Biology subjects

Yanagisawa, M.

Publications and source records attributed to Yanagisawa, M..

14 recordsLinked to original sources

bioRxiv2026

SIK3 links synaptic strength to sleep pressure through RhoA signaling

Sleep is regulated by homeostatic sleep pressure, which accumulates during wakefulness, triggering sleep, and dissipates during sleep, leading to wakefulness. Although recent evidence suggests that synaptic potentiation encodes sleep pressure, the intracellular mechanisms linking synaptic strength to sleep pressure remain unclear. Here we demonstrate that the sleep-promoting kinase SIK3 links synaptic strength and sleep pressure through RhoA signalling. We found that SIK3 is required for dendritic spine enlargement induced by chemical long-term potentiation (cLTP) in cultured cortical pyramidal neurons and positively regulates spine size in the prefrontal cortex in vivo. cLTP reduced RhoA activity in an SIK3-dependent manner during spine enlargement, whereas activation of RhoA reduced EEG delta density in vivo during non-rapid eye movement sleep (NREMS), an indicator of sleep pressure. SIK3 substrate screening identified several RhoA regulators including GEF-H1, and further biochemical analyses found that phosphorylation by SIK3 activates GEF-H1. However, contrary to our expectation, RhoA facilitation through pharmacological activation of GEF-H1 enlarged spine size in cultured neurons, and expression of constitutively active GEF-H1 increased NREMS delta density in a RhoA-dependent fashion. Together, these results indicate that SIK3 enlarges spine size and enhances NREMS delta density through two RhoA-dependent pathways, driving mutually opposing intermediate phenotypes. Our findings identify two clashing SIK3-RhoA signalling pathways that couple synaptic regulation to sleep homeostasis.

neuroscience↗

bioRxiv2026

Cell-sized droplet interfaces reorganize protein secondary structures through confinement-enhanced membrane interactions

Cell membranes can regulate protein organization, yet it remains unclear whether membrane-associated structural alterations primarily reflect membrane-induced destabilization or the reorganization of proteins that are already destabilized. How such membrane effects are amplified under cellular-scale confinement also remains poorly understood. Here, we investigate these questions using cell-sized lipid-coated droplets, where the high surface-area-to-volume ratio enhances the contribution of membrane interfaces. Native serum albumin and lysozyme showed little structural reorganization, whereas their thermally denatured forms exhibited pronounced $\beta$-sheet formation within small droplets when membrane interactions were attractive. For denatured albumin, $\beta$-sheet-rich organization increased progressively with protein--membrane attraction, while denatured lysozyme selectively formed a localized $\beta$-sheet-rich shell at a complementary anionic membrane. Circular dichroism spectroscopy independently supported the confinement-enhanced increase in $\beta$-sheet content of denatured albumin. Fluorescence recovery measurements further revealed strong interfacial arrest in both systems. Together, these results show that membrane interactions can promote structural reorganization of already destabilized proteins through electrostatic recruitment, while cell-sized confinement strongly amplifies the resulting membrane-dependent structural response. Our findings establish cell-sized droplet interfaces as a materials platform for spatially controlling protein structural organization through the interplay of interfacial chemistry and confinement.

biophysics↗

bioRxiv2025

Broadband electronic resonance coherent anti-Stokes/Stokes Raman scattering microscopy

Coherent Raman imaging (CRI) enables label-free chemical imaging based on intrinsic molecular vibrations, but its applicability is often limited by low sensitivity, hindering the detection of low-abundance biomolecules. While electronic resonance significantly enhances sensitivity, most resonance CRI implementations rely on narrowband excitation and/or detection, which limits spectral coverage and makes it difficult to distinguish target molecules from backgrounds. Here we address these challenges by developing broadband electronic-resonance coherent anti-Stokes/Stokes Raman scattering (BER-CARS/CSRS) microscopy. We show that BER-CARS/CSRS enables highly sensitive, label-free imaging of endogenous chromophores with broad spectral coverage of the entire fingerprint region. Specifically, we captured time-lapse images across the fingerprint region, visualizing low-abundance cytochromes alongside abundant biomolecules (lipids, proteins, nucleic acids) in living HEK293 cells. Furthermore, we applied the method to complex biological tissues, mapping distinct distributions of cytochromes in mouse brain slices, highlighting their characteristic localizations in the cortex and the cerebral ventricle wall. Our results demonstrate that BER-CARS/CSRS is a powerful platform for highly sensitive chemical imaging, from large-area tissue mapping to organelle-level dynamics. By coupling resonance enhancement with broadband fingerprinting, BER-CARS/CSRS enables dynamic, label-free phenotyping of mitochondrial and physiological states from single cells to tissues, opening a path to quantitative, slide-scale chemical histopathology and intraoperative margin assessment.

bioengineering↗

bioRxiv2025

Local niche-derived immunosuppressive CXCR2+ cells impair antiviral immunity

Symptom severity after viral infection varies among individuals, yet its mechanism remains poorly understood. We categorized mice into recovering and non-recovering groups based on body weight after infection of the same titer of vesicular stomatitis virus (VSV). We revealed that in the olfactory bulb (OB) where VSV initially expands, non-recovering mice exhibited an anti-inflammatory environment, suggesting its detrimental effect. Importantly, CXCR2+ cells resembling immunosuppressive myeloid-derived suppressor cells (MDSCs) were more abundant in the OB of non-recovering mice than in that of recovering mice after VSV infection. Depleting CXCR2+ MDSC-like cells from the brain increased inflammatory responses and the animals survival after infection. Furthermore, site-specific labeling indicated that a significant fraction of these cells in the OB originate from the skull-bone marrow (skull-BM) as well as circulation. This study reveals the lethal effects of CXCR2+ MDSC-like cells on local immune responses to viral infection, highlighting their therapeutic potential for antiviral defense.

immunology↗

bioRxiv2025

Membrane-enhanced repulsive interactions regulate protein diffusion in cell-size space

Intracellular molecular organization is often explained by attractive interactions driving clustering and phase separation. Although consideration of repulsive forces is essential in physics, their roles remain unclear in cellular contexts. Here, we demonstrated the fundamental role of repulsion in regulating protein diffusion within cell-size space. By analyzing negatively charged protein diffusion in bulk solutions and in cell-size spaces with membranes, we revealed that membrane-enhanced repulsion inhibited protein diffusion in cell-size spaces. This was due to the amplified electrostatic interactions among proteins because of the large membrane area-to-volume ratio. Notably, ATP, a cellular central energy source, further inhibited protein diffusion in cell-size spaces, whereas protein wave propagation on the membrane counteracted this inhibition. These findings suggest an active regulatory mechanism restoring molecular mobility by dynamically adjusting membrane-enhanced repulsive forces. Our study challenges the traditional emphasis on attractive interactions, highlighting repulsion as a critical tunable factor governing molecular transport and spatial organization in cells. TeaserProtein diffusion within crowded cell-size spaces is tuned by membrane-enhanced repulsion and its active regulation.

biophysics↗

bioRxiv2025

Deep Learning-coupled Proximity Proteomics to Deconvolve Kinase Signaling In Vivo

Deconvolving the substrates of hundreds of kinases linked to phosphorylation networks driving cellular behavior is a fundamental, unresolved biological challenge, largely due to the poorly understood interplay of kinase selectivity and substrate proximity. We introduce KolossuS, a deep learning framework leveraging protein language models to decode kinase-substrate specificity. KolossuS achieves superior prediction accuracy and sensitivity across mammalian kinomes, enabling proteome-wide predictions and evolutionary insights. By integrating KolossuS with CRISPR-based proximity proteomics in vivo, we capture kinase-substrate recognition and spatial context, obviating prior limitations. We show this combined framework identifies kinase substrates associated with physiological states such as sleep, revealing both known and novel Sik3 substrates during sleep deprivation. This novel integrated computational-experimental approach promises to transform systematic investigations of kinase signaling in health and disease.

cell biology↗

bioRxiv2024

Slow diffusion and signal amplification on membranes regulated by phospholipase D

Control of molecular diffusion is pivotal for highly fluidic membranes to serve as substrates for biochemical reactions and the self-assembly of molecular machinery driving membrane protrusions. Lateral diffusion in membranes depends on lipid composition, which is highly diverse and homeostatically controlled in living cells. Due to the complexity of the underlying processes, its impact on molecular diffusion remain largely unclear. In this study, we show that lipid diffusion in model membranes is markedly decreased in cytosolic extracts. The reduction in lipid diffusivity could be pharmacologically inhibited by targeting phospholipase D (PLD), and addition of PLD to membranes mimicked the reduction in diffusion. Phosphatidic acid, a direct product of PLD, diffused slowly in model membranes and reduced the diffusivity of surrounding lipids. Furthermore, we demonstrated that PLD specifically controls the lateral diffusion of a myristoylated protein in cells, possibly due to auxiliary electrostatic interactions between cationic residues located near the lipidated tail and anionic phospholipids. PLD controlled the size and lifetime of localized patches of phosphatidylinositol (3,4,5) triphosphates that specify regions of membrane protrusions. Overall, the results of this study suggest that PLD controls the lateral diffusion of certain membrane proteins, which play key roles in phosphoinositide signaling. Significance StatementIn living cells, many biochemical reactions occur in confined regions on the membranes, facilitating the local occurrence of specific events, such as membrane protrusion. This is puzzling from a physical perspective because the membrane is a two-dimensional fluidic structure that should allow molecules to spread freely. Herein, we found that the fluidity of artificial membranes was markedly reduced by adding extracts from the cell cytoplasm. A lipid-modifying protein phospholipase D (PLD) was found to be responsible for this and it regulates the diffusion of membrane proteins in cells. This study suggests the novel role of PLD as a regulator of molecular diffusion and its impact on phosphoinositide production that serves as an important signal for cell deformation.

biophysics↗

bioRxiv2023

Sequential Transitions of Male Sexual Behaviours Driven by Dual Acetylcholine-Dopamine Dynamics

The neural mechanisms regulating sequential transitions of male sexual behaviors, such as mounting, intromission, and ejaculation, in the brain remain unclear. Here, we report that dopamine (DA) and acetylcholine (ACh) dynamics in the ventral shell of the nucleus accumbens (vsNAc) closely aligns with serial transitions of sexual behaviors in male mice. During intromission, the vsNAc exhibits dual ACh-DA rhythms generated by reciprocal regulation between ACh and DA signaling via nicotinic acetylcholine (nAChR) and dopamine D2 (D2R) receptors. Knockdown of choline acetyl transferase (ChAT) or D2R in the vsNAc diminished the likelihood of intromission and ejaculation. Optogenetic manipulations reveal that DA signaling sustains male sexual behaviors by suppressing activities of D2RvsNAc neurons. Moreover, ACh signaling promotes the initiation of mounting and intromission, but also induces the intromission-to-ejaculation transition by triggering a slowdown of DA rhythm. Therefore, dual ACh-DA dynamics harmonize in the vsNAc to drive sequential transitions of male mating behaviors.

neuroscience↗

bioRxiv2022

Competitive membrane wetting of polymer blends in artificial cells initiates phase separation and promotes fractionation

Biomolecular condensates driven by liquid-liquid phase separation (LLPS) have received attention as novel activity regulators of living organisms. In intracellular LLPS, an important question is what type of biomolecules form condensates under what conditions. In this regard, possible interactions between biomolecules have been investigated. Recently, LLPS condensates have been reported to regulate the membrane structure upon wetting. However, the possibility of membrane wetting, in which the membrane conversely regulates the LLPS, remains unexplored. Using droplets of short polyethylene glycol and long dextran blends encapsulated with a lipid membrane, we demonstrate that membrane wetting regulates LLPS in cell-size spaces and alters the equilibrium state. In smaller droplets, the two-phase region expands beyond the bulk system, and the fractionation degree increases, particularly during the separation between short PEG and long dextran. We explain the space-size dependent LLPS based on the competitive membrane wetting between the polymers. Smaller droplets promote the membrane wetting of short PEG, which enhances the depletion force between long dextran molecules and finally induces LLPS. This shows that competition for membrane wettability among various molecules can regulate LLPS in cell-size spaces, rendering this LLPS principle feasible in living cells.

biophysics↗

bioRxiv2021

Stress-dependent dynamic and reversible formation of cytoskeleton-like filaments and gel-transition by tardigrade tolerance proteins.

Tardigrades are able to tolerate almost complete dehydration by entering a reversible ametabolic state called anhydrobiosis and resume their animation upon rehydration. Dehydrated tardigrades are exceptionally stable and withstand various physical extremes. Although trehalose and late embryogenesis abundant (LEA) proteins have been extensively studied as potent protectants against dehydration in other anhydrobiotic organisms, tardigrades produce high amounts of tardigrade-unique protective proteins. Cytoplasmic-abundant heat-soluble (CAHS) proteins are uniquely invented in the lineage of eutardigrades, a major class of the phylum Tardigrada and are essential for their anhydrobiotic survival. However, the precise mechanisms of their action in this protective role are not fully understood. In the present study, we first postulated the presence of tolerance proteins that form protective condensates via phase separation in a stress-dependent manner and searched for tardigrade proteins that reversibly form condensates upon dehydration-like stress. Through comprehensive analysis, we identified 336 such proteins, collectively dubbed "Desolvataion-induced ReversiblY condensing Proteins (DRYPs)". Unexpectedly, we rediscovered CAHS proteins as highly enriched in DRYPs, 3 of which were major components of DRYPs. We revealed that these CAHS proteins reversibly polymerize into many cytoskeleton-like filaments depending on hyperosmotic stress in cultured cells and undergo reversible gel-transition in vitro. CAHS filamentation increases cell stiffness to resist deformation and improves resistance to dehydration-like stress. The conserved putative helical C-terminal region is necessary and sufficient for filament formation by CAHS proteins, and mutations disrupting the secondary structure of this region impaired both the filament formation and the gel transition. On the basis of these results, we propose that CAHS proteins are novel cytoskeletal proteins that form filamentous networks and undergo gel-transition in a stress-dependent manner to provide on-demand physical stabilization of cell integrity against deformative forces during dehydration and also contribute to the exceptional physical stability in a dehydrated state.

biochemistry↗

bioRxiv2021

Real-time conversion of tissue-scale mechanical forces into an interdigitated growth pattern

The leaf epidermis is a dynamic biomechanical shell that integrates growth across spatial scales to influence organ morphology. Pavement cells, the fundamental unit of this tissue, morph irreversibly into highly lobed cells that drive planar leaf expansion. Here we define how tissue-scale cell wall tensile forces and the microtubule-cellulose synthase systems pattern interdigitated growth in real-time. A morphologically potent subset of cortical microtubules span the periclinal and anticlinal cell faces to pattern cellulose fibers that generate a patch of anisotropic wall. The result is local polarized growth that is mechanically coupled to the adjacent cell via a pectin-rich middle lamella, and this drives lobe formation. Finite element pavement cell models revealed cell wall tensile stress as an upstream patterning element that links cell- and tissue-scale biomechanical parameters to interdigitated growth. Cell lobing in leaves is evolutionarily conserved, occurs in multiple cell types, and is associated with important agronomic traits. Our general mechanistic models of lobe formation provide a foundation to analyze the cellular basis of leaf morphology and function.

cell biology↗

bioRxiv2019

Blood and Tissue Biomarker Analysis in Dogs with Osteosarcoma Treated with Palliative Radiation and Intra-Tumoral Autologous Natural Killer Cell Transfer

BackgroundWe have previously reported radiation-induced sensitization of canine osteosarcoma (OSA) to natural killer (NK) therapy, including results from a first-in-dog clinical trial. Here, we report correlative analyses of blood and tissue specimens for signals of immune activation in trial subjects.\n\nMethodsAmong 10 dogs treated with palliative radiotherapy (RT) and intra-tumoral adoptive NK transfer, we performed ELISA on serum cytokines, flow cytometry for immune phenotype of PBMCs, and PCR on tumor tissue for immune-related gene expression. We then queried The Cancer Genome Atlas (TCGA) to evaluate the association of cytotoxic/immune-related gene expression with human sarcoma survival.\n\nResultsUpdated survival analysis revealed five 6-month survivors, including one dog who lived 17.9 months. Using feeder line co-culture for NK expansion, we observed maximal activation of dog NK cells on day 17 - 19 post isolation with near 100% expression of granzyme B and NKp46 and high cytotoxic function in the injected NK product. Among dogs on trial, we observed a trend for higher baseline serum IL-6 to predict worse lung metastasis-free and overall survival (P=0.08). PCR analysis revealed low absolute gene expression of CD3, CD8, and NKG2D in untreated OSA. Among treated dogs, there was marked heterogeneity in the expression of immune-related genes pre- and post-treatment, but increases in CD3 and CD8 gene expression were higher among dogs that lived > 6 months compared to those who did not. Analysis of the TCGA confirmed significant differences in survival among human sarcoma patients with high and low expression of genes associated with greater immune activation and cytotoxicity (CD3e, CD8a, IFN-{gamma}, perforin, and CD122/IL-2 receptor beta).\n\nConclusionsUpdated results from a first-in-dog clinical trial of palliative RT and autologous NK cell immunotherapy for OSA illustrate the translational relevance of companion dogs for novel cancer therapies. Similar to human studies, analyses of immune markers from canine serum, PBMCs, and tumor tissue are feasible and provide insight into potential biomarkers of response and resistance.

immunology↗

bioRxiv2019

Cell-sized confinement controls generation and stability of a protein wave for spatiotemporal regulation in cells

Min system, which determines the cell division plane of bacteria, uses the localization change of protein (Min wave) emerged by a reaction-diffusion coupling. Although previous studies have shown that cell-sized space and boundaries modulate shape and speed of Min waves, its effects on Min wave emergence was still elusive. Here, by using a fully confined microsized space as a mimic of live cells, we revealed that confinement changes conditions for Min wave emergence. In the microsized space, an increase of surface-to-volume ratio changed the localization efficiency of proteins on membranes, and therefore, suppression of the localization change was necessary to produce stable Min wave generations. Furthermore, we showed that the cell-sized space more strictly limits parameters for wave emergence because confinement inhibits instability and excitability of the system. These results illuminate that confinement of reaction-diffusion systems works as a controller of spatiotemporal patterns in live cells.

biophysics↗

bioRxiv2018

Logistic regression analysis of multiple interosseous hand muscle activation using surface electromyography during isometric finger tasks

Intrinsic hand muscles are densely located in the hand, and the myoelectric observation from the surface is sometimes unreliable because of some outside influences that may interfere with the signals. In the present study, we evaluated the activities of multiple interosseous hand-muscles which densely located in the hand, through analyzing the surface electromyographic signals during finger-oriented tasks using univariate and multivariate logistic regression models. Ten healthy subjects participated in our experiment, and isometrically exercised each finger one by one in flexed form. The result of a univariate analysis with the power and amplitude domain predictor variables of the surface electromyographic signals showed significant consistency between the activated finger and the inserted finger of the dorsal interosseous muscles to the proximal phalanx (P < 0.001). Meanwhile, the results of a multivariate analysis showed a higher correlation of the regression model of the fourth dorsal interosseous muscle during the action of the ring finger using frequency-domain variables (the Nagelkerke R2 = 0.716 when the median frequency was used), compared to the model without the frequency-domain variables (the Nagelkerke R2 = 0.583). Our result showed that the logistic regression models have a particular possibility for the analysis of the surface electromyographic signals of densely located hand-muscle activities related to the finger-oriented tasks.

bioengineering↗