Biology subjects

Nakano, A.

Publications and source records attributed to Nakano, A..

At least 19 recordsLinked to original sources

bioRxiv2026

Contact of mobile cis-Golgi network with ER exit sites executes COPI-mediated receptor recycling

COPI-mediated retrograde transport is essential for maintaining homeostasis between the endoplasmic reticulum (ER) and the Golgi in eukaryotic cells. However, its spatiotemporal dynamics remain unclear. In budding yeast, ER export occurs through a hug-and-kiss mechanism, wherein mobile cis-Golgi network (CGN) elements transiently approach ER exit sites (ERES) to capture cargo before departing to deliver it to the Golgi. Here, using high-resolution 3D and 4D live imaging, we show that the COPI-mediated recycling of a cargo receptor from the CGN back to the ER occurs during the hug-and-kiss event. Furthermore, this recycling is complete before the CGN detaches from the ERES, suggesting that COPI-mediated retrograde transport is spatiotemporally coupled to ER export during a single and transient CGN-ERES association. Consistent with this model, when COPI function is impaired, the receptor fails to return efficiently to the ER and instead remains in the CGN after separation from the ERES. Additionally, COPI impairment prolonged CGN-ERES association, suggesting that COPI function also facilitates CGN detachment from ERES. Thus, we propose that rapid COPI-mediated recycling drives CGN maturation directly at the ERES interface, enabling the CGN to detach and proceed along the secretory pathway.

cell biology↗

bioRxiv2026

Topological defects and coherent myocardial chirality shape torsional heart contraction

The efficient pumping of the mammalian heart relies on torsional contractile motion generated by its highly ordered three-dimensional (3D) architecture of myocardial fibres. However, the topological principles governing how its complex geometry translates into contractile mechanics remain elusive. Here, we show that the mammalian heart forms a chiral nematic field, a biological analogue to 3D liquid crystals, whose topological organisation underlies its function. Analysis of 3D imaging data revealed disclination lines, continuous assemblies of topological defects characteristic of nematic systems, within the compact myocardium. Finite-element simulations reveal that these defects are not mere structural irregularities but can locally modulate contractile behaviour and reduce mechanical work. In heterotaxy hearts with reversed global anatomy (situs inversus), myocardial fibres retain a predominantly counter-clockwise twist, similar to that of the normal heart, but with a small clockwise component near the base. This decoupling of tissue-level chirality from systemic left-right patterning suggests that cardiac twist is an intrinsic property of the myocardial fibre. Mechanical simulations of situs inversus heart demonstrate that the coherence of transmural chirality, rather than its specific orientation, is critical for contractile efficiency. Together, these findings establish the heart as a topological material and reveal how organised chiral fields generate robust organ-level mechanical function.

biophysics↗

bioRxiv2025

In situ structure determination of Respiratory Supercomplexes and ATP synthase oligomers in mammalian mitochondrial inner membrane.

To understand how membrane protein complexes function within biological membranes, it is essential to determine their structure in their natural membrane environment. Here, we employed cryoEM structure analysis to elucidate the structures of ATP synthase FoF1 and respiratory Supercomplexes (SCs) on sub-mitochondrial particles (SMPs) isolated from bovine heart mitochondria. On SMPs, the majority of FoF1 was identified as dimers bound by the regulatory factor dimeric IF1. In addition, a tetrameric structure formed by association of FoF1 IF1 dimers and with a linear arrangement of the F1 head were also identified. These structures induced a steep membrane curvature, indicating the presence of a structure on SMPs similar to that found on the tips of mitochondrial cristae. High-resolution structures of the respiratory complexes were also determined, and sub-class structures of both CI and CIII2 were resolved. Most SCs were of the CI1CIII2CIV3 structure, although the presence of the CI2CIII2CIV6 mega complex was also identified. Our study enabled rapid in situ structural determination of SCs and FoF1 ATP synthase from small amount of membrane fractions, paving the way for elucidation of the molecular basis of metabolic disorders and mitochondrial diseases at the level of higher-order architecture.

biochemistry↗

bioRxiv2025

A planar dimer of bovine ATP synthase

Mammalian mitochondrial ATP synthase typically organizes into rows of V-shaped dimers that impose significant membrane curvature essential for mitochondrial cristae formation. Using gentle, column-free purification combined with single-particle cryo-electron microscopy, we have identified a previously unrecognized planar dimeric form of bovine ATP synthase exhibiting minimal membrane bending. This planar dimer is characterized structurally by antiparallel arrangement of two ATP synthase complexes linked by a straight conformation of the inhibitory factor 1 (IF1), a sharp contrast to the kinked IF1 observed in tetrameric assemblies. Molecular dynamics simulations confirm that transitioning between straight and kinked IF1 conformations occurs without significant energetic barriers. The planar dimer also displays distinct peripheral stalk positioning relative to its adjacent subunit. These structural divergences suggest a specialized functional role and localization distinct from the canonical sharply membrane bending ATP synthase oligomers, providing structural support for a model of membrane curvature-driven division of labor within mitochondrial ATP synthase populations.

biophysics↗

bioRxiv2024

Brassinosteroids and Flavonols Confer Temperature Stress Tolerance to Pollen Tube Germination and Growth

Successful reproduction is a prerequisite for the maintenance of species in the era of climate change. In agriculture, temperature has a significant impact on pollen tube elongation and attraction, and consequently, on the efficiency of fruiting and crop yields. Brassinosteroids (BRs) are plant steroid hormones involved in pollen tube germination, elongation, and capacitation. To elucidate the effect of BR signaling in pistils during the reproductive process, transcriptome analysis of BR receptor (BRI1) mutant pistils was performed, which indicated that genes involved in flavonol biosynthesis were downregulated in bri1 pistils. Flavonols are essential for pollen tube growth in some plants and facilitate pollen tube elongation under high-temperature conditions. To investigate the effects of BR and flavonols on pollen tubes under temperature stress, in vitro assays were performed using both the compounds. Brassinolide (BL), which is the most active BR, promoted pollen tube germination at high, low, and optimal temperatures. Germination was further enhanced by the flavonol, quercetin, in the presence of BL especially at low temperatures. in vivo analysis supported the observation that when BL and quercetin were supplied concurrently, the pollen tube guidance rate was rescued at low temperatures. Quercetin enhanced pollen tube elongation under all the conditions tested at an optimal concentration of 15 M. BL elevated reactive oxygen species (ROS) under all temperature conditions, whereas quercetin regulated ROS synergistically with BR. Thus, BRs and flavonols supplied by pistils are suggested to function in pollen tube germination and elongation through the maintenance of ROS homeostasis.

plant biology↗

bioRxiv2024

Human formin FHOD3-mediated actin elongation is required for sarcomere integrity in cardiomyocytes

Contractility and cell motility depend on accurately controlled assembly of the actin cytoskeleton. Formins are a large group of actin assembly proteins that nucleate and elongate new actin filaments. Some formins may cap filaments while others sever or bundle filaments. The Formin HOmology Domain-containing protein (FHOD)-family of formins is critical to the formation of the fundamental contractile unit in muscle, the sarcomere. Specifically, mammalian FHOD3L plays an essential role in cardiomyocytes. Despite our knowledge of FHOD3Ls importance in cardiomyocytes, its biochemical and cellular activities remain poorly understood. It was proposed that FHOD-family formins act by capping and bundling, as opposed to assembling new filaments. Here, we demonstrate that FHOD3L nucleates actin and rapidly but briefly elongates filaments after temporarily pausing elongation. We designed function-separating mutants that enabled us to distinguish which biochemical roles are required in the cell. We found that human FHOD3Ls elongation activity, but not its nucleation, capping, or bundling activity, is necessary for proper sarcomere formation and contractile function in neonatal rat ventricular myocytes. The results of this work provide new insight into the mechanisms by which formins build specific structures and will contribute to knowledge regarding how cardiomyopathies arise from defects in sarcomere formation and maintenance.

cell biology↗

bioRxiv2024

A non-canonical CDK, Pho85 regulates the restart of the cell-cycle following stress

Environmental stress induces an arrest of the cell cycle. Thus, release from this arrest is essential for cell survival. The cell-cycle-arrest occurs via the down regulation of the cyclins that drive the main cyclin dependent kinase, CDK1/Cdc28. However, it was not clear how cells escape this potentially fatal arrest. Here we show that prior to the restoration of CDK1/Cdc28 cyclins, a non-canonical CDK, Pho85, initiates a cascade to restart the cell cycle. We demonstrate that following stress, Pho85 phosphorylates the Sch9 kinase, which in turn directly phosphorylates the transcriptional inhibitor Whi5, the yeast analog of RB1/retinoblastoma, and a CDK1 target. This promotes Whi5 translocation from the nucleus, and the release of the stress-induced arrest at G1 phase. In addition, we find that in parallel with Pho85, CDK1/Cdc28 also plays a role in the control of Whi5. Together, these findings provide insights into how cells re-enter the cell cycle during recovery from stress and reveal that a non-canonical CDK and cyclin takes on essential roles and acts via a pathway that functions in parallel with CDK1/Cdc28.

molecular biology↗

bioRxiv2024

Brassinosteroids function as the plant male and female reproductive hormone coordinating gene expression.

Brassinosteroids (BRs) are steroid hormones identified in plants. Besides promoting cell elongation and division, BRs facilitate the development of both male and female reproductive tissues. In animals, reproductive steroid hormones play an essential role in reproductive tissue development by regulating gene expression. Here, we focused on the function of BRs during fertilization. We measured the content of biologically active BRs, brassinolide (BL) and castasterone (CS), in the reproductive tissues of Arabidopsis thaliana. Both BL and CS accumulated abundantly in pollen grains and in larger amounts in pistils than in leaves. To evaluate BL function during fertilization, we used an in vitro guidance assay with exogenously applied BL. Although pollen tubes need to be elongated through the pistils for efficient capacitation, BL treatment promoted pollen tube capacitation and improved attraction to ovules in vitro. Transcriptome analysis demonstrated that BL treatment induced the expression of half of the genes expressed in pollen tubes that elongated through the pistils. These results indicated that BL supplied from pistils is a key factor for pollen tube capacitation. However, using the bri1 mutant for the guidance assay resulted in reduced pollen tube capacitation, suggesting that BRI1-signaling in pistils is also important. Furthermore, BRs act on ovules. Exogenous BL application to ovules maintained guidance capacity by promoting the expression of small secreted proteins involved in pollen tube attraction and gamete fusion. Overall, BRs play a significant role as male and female reproductive hormones throughout the plant fertilization process.

plant biology↗

bioRxiv2023

Transgenic mice overexpressing Pitx2 in the atria develop tachycardia-bradycardia syndrome

Sinoatrial node (SAN) dysfunction often accompanies supraventricular tachyarrhythmias such as atrial fibrillation (AF), which is referred to as tachycardia-bradycardia syndrome (TBS). Although there have been many studies on electrical remodeling in TBS, the regulatory mechanisms that cause electrical remodeling in the SAN and atrial muscles by chronic bradycardia or tachycardia have not yet been fully investigated. Here we hypothesized that Pitx2c, a transcription factor that plays a central role in the late aspects of left-right asymmetric morphogenesis, regulates an interrelationship between the SAN and the atrial muscles and is involved in TBS-like pathology. To test this hypothesis, we generated transgenic mice overexpressing Pitx2c specifically in the atria (OE mice). Although Pitx2c is normally expressed only in left atria, the expression levels of Pitx2c protein in the right atria were significantly increased to similar levels of those in the left atria of non-transgenic control mice (WT). We found that the heart rate of OE mice was significantly variable although the average heart rate was similar between WT and OE mice. Electrophysiological examination showed that OE mice exhibited prolonged SAN recovery time and higher AF inducibility. In addition, recording of the atrial monophasic action potential duration using a Langendorff perfusion system demonstrated shorter action potential duration in OE atria. Histological analysis revealed that SAN-specific ion channel HCN4-positive cells were hardly detected in the SAN of OE mice, along with ectopic expression in the right atria. Furthermore, transcription factors associated with sinus node formation were down-regulated in the right atria of OE mice. Therefore, SAN dysfunction by Pitx2 dysregulation predisposed OE mice to a TBS-like phenotype. We conclude that Pitx2c is a key regulator that defines SAN function in the atria.

developmental biology↗

bioRxiv2023

Rapid Vacuolar Sorting of the Borate Transporter BOR1 Requires the Adaptor Protein Complex AP-4 in Arabidopsis

Plants maintain nutrient homeostasis by controlling the activities and abundance of nutrient transporters. In Arabidopsis thaliana, the borate (B) transporter BOR1 plays a role in the efficient translocation of B under low-B conditions. BOR1 undergoes polyubiquitination in the presence of sufficient B and is then transported to the vacuole via multivesicular bodies (MVBs) to prevent B accumulation in tissues at a toxic level. A previous study indicated that BOR1 physically interacts with subunits of adaptor protein complexes AP-3 and AP-4, both involved in vacuolar sorting pathways. In this study, we investigated the roles of AP-3 and AP-4 subunits in BOR1 trafficking in Arabidopsis. The lack of AP-3 subunits did not affect either vacuolar sorting or polar localization of BOR1-GFP, whereas the absence of AP-4 subunits resulted in a delay in high-B-induced vacuolar sorting without affecting polar localization. Super-resolution microscopy revealed a rapid sorting of BOR1-GFP into AP-4-positive spots in the trans-Golgi network (TGN) upon high-B supply. These results indicate that AP-4 is involved in sequestration of ubiquitinated BOR1 into a TGN-specific subdomain "vacuolar-trafficking zone," and is required for efficient sorting to MVB and vacuole. Our findings elucidate the rapid vacuolar sorting process facilitated by AP-4 in plant nutrient transporters. Subject areas(7) Membrane and transport

plant biology↗

bioRxiv2023

RudLOV-a new optically synchronized cargo transport method reveals unexpected effect of dynasore

Live imaging of secretory cargoes is a powerful method for understanding the mechanisms of membrane trafficking. Inducing the synchronous release of cargoes from an organelle is a key for enhancing microscopic observation. We developed an optical cargo-releasing method named as retention using dark state of LOV2 (RudLOV), which enables exceptional spatial, temporal, and quantity control during cargo release. A limited amount of cargo-release using RudLOV successfully visualized cargo cisternal-movement and cargo-specific exit sites on the Golgi/trans-Golgi network. Moreover, by controlling the timing of cargo-release using RudLOV, we revealed the canonical and non-canonical effects of the well-known dynamin inhibitor dynasore, which inhibits early-Golgi but not late-Golgi transport and exit from the trans-Golgi network where dynamin-2 is active. Accumulation of COPI vesicles at the cis-side of the Golgi stacks in dynasore-treated cells suggests that dynasore targets COPI-uncoating/tethering/fusion machinery in the early-Golgi cisternae or endoplasmic reticulum but not in the late-Golgi cisternae. These results provide insight into the cisternal maturation of Golgi stacks.

cell biology↗

bioRxiv2023

ER-to-Golgi trafficking via a dynamic intermediate cis-Golgi tubular network in Arabidopsis

Endoplasmic Reticulum (ER)-to-Golgi trafficking is a central process of the secretory system of eukaryotic cells that ensures proper spatiotemporal sorting of proteins and lipids1-5. However, the nature of the ER-Golgi Intermediate Compartments (ERGIC) and the molecular mechanisms mediating the transition between the ERGIC and the Golgi, as well as the universality of these processes amongst Eukaryotes, remain undiscovered. Here, we took advantage of the plant cell system in which the Golgi is highly dynamic and in close vicinity to the ER6-9. We discovered that the ERGIC is composed from at least two distinct subpopulations of cis-Golgi. A subpopulation is a reticulated tubulo-vesicular network mostly independent from the Golgi, highly dynamic at the ER-Golgi interface and crossed by ER-induced release of luminal cargos at early stage. Another subpopulation is more stable, cisterna-like and mostly associated to the Golgi. Our results identified that the generation and dynamics of the ER-Golgi intermediate tubulo-vesicular network is regulated by the acyl-chain length of sphingolipids as well as the contacts it establishes with existing Golgi cisternae. Our study is a major twist in the understanding of the Golgi by identifying that the ERGIC in plants is a Golgi-independent highly dynamic tubular network from which arise more stable cisternae-like Golgi structures. This novel model presents a mechanism for early secretory trafficking adapted to respond to developmental and environmental stimuli, including susceptibility or resistance to diseases, autophagy or cell-reprograming.

cell biology↗

bioRxiv2023

Rtf1-dependent transcriptional pausing regulates cardiogenesis

Transcriptional pause-release critically regulates cellular RNA biogenesis, yet how dysregulation of this process impacts embryonic development is not fully understood. Rtf1 is a multifunctional transcription regulatory protein involved in modulating promoter-proximal pausing of RNA polymerase II. Using zebrafish and mouse as model systems, we show that Rtf1 activity is essential for the differentiation of the myocardial lineage from mesoderm. Ablation of rtf1 impairs the formation of nkx2.5+/tbx5a+ cardiac progenitor cells, resulting in the development of embryos without cardiomyocytes. Structure-function analysis demonstrates that Rtf1s cardiogenic activity requires its Plus3 domain, which confers interaction with the pausing/elongation factor Spt5. In Rtf1-deficient embryos, the occupancy of RNA Pol II at transcription start sites was reduced relative to downstream occupancy, suggesting a reduction in transcriptional pausing. Intriguingly, attenuating pause release by pharmacological inhibition or morpholino targeting of CDK9 improved RNA polymerase II occupancy at the transcription start sites of key cardiac genes and restored cardiomyocytes in Rtf1-deficient embryos. Thus, our findings demonstrate the crucial role that Rtf1-mediated transcriptional pausing plays in controlling the precise spatio-temporal transcription programs that govern early heart development.

developmental biology↗

bioRxiv2023

Presence of the ER-Golgi intermediate compartment that matures into the Golgi in yeast

Cargo traffic through the Golgi apparatus is mediated by cisternal maturation, but it remains largely unclear how the cis-cisternae, the earliest Golgi sub-compartment, is generated. Here, we use high-speed, high-resolution confocal microscopy to analyze the spatiotemporal dynamics of a diverse set of proteins that reside in and around the Golgi in budding yeast. We find many mobile punctate structures that harbor yeast counterparts of mammalian endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC) proteins, which we term "yeast ERGIC". It occasionally attaches onto the ER exit sites and gradually matures into the cis-Golgi. Upon treatment with the Golgi-disrupting agent brefeldin A, the ERGIC proteins form larger aggregates corresponding to the Golgi entry core compartment in plants, while cis- and medial-Golgi proteins are absorbed into the ER. We further analyze the dynamics of several late Golgi proteins. Together with our previous studies, we demonstrate a detailed spatiotemporal profile of the cisternal maturation process from ERGIC to Golgi and further to the trans-Golgi network. SummaryTojima et al. perform spatiotemporal mapping of a variety of proteins residing in and around the Golgi apparatus in budding yeast using super-resolution live imaging microscopy. They identified the ER-Golgi intermediate compartment that matures into the Golgi.

cell biology↗

bioRxiv2023

Remodeling of the secretory pathway is coordinated with de novo membrane formation in budding yeast gametogenesis

Gametogenesis in budding yeast involves a large-scale rearrangement of membrane traffic to allow de novo formation of a membrane, called the prospore membrane (PSM). However, the mechanism underlying this event is not fully elucidated. Here, we show that the number of endoplasmic reticulum exit sites (ERES) per cell fluctuates and switches from decreasing to increasing upon the onset of PSM formation. Reduction in ERES number, presumably accompanying a transient stall in membrane traffic, resulting in the loss of preexisting Golgi apparatus from the cell, was followed by local ERES regeneration, leading to Golgi reassembly in nascent spores. We have revealed that protein phosphatase-1 (PP-1) and its development-specific subunit, Gip1, promote ERES regeneration through Sec16 foci formation. Furthermore, sed4{Delta}, a mutant with impaired ERES formation, showed defects in PSM growth and spore formation. Thus, ERES regeneration in nascent spores facilitates the segregation of membrane traffic organelles, leading to PSM growth.

cell biology↗

bioRxiv2023

Atlas of fetal metabolism during mid-to-late gestation and diabetic pregnancy

Mounting evidence supports an instructive role for metabolism in stem cell fate decisions. However, much is yet unknown about how fetal metabolism changes during mammalian development and how altered maternal metabolism shapes fetal metabolism. Here, we present a descriptive atlas of in vivo fetal murine metabolism during mid-to-late gestation in normal and diabetic pregnancy. Using 13C-glucose and LC-MS, we profiled the metabolism of fetal brains, hearts, livers, and placentas harvested from pregnant dams between embryonic days (E)10.5 and 18.5. Comparative analysis of our large metabolomics dataset revealed metabolic features specific to fetal tissues developed under a hyperglycemic environment as well as metabolic signatures that may denote developmental transitions during euglycemic development. We observed sorbitol accumulation in fetal tissues and altered neurotransmitter levels in fetal brains isolated from dams with maternal hyperglycemia. Tracing 13C-glucose revealed disparate nutrient sourcing in fetuses depending on maternal glycemic states. Regardless of glycemic state, histidine-derived metabolites accumulated during late development in fetal tissues and maternal plasma. Our rich dataset presents a comprehensive overview of in vivo fetal tissue metabolism and alterations occurring as a result of maternal hyperglycemia.

cell biology↗

bioRxiv2022

Mechanism of ATP hydrolysis dependent rotation of ATP synthases

F1 domain of ATP synthase is a rotary ATPase complex in which rotation of central {gamma}-subunit proceeds in 120{degrees} steps against a surrounding 3{beta}3 fueled by ATP hydrolysis. How the ATP hydrolysis reactions occurring in three catalytic {beta} dimers are coupled to mechanical rotation is a key outstanding question. Here we describe catalytic intermediates of the F1 domain during ATP mediated rotation captured using cryo-EM. The structures reveal that three catalytic events and the first 80{degrees} rotation occur simultaneously in F1 domain when nucleotides are bound at all the three catalytic {beta} dimers. The remaining 40{degrees} rotation of the complete 120{degrees} step is driven by completion of ATP hydrolysis at D{beta}D, and proceeds through three sub-steps (83{degrees}, 91{degrees}, 101{degrees}, and 120{degrees}) with three associated conformational intermediates. All sub-steps except for one between 91{degrees} and 101{degrees} associated with phosphate release, occur independently of the chemical cycle, suggesting that the 40{degrees} rotation is largely driven by release of intramolecular strain accumulated by the 80{degrees} rotation. Together with our previous results, these findings provide the molecular basis of ATP driven rotation of ATP synthases.

biophysics↗

bioRxiv2022

Golgi retention and oncogenic KIT signaling via PLCγ2-PKD2-PI4KIIIβ activation in GIST cells

Most gastrointestinal stromal tumors (GISTs) develop due to gain-of-function mutations in the tyrosine kinase, KIT. We recently showed that mutant KIT mislocalizes to the Golgi area and initiates uncontrolled signaling. However, the molecular mechanisms underlying its Golgi retention remain unknown. Here, we show that protein kinase D2 (PKD2) is activated by the mutant, which causes KITs Golgi retention. In PKD2-inhibited cells, KIT migrates from the Golgi region to lysosomes and subsequently undergoes degradation. Importantly, delocalized KIT is unable to trigger downstream activation. In the Golgi area, KIT activates the PKD2-phosphatidylinositol 4-kinaseIII{beta} (PKD2-PI4KIII{beta}) pathway through phospholipase {gamma}2 (PLC{gamma}2) to generate a PI4P-rich membrane domain, where the AP1-GGA1 complex is aberrantly recruited. Disruption of any factors in this cascade results in KIT release from the Golgi region, indicating that these PKD2-related pathways are responsible for the Golgi retention of KIT. Our findings unveil the molecular mechanisms underlying KIT mislocalization and provide evidence for a new strategy for inhibition of oncogenic signaling.

cancer biology↗