CaV1.3 channels had elevated depolarization-induced dendritic Ca2+ entry and impaired NO-LTD. Extracellular uncaging of glutamate and intracellular uncaging of cGMP suggested that this Ca2+-dependent regulation of PDE1 activity allowed for local regulation of dendritic NO signaling. This inference was supported by simulation of SPN dendritic integration, which revealed that dendritic spikes engaged PDE1 in a branch-specific manner. In a mouse model of Parkinsons disease (PD), NO-LTD was absent not because of a postsynaptic deficit in NO signaling machinery, but rather due to impaired interneuronal NO release. Re-balancing intrastriatal neuromodulatory signaling in the PD model restored NO release and NO-LTD. Taken together, these studies provide novel insights into the mechanisms governing NO-LTD in SPN and its role in psychomotor disorders, like PD.

neuroscience↗

bioRxiv2023

Structural evidence for elastic tethers connecting separating chromosomes in crane-fly spermatocytes

Different types of anaphase bridges have been reported to form between segregating chromosomes during cell division. Previous studies using laser microsurgery suggested that elastic tethers connect the telomeres of separating anaphase chromosomes in many animal meiotic and mitotic cells. However, structural evidence is lacking for their existence. In this study, we correlated live imaging with electron tomography of crane-fly primary spermatocytes to examine whether visible structures connect separating telomeres in meiosis I. We found structures extending between separating telomeres in all stages of anaphase, from very early anaphase to late anaphase. Ultrastructural analysis revealed that the structures consist of two components: one is darkly stained, looking somewhat like chromatin, whereas the other is more lightly stained, appearing filamentous with internal thinner filaments. While in early anaphase both structures extend between telomeres, in later anaphase the darker structure extends shorter distances from the telomeres but the lighter structure still extends between the separating telomeres. From these observations, we deduced that these structures represent the tethers inferred from the laser-cutting experiments in crane-fly spermatocytes. Since the presence of elastic tethers are suggested in a variety of animal cells, they probably are present during anaphase in all animal cells.

cell biology↗

bioRxiv2023

The junctions connecting the endoplasmic reticulum to the nuclear envelope are constricted and remodelled during the cell cycle

The endoplasmic reticulum (ER) is physically connected to the nucleus by junctions with the outer membrane of the nuclear envelope (NE). The ER-NE junctions are essential for supplying the NE with lipids and proteins synthesized in the ER. However, little is known about the structure of these ER-NE junctions. Here, we systematically studied the ultrastructure of ER-NE junctions in cryo-fixed mammalian cells staged in anaphase, telophase, and interphase by correlating live cell imaging with three-dimensional electron microscopy. Strikingly, our results revealed that ER-NE junctions in interphase cells have a pronounced hourglass shape with a constricted neck of 7-20 nm width. This morphology is significantly distinct from that of junctions among the ER, and it emerges as early as telophase. The highly constricted ER-NE junctions are seen in several mammalian cell types, but not in budding yeast. We speculate that the unique and highly-constricted ER-NE junctions are regulated via novel mechanisms that contribute to ER-to-NE lipid and protein traffic in higher eukaryotes.

cell biology↗

bioRxiv2021

A chromatin phase transition protects mitotic chromosomes against microtubule perforation

Dividing eukaryotic cells package extremely long chromosomal DNA molecules into discrete bodies to enable microtubule-mediated transport of one genome copy to each of the newly forming daughter cells1-3. Assembly of mitotic chromosomes involves DNA looping by condensin4-8 and chromatin compaction by global histone deacetylation9-13. While condensin confers mechanical resistance towards spindle pulling forces14-16, it is not known how histone deacetylation affects material properties and segregation mechanics of mitotic chromosomes. Here, we show how global histone deacetylation at the onset of mitosis induces a chromatin-intrinsic phase transition that endows chromosomes with specific characteristics necessary for their precise movement during cellular division. Deacetylation-mediated compaction of chromatin forms a structure dense in negative charge and allows mitotic chromosomes to resist perforation by microtubules as they are pushed to the metaphase plate. Hyperacetylated mitotic chromosomes lack a defined surface boundary, are frequently perforated by microtubules, and are prone to missegregation. Our study highlights the different contributions of DNA loop formation and chromatin-intrinsic phase separation to genome segregation in dividing cells.

cell biology↗

bioRxiv2021

A quantitative map of nuclear pore assembly reveals two distinct mechanisms

Understanding how the nuclear pore complex (NPC) assembles is of fundamental importance to grasp the mechanisms behind its essential function and understand its role during evolution of eukaryotes1-4. While at least two NPC assembly pathways exist, one during exit from mitosis and one during nuclear growth in interphase, we currently lack a quantitative map of the molecular assembly events. Here, we use fluorescence correlation spectroscopy (FCS) calibrated live imaging of endogenously fluorescently-tagged nucleoporins to map the changes in composition and stoichiometry of seven major modules of the human NPC during its assembly in single dividing cells. This systematic quantitative map reveals that the two assembly pathways employ strikingly different molecular mechanisms, inverting the order of addition of two large structural components, the central ring complex and nuclear filaments. The dynamic stoichiometry data underpinned integrative spatiotemporal modeling of the NPC assembly pathway, predicting the structures of postmitotic NPC assembly intermediates.

cell biology↗

bioRxiv2020

A synthetic synaptic organizer protein restores glutamatergic neuronal circuits

Neuronal synapses undergo structural and functional changes throughout life, essential for nervous system physiology. However, these changes may also perturb the excitatory/inhibitory neurotransmission balance and trigger neuropsychiatric and neurological disorders. Molecular tools to restore this balance are highly desirable. Here, we report the design and characterization of CPTX, a synthetic synaptic organizer combining structural elements from cerebellin-1 and neuronal pentraxin-1 to interact with presynaptic neurexins and postsynaptic AMPA-type ionotropic glutamate receptors. CPTX induced the formation of excitatory synapses in vitro and in vivo and restored synaptic functions, motor coordination, spatial and contextual memories, and locomotion in mouse models for cerebellar ataxia, Alzheimers disease and spinal cord injury, respectively. Thus, CPTX represents a prototype for novel structure-guided biologics that can efficiently repair or remodel neuronal circuits. One Sentence SummaryStructural biology information was used to design CPTX, a synthetic protein that induces functional excitatory synapses and restores normal behaviors in mouse models of neurological diseases.

neuroscience↗

bioRxiv2019

Chemogenetic control of nanobodies

We introduce an engineered nanobody whose affinity to green fluorescent protein (GFP) can be switched on and off with small molecules. By controlling the cellular localization of GFP fusion proteins, the engineered nanobody allows to study their role in basic biological processes, an approach that should be applicable to numerous previously described GFP fusions. We also outline how the binding affinities of other nanobodies can be controlled by small molecules.

synthetic biology↗

bioRxiv2019

Epigenetic Effects of Exposure to Insecticide on Early Differentiation of Mouse Embryonic Stem Cells

Increasing evidence indicates that insecticides induce various diseases via DNA methylation. DNA methylation plays an important role during cell differentiation and exhibits its greatest vulnerability to environmental factors during embryogenesis. Therefore, it is important to evaluate the effects on DNA methylation at the early stage of cell differentiation to understand developmental toxicity. However, DNA methylation induced by insecticides and the associated effects on cell differentiation are unclear. In this research, we introduced a high-content approach utilizing mouse embryonic stem cells harboring enhanced green fluorescent protein fused with methyl CpG-binding protein to evaluate global DNA methylation induced by various insecticides. DNA methylation was assessed in 22 genes after pesticide exposure to investigate the relationships with biological processes such as cell cycle, cell apoptosis, and cell differentiation. Exposure to acetamiprid, imidacloprid, carbaryl, and o,p'-DDT increased the granular intensity, indicating their global DNA-methylating effects. Exposure to imidacloprid decreased DNA methylation in genes such as Cdkn2a, Dapk1, Cdh1, Mlh1, Timp3, and Rarb, indicating the potential influence of the DNA methylation pattern on cell differentiation. We developed a promising approach for evaluating global DNA methylation, and our findings suggested that imidacloprid might exhibit developmental effects through DNA methylation pattern.

pharmacology and toxicology↗

bioRxiv2018

Soil nutrient stoichiometry affects the initial response of microbial community to trophic perturbation.

Soil microbes are drivers of global ecosystem functionality and are continuously subjected to external perturbations. It is fundamental for ecologists and environmental scientists to understand and further predict the microbes responses to these perturbations. A major and ubiquitous perturbation is the addition of chemical nutrients, including fertilizers and animal urine, to soil. Recent biogeographical studies suggest that soil nutrient stoichiometry (i.e., nutritional balance) determines microbial community structure and its functions with regard to material circulation. Given this information, here, we show that soil nutrient stoichiometry, or the bioavailable C:P ratio, determines the impact of nutrient addition on the soils microbial communities. We sampled two soils with similar carbon and nitrogen concentrations but with a 20-fold difference in phosphorus bioavailability. Soil microcosms with carbon and nitrogen amendments were constructed for both the soils. The phosphorus-depleted soil received prolonged effect from carbon and nitrogen amendments: the phosphatase activity gradually increased over a 24-day incubation period and the microbial community structure did not present recovery to its initial state. In contrast, in the other soil, both phosphatase activity and microbial community structure gradually returned to those of the control samples. Phosphorus depletion mitigated carbon and nitrogen intake; therefore, the effects of carbon and nitrogen amendment lasted longer. Our results demonstrate that nutritional stoichiometry is a strong predictor of microbial community dynamics in response to trophic perturbation, particularly when considering the length of time the trait of perturbation persists in the soil.

ecology↗

bioRxiv2017

Postmitotic Nuclear Pore Assembly Proceeds By Radial Dilation Of Small ER Membrane Openings

The nuclear envelope has to be reformed after mitosis to create viable daughter cells with closed nuclei. How membrane sealing of DNA and assembly of nuclear pore complexes (NPCs) are achieved and coordinated is poorly understood. Here, we reconstructed nuclear membrane topology and structure of assembling NPCs in a correlative three dimensional electron microscopy time-course of dividing human cells. Our quantitative ultrastructural analysis shows that nuclear membranes form from highly fenestrated ER sheets, whose shrinking holes are stabilized and then dilated into NPCs during inner ring complex assembly, forming thousands of transport channels within minutes. This mechanism is fundamentally different from interphase NPC assembly and explains how mitotic cells can rapidly establish a closed nuclear compartment while making it transport-competent at the same time.

cell biology↗