Biology subjects

Yagi, H.

Publications and source records attributed to Yagi, H..

17 recordsLinked to original sources

bioRxiv2026

CaMKII-LUZP1 signaling couples cytoskeletal acetylation and autophagy to drive neuronal plasticity

Cytoskeletal acetylation and autophagy are fundamental drivers of neuronal plasticity, yet how these pathways are coordinated across subcellular compartments remains unknown. Here, we identify LUZP1 as a signaling organizer that couples cytoskeletal acetylation to autophagy in hippocampal neurons. LUZP1 deficiency impaired neurite outgrowth and dendritic spine maturation while promoting ciliary elongation; these phenotypes were partially mirrored in neuron-specific Luzp1 knockout mice, which also showed altered locomotor behavior. Mechanistically, LUZP1 promoted neurite extension by enhancing ATAT1-dependent -tubulin acetylation while driving spine maturation and limiting ciliary growth by restraining HDAC6-dependent cortactin (CTTN) deacetylation. An acetylation-mimetic CTTN mutant rescued both spine and ciliary defects caused by LUZP1 deficiency. In parallel, blocking autophagy-dependent OFD1 degradation attenuated ciliary elongation, linking CTTN deacetylation to increased autophagy under LUZP1-deficient conditions. Finally, activated CaMKII associated with LUZP1 and selectively enhanced its interaction with HDAC6 and CTTN, coupling neuronal activity to cytoskeletal remodeling. Together, these findings identify a CaMKII-LUZP1 pathway that integrates cytoskeletal acetylation with autophagy to coordinate neuronal morphogenesis and ciliary homeostasis. HighlightsO_LILUZP1 couples cytoskeletal acetylation to autophagy in hippocampal neurons C_LIO_LILUZP1 promotes neurite extension through ATAT1-dependent -tubulin acetylation C_LIO_LILUZP1 restrains HDAC6-CTTN signaling to drive spine maturation and limit ciliary growth C_LIO_LICaMKII selectively strengthens the LUZP1-HDAC6-CTTN pathway C_LI

neuroscience↗

bioRxiv2026

Dynamic organizational strategies of multidomain glycosyltransferases revealed by high-speed AFM and solution biophysics

Glycosyltransferases often contain multiple structural modules that contribute to substrate recognition, catalytic coordination, and higher-order molecular organization. However, how multidomain glycosyltransferases dynamically organize their catalytic domains in solution remains poorly understood. Here, we investigated the assembly states and conformational dynamics of POMGNT2, LARGE1, K4CP, and L137 using high-speed atomic force microscopy (HS-AFM) integrated with complementary solution biophysical analyses. POMGNT2 formed a stable dimeric architecture with limited large-scale conformational fluctuation, consistent with its role in site-selective substrate recognition. In contrast, LARGE1 and K4CP exhibited concentration-dependent and heterogeneous assembly behavior. K4CP displayed pronounced open-closed interdomain motion and substrate-dependent conformational compaction, indicating dynamic catalytic-domain reorganization during glycan elongation. By comparison, the mimivirus glycosyltransferase candidate L137 predominantly behaved as a monomeric species under the tested conditions. These findings demonstrate that multidomain glycosyltransferases employ diverse dynamic organizational strategies ranging from rigid recognition architectures to highly flexible and reversible catalytic assemblies. Our results further suggest that glycosyltransferase function is governed not only by catalytic-domain structure, but also by dynamic conformational coordination adapted to distinct catalytic demands.

biophysics↗

bioRxiv2026

Arabidopsis hydathodes contain a dense and heterogeneous epithem for apoplastic fluid release

Hydathodes are specialized leaf tissues that mediate guttation, the release of liquid water droplets from vascular plants. They consist of xylem endings, water pores, and the epithem. The epithem comprises small cells whose cell types and functional properties remain poorly understood. While molecular genetic information on hydathodes has accumulated--particularly in the model plant Arabidopsis thaliana through recent RNA-seq analyses--high-resolution anatomical insights into their spatial organization remain limited. In this study, we aimed to characterize the specialized anatomy of Arabidopsis hydathodes using complementary imaging approaches. A fluorescent dye taken up from roots stained hydathodes, consistent with a connection between hydathodes and the vasculature. Microfocus-X-ray computed tomography revealed that the hydathodes protrude from the abaxial leaf surface and are more densely packed internally than surrounding tissues. Light microscopy showed that the epithem was heterogeneous, with elongated cells proximally and rounded cells distally. We found that the epithem was surrounded by cells (hereafter referred to as "boundary cells") with plastids that resembled those of mesophyll cells. Transmission electron microscopy showed that the organelles in epithem cells were immature, and the apoplast contained electron-dense material. Finally, using transgenic plants expressing a secreted fluorescent marker protein, we detected GFP in guttation droplets, indicating that the droplets can contain apoplastic material. Together, these findings reveal that Arabidopsis hydathodes possess a dense and spatially heterogeneous epithem associated with the release of apoplastic fluid.

plant biology↗

bioRxiv2024

Evolutionary acquisition of a primitive light-dependent nuclear relocation in Marchantia polymorpha

The terrestrialization of plants was accompanied by exposure to several environmental stresses. Adaptation to these stresses required numerous changes at the cellular and molecular level. One such adaptation in the leaves of Arabidopsis thaliana is the movement of cell nuclei to avoid UV damage. In the dark, the nuclei locate to the bottom walls of leaf cells to distance genetic material from external stresses, but in response to intense blue light (an indication of the presence of UV), they move to the side walls to escape UV-induced DNA damage1. The movement is driven by the photoreceptor phototropin and the actin cytoskeleton2. However, how this protective mechanism evolved in land plants remains unclear. Here, we show that in the liverwort Marchantia polymorpha, nuclei show a similar, but less stable movement in response to intense blue light. In the dark, M. polymorpha positioned nuclei on the upper walls of epidermal cells in young thalli, but in response to intense blue light, the nuclei immediately moved to the side walls, similar to A. thaliana. However, the movement was transient and the nuclei returned to the upper walls through both the actin and microtubule cytoskeletons. Unlike A. thaliana, M. polymorpha responded to prolonged (> 1 day) exposure to low light by moving nuclei from the upper to the side walls through both the actin and microtubule cytoskeletons and two photoreceptors (phototropin and phytochrome). However, no light-dependent nuclear relocation was observed in charophyte algae, suggesting that light-dependent nuclear relocation was initially established in the common ancestor of land plants as a result of terrestrialization and then diverged during land plant evolution.

plant biology↗

bioRxiv2023

Pathogenic mechanisms underlying adverse neurodevelopmental outcome in congenital heart disease

Poor neurodevelopment is often observed with congenital heart disease (CHD), especially with mutations in chromatin modifiers. Here analysis of mice with hypoplastic left heart syndrome (HLHS) arising from mutations in Sin3A associated chromatin modifier Sap130, and adhesion protein Pcdha9, revealed neurodevelopmental and neurobehavioral deficits reminiscent of those in HLHS patients. Microcephaly was associated with impaired cortical neurogenesis, mitotic block, and increased apoptosis. Transcriptional profiling indicated dysregulated neurogenesis by REST, altered CREB signaling regulating memory and synaptic plasticity, and impaired neurovascular coupling modulating cerebral blood flow. Many neurodevelopmental/neurobehavioral disease pathways were recovered, including autism and cognitive impairment. These same pathways emerged from genome-wide DNA methylation and Sap130 chromatin immunoprecipitation sequencing analyses, suggesting epigenetic perturbation. Mice with Pcdha9 mutation or forebrain-specific Sap130 deletion without CHD showed learning/memory deficits and autism-like behavior. These novel findings provide mechanistic insights indicating the adverse neurodevelopment in HLHS may involve cell autonomous/nonautonomous defects and epigenetic dysregulation and suggest new avenues for therapy.

developmental biology↗

bioRxiv2023

The Arabidopsis katamari2 Mutant Exhibits a Hypersensitive Seedling Arrest Response at the Phase Transition from Heterotrophic to Autotrophic Growth

Young seedlings use nutrients stored in the seeds to grow and acquire photosynthetic potential. This process, called seedling establishment, involves a developmental phase transition from heterotrophic to autotrophic growth. Some membrane-trafficking mutants of Arabidopsis thaliana (Arabidopsis), such as the katamari2 (kam2) mutant, exhibit growth arrest during seedling development, with a portion of individuals failing to develop true leaves on sucrose-free solid medium. However, the reason for this seedling arrest is unclear. In this study, we show that seedling arrest is a temporal growth arrest response that occurs not only in kam2 but also in wild-type Arabidopsis; however, the threshold for this response is lower in kam2 than in the wild type. A subset of the arrested kam2 seedlings resumed growth after transfer to fresh sucrose-free medium. Growth arrest in kam2 on sucrose-free medium was restored by increasing the gel concentration of the medium or covering the surface of the medium with a perforated plastic sheet. Wild-type Arabidopsis seedlings were also arrested when the gel concentration of sucrose-free medium was reduced. RNA sequencing revealed that transcriptomic changes associated with the rate of seedling establishment were observed as early as 4 days after sowing. Our results suggest that the growth arrest of both kam2 and wild-type seedlings is an adaptive stress response and is not simply caused by the lack of a carbon source in the medium. This study provides a new perspective on an environmental stress response under unfavorable conditions during the phase transition from heterotrophic to autotrophic growth in Arabidopsis.

plant biology↗

bioRxiv2023

UGGT1/2-mediated reglucosylation of N-glycan competes with ER-associated degradation of unstable and misfolded glycoproteins

How the fate (folding versus degradation) of glycoproteins is determined in the endoplasmic reticulum (ER) is an intriguing question. Monoglucosylated glycoproteins are recognized by lectin chaperones to facilitate their folding, whereas glycoproteins exposing well-trimmed mannoses are subjected to glycoprotein ER-associated degradation (gpERAD); we have elucidated how mannoses are sequentially trimmed by EDEM family members (George et al., 2020, 2021 eLife). Although reglucosylation by UGGT was previously reported to have no effect on substrate degradation, here we directly tested this notion using cells with genetically disrupted UGGT1/2. Strikingly, the results showed that UGGT1 delayed the degradation of misfolded substrates and unstable glycoproteins including ATF6. An experiment with a point mutant of UGGT1 indicated that the glucosylation activity of UGGT1 was required for the inhibition of early glycoprotein degradation. These and overexpression-based competition experiments suggested that the fate of glycoproteins is determined by a tug-of-war between structure formation by UGGT1 and degradation by EDEMs. We further demonstrated the physiological importance of UGGT1, since ATF6 cannot function properly without UGGT1. Thus, our work strongly suggests that UGGT1 is a central factor in ER protein quality control via regulation of both glycoprotein folding and degradation.

cell biology↗

bioRxiv2023

Cardiomyocyte Targeting Peptide to Deliver Amiodarone

BackgroundAmiodarone is underutilized due to significant off-target toxicities. We hypothesized that targeted delivery to the heart would lead to lowering of dose by utilizing a cardiomyocyte targeting peptide (CTP), a cell penetrating peptide identified by our prior phage display work. MethodsCTP was synthesized thiolated at the N-terminus, conjugated to amiodarone via Schiff base chemistry, HPLC purified and confirmed with MALDI/TOF. Stability of the conjugate was assessed using serial HPLCs. Guinea pigs (GP) were injected intraperitoneally daily with vehicle (7 days), amiodarone (7 days; 80mg/Kg), CTP-amiodarone (5 days;26.3mg/Kg), or CTP (5 days; 17.8mg/Kg), after which GPs were euthanized, hearts excised, perfused on a Langendorff apparatus with Tyrodes solution and blebbistatin (5M) to minimize contractions. Voltage (RH237) and Ca2+-indicator dye (Rhod-2/AM) were injected, fluorescence from the epicardium split and focused on two cameras capturing at 570-595nm for cytosolic Ca2+ and 610-750nm wavelengths for voltage. Subsequently, hearts were paced at 250ms with programmed stimulation to measure changes in conduction velocities (CV), action potential duration (APD) and Ca2+ transient durations at 90% recovery (CaTD90). mRNA was extracted from all hearts and RNA sequencing performed with results compared to control hearts. ResultsCTP-amiodarone remained stable for up to 21 days at 37{degrees}C. At [~]1/15th of the dose of amiodarone, CTP-amiodarone decreased CV in hearts significantly compared to control GPs (0.92{+/-}0.05 vs. 1.00{+/-}0.03m/s, p=0.0007), equivalent to amiodarone alone (0.87{+/-}0.08ms, p=0.0003). Amiodarone increased APD (192{+/-}5ms vs. 175{+/-}8ms for vehicle, p=0.0025), while CTP-amiodarone decreased it significantly (157{+/-}16ms, p=0.0136) similar to CTP alone (155{+/-}13ms, p=0.0039). Both amiodarone and CTP-amiodarone significantly decreased calcium transients compared to controls. CTP-amiodarone and CTP decreased CaTD90 to an extent greater than amiodarone alone (p<0.001). RNA-seq showed that CTP alone increased the expression of DHPR and SERCA2a, while decreasing expression of proinflammatory genes NF-kappa B, TNF-, IL-1{beta}, and IL-6. ConclusionsOur data suggests that CTP can deliver amiodarone to cardiomyocytes at [~]1/15th the total molar dose of amiodarone needed to produce comparable slowing of CVs. The ability of CTP to decrease AP durations and CaTD90 may be related to its increase in expression of Ca-handling genes, and merits further study.

molecular biology↗

bioRxiv2023

Spatial transcriptome profiling uncovers metabolic regulation of left-right patterning

Left-right patterning disturbance can cause severe birth defects, but it remains least understood of the three body axes. We uncovered an unexpected role for metabolic regulation in left-right patterning. Analysis of the first spatial transcriptome profile of left-right patterning revealed global activation of glycolysis, accompanied by right-sided expression of Bmp7 and genes regulating insulin growth factor signaling. Cardiomyocyte differentiation was left-biased, which may underlie the specification of heart looping orientation. This is consistent with known Bmp7 stimulation of glycolysis and glycolysis suppression of cardiomyocyte differentiation. Liver/lung laterality may be specified via similar metabolic regulation of endoderm differentiation. Myo1d, found to be left-sided, was shown to regulate gut looping in mice, zebrafish, and human. Together these findings indicate metabolic regulation of left-right patterning. This could underlie high incidence of heterotaxy-related birth defects in maternal diabetes, and the association of PFKP, allosteric enzyme regulating glycolysis, with heterotaxy. This transcriptome dataset will be invaluable for interrogating birth defects involving laterality disturbance.

developmental biology↗

bioRxiv2022

Inverse Agonist Activity of Angiotensin II Receptor Blocker Is Crucial for Prevention of Aortic Aneurysm Formation in Marfan Syndrome

BackgroundMarfan syndrome (MFS), an inherited disorder caused by FBN1 gene mutations, causes fatal aortic aneurysm. Selective angiotensin II (Ang II) type 1 (AT1) receptor blockade is a preventive option for patients with MFS aortopathy, and recent clinical studies demonstrated that the inhibitory effect of an AT1 receptor blocker (ARB) losartan on aortic aneurysm growth is equivalent to that of {beta}-blockers. At present, several ARBs are clinically available, and they have drug-specific differences in pharmacological properties. Especially, inverse agonism of ARBs has potential benefits for cardiovascular protection, but its impact on MFS aortopathy remains poorly understood. MethodsCandesartan-7H is a candesartan derivative which lacks the carboxyl group critical for inverse agonist activity and works as a neutral antagonist for AT1 receptor. Candesartan cilexetil (1 mg/kg/day), candesartan-7H (1 mg/kg/day or 20 mg/kg/day), or vehicle was administered to Fbn1C1041G/+ mice, and aortic aneurysm formation was analyzed using echocardiography, histological staining, and in situ MMP assay. Activation of TGF-{beta} signaling and mechanosensitive signaling was studied using western blot and immunohistochemical analysis. ResultsCandesartan cilexetil (1 mg/kg/day) and candesartan-7H (20 mg/kg/day) lowered blood pressures equally in Fbn1C1041G/+ mice, but that candesartan-7H (1 mg/kg/day) did not. Aortic aneurysmal progression in association with aortic wall thickening, degeneration of elastic fibers, deposition of collagen, MMP activation, and TGF-{beta} signaling activation in Fbn1C1041G/+ mice was significantly suppressed by treatment with candesartan cilexetil (1 mg/kg/day), but not by candesartan-7H even at 20 mg/kg/day. In addition, candesartan cilexetil, but not candesartan-7H, inhibited up-regulation of mechanical stress-responsive transcriptional factor Egr-1 in ascending aorta of Fbn1C1041G/+ mice. ConclusionsOur findings support a crucial role of inverse agonist activity of ARB for prevention of mechanical stress-induced AT1 receptor activation and aortic aneurysm formation in MFS mice.

pharmacology and toxicology↗

bioRxiv2022

Orchestration of Proteins in cyanobacterial Circadian Clock System 1

Circadian rhythm by Cyanobacteria is one of the simplest biological clocks: the clock consists of only three proteins, KaiA, KaiB and KaiC. Their oligomers, KaiA dimer (A2), KaiB tetramer (B4) and KaiC hexamer (C6) oscillate an association- disassociation cycle with 24hr period. In a widely accepted model, the oscillation process is as follows. From the viewpoint of a base unit (C6), C6 homo-oligomer [->] A2C6 complex [->] B6C6 complex [->] AnB6C6 complex (n[≤]12) [->]C6 homo-oligomer. In this study, Small-Angle X-ray Scattering, Contrast Matching-Small-Angle Neutron Scattering, Analytical Ultracentrifuge and phosphorylation-analysis PAGE measurements were performed to reveal the kinetics not only of KaiC hexamer but also of all components in a working Kai clock. The complementary analysis disclosed that the oscillation is not the single process as the widely accepted model but composed with synchronized multiple association-dissociation reactions between components. Namely, there are various reactions between components, which proceed simultaneously, in a working Kai-clock.

biophysics↗

bioRxiv2022

Aberrant mechanosensitive signaling underlies activation of vascular endothelial xanthine oxidoreductase that promotes aortic aneurysm formation in Marfan syndrome

Marfan syndrome (MFS) is an inherited connective tissue disorder caused by mutations in the FBN1 gene encoding fibrillin-1, a matrix component of extracellular microfibrils. The main cause of morbidity and mortality in MFS is thoracic aortic aneurysm and dissection, but the underlying mechanisms remain undetermined. We found a significant increase in reactive oxygen species (ROS) generation in ascending aorta of MFS patients and MFS mice harboring the Fbn1 mutation (C1039G), which was associated with up-regulation of xanthine oxidoreductase (XOR) protein in aortic endothelial cells (ECs). Mechanosensitive signaling involving focal adhesion kinase (FAK)-p38 mitogen-activated protein kinase (MAPK) and early growth response-1 (Egr- 1) was aberrantly activated in ascending aorta of Fbn1C1039G/+ mice, and mechanical stress on human aortic ECs up-regulated XOR expression through FAK-p38 MAPK activation and Egr-1 up-regulation. Inhibition of XOR function by ECs-specific disruption of Xdh gene or by systemic administration of XOR inhibitor febuxostat in Fbn1C1039G/+ mice suppressed ROS generation, FAK-p38 MAPK activation, and Egr-1 up-regulation, leading to attenuation of aortic aneurysm formation. These findings unveil aberrant mechanosensitive signaling in vascular ECs triggering endothelial XOR activation and ROS generation as a culprit underlying the pathogenesis of aortic aneurysm formation in MFS, and highlight a drug repositioning approach using a uric acid lowering drug febuxostat as a potential therapy for MFS.

pathology↗

bioRxiv2021

Cancer malignancy is correlated with up-regulation of PCYT2-mediated glycerol phosphate modification of α-dystroglycan

The dystrophin-glycoprotein complex connects the cytoskeleton with base membrane components such as laminin through unique O-glycans displayed on -dystroglycan (-DG). Genetic impairment of elongation of these glycans causes congenital muscular dystrophies. We previously identified that glycerol phosphate (GroP) can cap the core part of the -DG O-glycans and terminate their further elongation. This study examined the possible roles of the GroP modification in cancer malignancy, focusing on colorectal cancer. We found that the GroP modification critically depends on PCYT2, which serves as CDP-Gro synthase. Furthermore, we identified a significant positive correlation between cancer progression and GroP modification, which also correlated positively with PCYT2 expression. Moreover, we demonstrate that GroP modification promotes the migration of cancer cells. Based on these findings, we propose that the GroP modification by PCYT2 disrupts the glycan-mediated cell adhesion to the extracellular matrix and thereby enhances cancer metastasis. Thus, the present study suggests the possibility of novel approaches for cancer treatment by targeting the PCYT2-mediated GroP modification.

biochemistry↗

bioRxiv2021

iPSC modeling shows uncompensated mitochondrial mediated oxidative stress underlies early heart failure in hypoplastic left heart syndrome

Hypoplastic left heart syndrome (HLHS) is a severe congenital heart defect with 30% mortality from heart failure (HF) in the first year of life, but why only some patients suffer early-HF and its cause remain unknown. Modeling using induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) showed early-HF patient iPSC-CM have increased apoptosis, redox stress, and failed antioxidant response. This was associated with mitochondrial permeability transition pore (mPTP) opening, mitochondrial hyperfusion and respiration defects. Whereas iPSC-CM from patients without early-HF had hyper-elevated antioxidant response with increased mitochondrial fission and mitophagy. Single cell transcriptomics showed dichotomization by HF outcome, with mitochondrial dysfunction and endoplasmic reticulum (ER) stress associated with early-HF. Importantly, oxidative stress and apoptosis associated with early HF were rescued by sildenafil inhibition of mPTP opening or TUDCA suppression of ER stress. Together these findings demonstrate a new paradigm for modeling clinical outcome in iPSC-CM, demonstrating uncompensated mitochondrial oxidative stress underlies early HF in HLHS.

cell biology↗

bioRxiv2021

Multi-state structure determination and dynamics analysis reveals a new ubiquitin-recognition mechanism in yeast ubiquitin C-terminal hydrolase

Despite accumulating evidence that protein dynamics is indispensable for understanding the structural basis of biological activities, it remains challenging to visualize the spatial description of the dynamics and to associate transient conformations with their molecular functions. We have developed a new NMR protein structure determination method for the inference of multi-state conformations using multiple types of NMR data, including paramagnetic NMR and residual dipolar couplings, as well as conventional NOEs. Integration of these data in the structure calculation permits delineating accurate ensemble structures of biomacromolecules. Applying the method to the protein yeast ubiquitin hydrolase 1 (YUH1), we find large dynamics of its N-terminus and crossover loop surrounding the active site for ubiquitin-recognition and proteolysis. The N-terminus gets into and out of the crossover loop, suggesting their underlying functional significance. Our results, including those from biochemical analysis, show that large motion surrounding the active site contributes strongly to the efficiency of the enzymatic activity.

biophysics↗

bioRxiv2020

Hyperactivation of L-lactate oxidase by liquid-liquid phase separation

Liquid droplets formed by liquid-liquid phase separation are attracting attention as functional states of proteins in living cells. Liquid droplets are thought to activate enzymatic reactions by assembling the required molecules. Thus, liquid droplets usually increase the affinity of an enzyme to its substrates, leading to decreased KM values. In this study, we demonstrate a new mechanism of enzyme activation in the droplets using Llactate oxidase (LOX). In the presence of poly-L-lysine (PLL), LOX formed droplets with diameters of hundreds of nanometers to tens of micrometers, stabilized by electro-static interaction. The enzyme activity of LOX in the droplets was significantly enhanced by a fourfold decrease in KM and a tenfold increase in kcat. To our knowledge, this represents the first report for increasing kcat by the formation of the liquid droplet. Interestingly, the conformation of LOX changed in the liquid droplet, probably leading to increased kcat value. Understanding enzyme activation in the droplets provides essential information about enzyme function in living cells in addition to biotechnology applications.

biochemistry↗

bioRxiv2018

An unconventional myosin, myosin 1d regulates Kupffer’s vesicle morphogenesis and laterality

INTRODUCTION INTRODUCTION RESULTS MATERIALS AND METHODS AUTHOR CONTRIBUTIONS REFERENCES Establishing left-right (LR) asymmetry is a fundamental process essential for arrangement of visceral organs during development. In vertebrates, motile cilia driven fluid flow in the left-right organizer (LRO) is essential for initiating symmetry breaking event1-3. Without a definite LRO structure in invertebrates, LR asymmetry is initiated at a cellular level by actin-myosin driven chirality4, 5. In Drosophila, myosin1D drives tissue-specific chirality in hind-gut looping6, 7. Here, we show that myosin 1d (myo1d) is essential for establishing LR asymmetry in zebrafish. Using super-r ...

developmental biology↗