Biology subjects

Imai, K.

Publications and source records attributed to Imai, K..

14 recordsLinked to original sources

bioRxiv2026

Evolutionary diversification of the nuclear pore complex in Entamoeba histolytica reveals conserved and lineage-specific nucleoporins

Nuclear pore complexes (NPCs) are the gateways for macromolecular exchange between the nucleus and cytoplasm. Although NPC architecture is broadly conserved across eukaryotes, substantial lineage-specific diversification has emerged, particularly among divergent protists. Here, we investigated the NPC of Entamoeba histolytica, an evolutionarily divergent amoebozoan and the causative agent of amebiasis. Using the FG-repeat nucleoporin EhNup98-like as bait in affinity purification coupled with mass spectrometry, we identified various associated proteins, including a previously uncharacterized candidate nucleoporin with similarity to Nup53/Nup35. Reciprocal proteomic analysis of EhNup53-like further recovered a broader repertoire of candidate NPC components, including proteins corresponding to conserved nucleoporins as well as several uncharacterized nuclear pore-associated proteins. Structural analyses indicated that this EhNup53-like protein retains a conserved RNA recognition motif (RRM)-like fold despite extensive primary-sequence divergence, while displaying lineage-specific features, including an expanded repeat-rich region and truncation of the C-terminal region typically associated with Nup155 binding. Together, these findings expand the known candidate nucleoporins in E. histolytica and reveal NPC-associated networks that combines recognizable conserved components with extensively remodeled and poorly characterized proteins. Our study highlights the evolutionary plasticity of the NPC and provides a framework for understanding how nuclear pore architecture has diversified across deeply divergent eukaryotic lineages.

cell biology↗

bioRxiv2026

Characterization of the trimeric TOM complex by HS-AFM single-molecule analysis

The translocase of the outer mitochondrial membrane (TOM) complex is the main entry gate for mitochondrial proteins. Approximately 99 % of mitochondrial proteins are synthesized as precursor proteins (preproteins) in the cytosol and subsequently translocated into mitochondria through the TOM complex. The TOM complex exists in a dynamic equilibrium among multiple assembly states through spatial rearrangements of its subunits. The recent cryo-electron microscopy (cryo-EM) studies revealed near-atomic structures of the TOM core dimer, whereas previous biochemical studies indicated the TOM complex functions as a trimer in intact mitochondria. However, the relationship between the core dimer and the functional trimer remains unclear. In the present study, we analyzed the dynamics of the TOM complex using high-speed atomic force microscopy (HS-AFM) to investigate the assembly states and conformation transitions of the TOM complexes. We demonstrated that purified yeast TOM complexes predominantly adopt a trimeric organization but dynamically dissociate into dimeric and monomeric states during HS-AFM observation. The trimeric particles observed by HS-AFM exhibited spherical molecular shapes consistent with a trimeric structural model proposed from previous crosslinking analyses. In contrast, the dissociated dimeric particles closely resembled the dimensions of the TOM core-dimer structures determined by cryo-EM. Furthermore, HS-AFM analyses provided insight into the spatial arrangement of the Tom20 receptor, consistent with previous models of the trimeric TOM complex. These observations enabled characterization of the trimeric TOM complex in vitro and provide a foundation for future structural and functional analyses of TOM complex assembly. Significance statementThe translocase of the mitochondrial outer membrane (TOM) complex is the universal entry gate for almost all mitochondrial proteins, yet its native organization has remained elusive. By combining high-speed atomic force microscopy with cryo-electron microscopy, we provide evidence that the yeast TOM complex predominantly adopts a trimeric assembly with three protein-conducting channels, rather than the dimeric architecture proposed by previous structural studies. Furthermore, single-molecule imaging directly visualized interactions with mitochondrial precursor proteins and receptor-specific antibodies, enabling the experimental identification of receptor subunits within the trimeric TOM complex. Together, these findings uncover previously unrecognized structural and dynamic features of the TOM complex and provide a framework for understanding the molecular mechanism of mitochondrial protein import.

biochemistry↗

bioRxiv2024

Integrated hepatic ferroptosis gene signature dictates pathogenic features of ferroptosis

Background & AimsFerroptosis, a distinctive form of cell death induced by iron-dependent lipid peroxidation, is implicated in various biological processes, including liver diseases. Establishing an iron overload-induced ferroptosis model and identifying hepatic gene signatures associated with ferroptosis are crucial for understanding its role in liver pathogenesis. MethodsF-box and leucine-rich repeat protein 5 (FBXL5) is a substrate-recognition component of the SCF E3 ligase complex that restricts intracellular iron levels. In this study, we used liver-specific Fbxl5-null mice to establish an iron overload-induced ferroptosis model. Transcriptome analysis identified genes involved in hepatic ferroptosis. Integrating these gene signatures with another ferroptosis model enabled the assessment of ferroptosis-related pathology in murine liver injury models and in 174 patients undergoing liver resection surgery. ResultsIron overload induced severe liver damage in liver-specific Fbxl5-null mice, characterized by elevated liver enzymes, histopathological changes, and lipid peroxidation. Transcriptome analysis revealed a distinct set of genes associated with hepatic ferroptosis response. Generating a gene signature for evaluating ferroptosis enhanced the understanding of ferroptosis-related pathologies in liver diseases. Iron overload exacerbated liver damage in murine ischemia-reperfusion injury models via ferroptosis induction. In human patients, elevated serum iron levels correlated with sustained post-operative liver damage, indicating heightened susceptibility to ferroptosis. ConclusionHere, a murine model of iron overload-induced hepatic ferroptosis was established, and a gene signature indicative of hepatic ferroptosis response in both mice and humans was identified. These findings underscore the role of ferroptosis in liver injury progression and suggest potential therapeutic targets for liver disease intervention. HIGHLIGHTSO_LILiver-specific Fbxl5 knockout mice provide an iron-induced ferroptotic injury model C_LIO_LIIntegrated gene signature of iron- and acetaminophen-induced liver injury dictates ferroptosis C_LIO_LIIron overload aggravates hepatic ischemia-reperfusion injury in mice C_LIO_LIPatients with high iron levels show delayed post-operative liver damage recovery C_LI IMPACT AND IMPLICATIONSOur study elucidated the critical role of iron in liver disease pathogenesis and ischemia-reperfusion injury (IRI). By establishing a murine model of iron overload-induced ferroptosis, we confirmed that iron overload exacerbated hepatic IRI, underscoring the importance of ferroptosis in liver damage. Additionally, the development of an integrated gene signature for hepatic ferroptosis response provides a valuable tool for evaluating ferroptosis in liver diseases. Via analysis of patient data, we also highlighted the clinical relevance of ferroptosis in post-operative liver damage, offering insights into potential therapeutic strategies targeting iron and ferroptosis to improve outcomes in patients with liver diseases.

molecular biology↗

bioRxiv2024

Topological data analysis based on mixed-solvent molecular dynamics simulations enhance cryptic pocket detection

Some functional proteins undergo conformational changes to expose hidden binding sites when a binding molecule approaches their surface. Such binding sites are called cryptic sites and are important targets for drug discovery. However, it is still difficult to correctly predict cryptic sites. Therefore, we introduce a new method, CrypToth, for the precise identification of cryptic sites utilizing the persistent homology method. This method integrates topological data analysis and mixed-solvent molecular dynamics (MSMD) simulations. To identify hotspots corresponding to cryptic sites, we conducted MSMD simulations using six probes with different chemical properties: benzene, isopropanol, phenol, imidazole, acetonitrile, and ethylene glycol. Subsequently, we applied our topological data analysis method to rank hotspots based on the possibility of harboring cryptic sites. Evaluation of CrypToth using nine target proteins containing well-defined cryptic sites revealed its superior performance compared to recent machine-learning methods. As a result, in 7 out of 9 cases, hotspots associated with cryptic sites were ranked highest. CrypToth can explore hotspots on the protein surface favorable to ligand binding using MSMD simulations with six different probes and then identify hotspots corresponding to cryptic sites by assessing the proteins conformational variability using the topological data analysis. This synergistic approach facilitates the prediction of cryptic sites with high accuracy.

bioinformatics↗

bioRxiv2024

A fatal case of peritonitis caused by Dysgonomonas capnocytophagoides harboring the novel metallo-beta-lactamase gene blaDYB-1

Dysgonomonas capnocytophagoides shows multidrug resistance to antibiotics and causes opportunistic infections in immunocompromised hosts. We report a fatal case of peritonitis caused by D. capnocytophagoides in Japan. We identified a novel chromosomally encoded class B1 metallo-beta-lactamase gene designated blaDYB-1 and an ermFS gene that contributed to multidrug resistance.

microbiology↗

bioRxiv2023

Plastic vasomotion entrainment

The presence of global synchronization of vasomotion induced by oscillating visual stimuli was identified in the mouse brain. Endogenous autofluorescence was used and the vessel "shadow" was quantified to evaluate the magnitude of the frequency-locked vasomotion. This method allows vasomotion to be easily quantified in non-transgenic wild-type mice using either the wide-field macro-zoom microscopy or the deep-brain fiber photometry methods. Vertical stripes horizontally oscillating at a low temporal frequency (0.25 Hz) were presented to the awake mouse and oscillatory vasomotion locked to the temporal frequency of the visual stimulation was induced not only in the primary visual cortex but across a wide surface area of the cortex and the cerebellum. The visually induced vasomotion adapted to a wide range of stimulation parameters. Repeated trials of the visual stimulus presentations resulted in the entrainment of the amplitude of the vasomotion. Horizontally oscillating visual stimulus is known to induce horizontal optokinetic response (HOKR). The amplitude of the eye movement is known to increase with repeated training sessions and the flocculus region of the cerebellum is known to be essential for this learning to occur. Here, we show a strong correlation between the average HOKR performance gain and the vasomotion entrainment magnitude in the cerebellar flocculus. Therefore, the plasticity of vasomotion and neuronal circuits appeared to occur in parallel. Efficient energy delivery by the entrained vasomotion may contribute to meeting the energy demand for increased coordinated neuronal activity and the subsequent neuronal circuit reorganization.

neuroscience↗

bioRxiv2023

The similarities in microbial and chemical patterns of fermentation in two open environments were promoted by using 150-year-old Nukadoko as starters.

Nukadoko, a fermented rice bran employed in traditional Japanese pickling, uses lactic acid bacteria to ferment vegetables. Here, we report the microbial and chemical data of a mixture of matured 150-year-old nukadoko and commercially available rice bran placed in two open environments over 29 days. Across the two environments, Loigolactobacillus was identified as the dominant microbial genera in the later stages of fermentation in nukadoko. The period of increase in the relative abundance of Loigolactobacillus correlated with a decrease in pH and Oxidation-Reduction Potential (ORP) values. While the two environments showed a difference in the rate of change in microbial diversity, they shared the common process through which Loigolactobacillus outcompeted adventitious bacteria in nukadoko, as indicated by the alpha and beta diversity index. Thus, the similarities in microbial and chemical data across two open environments during fermentation using starters indicate that starters contribute to the stability of fermentation in open environments.

microbiology↗

bioRxiv2022

Comprehensive multiomic profiling of somatic mutations in malformations of cortical development

Malformations of cortical development (MCD) are neurological conditions displaying focal disruption of cortical architecture and cellular organization arising during embryogenesis, largely from somatic mosaic mutations. Identifying the genetic causes of MCD has been a challenge, as mutations remain at low allelic fractions in brain tissue resected to treat epilepsy. Here, we report a genetic atlas from 317 brain resections, identifying 69 mutated genes through intensive profiling of somatic mutations, combining whole-exome and targeted-amplicon sequencing with functional validation and single-cell sequencing. Genotype-phenotype correlation analysis elucidated specific MCD gene sets associating distinct pathophysiological and clinical phenotypes. The unique spatiotemporal expression patterns identified by comparing single-nucleus transcriptional sequences of mutated genes in control and patient brains implicate critical roles in excitatory neurogenic pools during brain development, and in promoting neuronal hyperexcitability after birth.

genetics↗

bioRxiv2022

Entamoeba histolytica EHD1 is involved in mitosome-endosome contact

Inter-organellar crosstalk is often mediated by membrane contact sites (MCSs), which are zones where participating membranes come within a proximity of 30 nm. MCSs have been found in organelles including the endoplasmic reticulum, Golgi bodies, endosomes, and mitochondria. Despite its seeming ubiquity, reports of MCS involving mitochondrion-related organelles (MROs) present in a few anaerobic parasitic protozoa remain lacking. Entamoeba histolytica, the etiological agent of amoebiasis, possesses an MRO called mitosome. We previously discovered several Entamoeba-specific transmembrane mitosomal proteins (ETMPs) from in silico and cell biological analyses. One of them, ETMP1 (EHI_175060), was predicted to have one transmembrane domain and two coiled-coil regions, and was demonstrated to be mitosome membrane-integrated based on carbonate fractionation and immunoelectron microscopy (IEM) data. Immunoprecipitation analysis detected a candidate interacting partner, EH-domain containing protein (EHD1, EHI_105270). We expressed HA-tagged EHD1 in E. histolytica and subsequent immunofluorescence and IEM data indicated an unprecedented MCS between the mitosome and the endosome. Live imaging of GFP-EHD1 expressing strain demonstrated that EHD1 is involved in early endosome formation, and is observed in MCS between endosomes of various sizes. In vitro assay using recombinant His-EHD1 demonstrated ATPase activity. MCSs are involved in lipid transfer, ion homeostasis, and organelle dynamics. The serendipitous discovery of the ETMP1 interacting partner EHD1, led to the observation of the mitosome-endosome contact site in E. histolytica. It opened a new view of how the relic mitochondria of Entamoeba may likewise be involved in organelle crosstalk, a conserved feature of mitochondria and other organelles in general.

cell biology↗

bioRxiv2021

Discovery of a conserved rule behind the assembly of β-barrel membrane proteins.

Outer membrane proteins (OMPs) are essential components of the outer membrane of Gram-negative bacteria. In terms of protein targeting and assembly, the current dogma holds that a "{beta}-signal" imprinted in the final {beta}-strand of the OMP engages the {beta}-barrel assembly machinery (BAM complex) to initiate membrane insertion and assembly of the OMP into the outer membrane. Here, we revealed an additional rule, that signals equivalent to the {beta}-signal are repeated in other, internal {beta}-strands within bacterial OMPs, by peptidomimetic and mutational analysis. The internal signal is needed to promote the efficiency of the assembly reaction of these OMPs. BamD, an essential subunit of the BAM complex, recognizes the internal signal and the {beta}-signal, arranging several {beta}-strands and partial folding for rapid OMP assembly. The internal signal-BamD ordering system is not essential for bacterial viability but is necessary to retain the integrity of the outer membrane against antibiotics and other environmental insults. TEASERBacterial outer membrane proteins are recognized and bound by BamD at specific signals located in multiple {beta}-strands at the C-terminus of these proteins.

biochemistry↗

bioRxiv2021

Preservation of collagen in the soft tissues of frozen mammoths

We investigated the characteristics of extracellular matrix (ECM) in the soft tissue of two frozen baby woolly mammoths (Mammuthus primigenius) that died and were buried in Siberian permafrost approximately 40,000 years ago. Morphological and biochemical analyses of mammoth lung and liver demonstrated that those soft tissues were preserved at the gross anatomical and histological levels. The ultrastructure of ECM components, namely a fibrillar structure with a collagen-characteristic pattern of cross-striation, was clearly visible with transmission and scanning electron microscopy. Type I and type IV collagens were detected by immunohistochemical observation. Quantitative amino acid analysis of liver and lung tissues of the baby mammoths indicated that collagenous protein is selectively preserved in these tissues as a main protein. Type I and type III collagens were detected as major components by means of liquid chromatography-mass spectrometry analysis after digestion with trypsin. These results indicate that the triple helical collagen molecule, which is resistant to proteinase digestion, has been preserved in the soft tissues of these frozen mammoths for 40,000 years.

paleontology↗

bioRxiv2020

Naturally mutated spike proteins of SARS-CoV-2 variants show differential levels of cell entry

The causative agent of the coronavirus disease 2019 (COVID-19) pandemic, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is steadily mutating during continuous transmission among humans. Such mutations can occur in the spike (S) protein that binds to the angiotensin-converting enzyme-2 (ACE2) receptor and is cleaved by transmembrane protease serine 2 (TMPRSS2). However, whether S mutations affect SARS-CoV-2 infectivity remains unknown. Here, we show that naturally occurring S mutations can reduce or enhance cell entry via ACE2 and TMPRSS2. A SARS-CoV-2 S-pseudotyped lentivirus exhibits substantially lower entry than SARS-CoV S. Among S variants, the D614G mutant shows the highest cell entry, as supported by structural observations. Nevertheless, the D614G mutant remains susceptible to neutralization by antisera against prototypic viruses. Taken together, these data indicate that the D614G mutation enhances viral infectivity while maintaining neutralization susceptibility.

microbiology↗

bioRxiv2020

A single-cell analysis of the molecular lineage of chordate embryogenesis

In multicellular organisms, a single zygote develops along divergent lineages to produce distinct cell types. What governs these processes is central to the understanding of cell fate specification and stem cell engineering. Here we used the protochordate model Ciona savignyi to determine gene expression profiles of every cell of single embryos from fertilization through the onset of gastrulation and provided a comprehensive map of chordate early embryonic lineage specification. We identified 47 cell types across 8 developmental stages up to the 110-cell stage in wild type embryos and 8 fate transformations at the 64-cell stage upon FGF-MAPK inhibition. The identities of all cell types were evidenced by in situ expression pattern of marker genes and expected number of cells based on the invariant lineage. We found that, for the majority of asymmetrical cell divisions, the bipotent mother cell shows predominantly the gene signature of one of the daughter fates, with the other daughter being induced by subsequent signaling. Our data further indicated that the asymmetric segregation of mitochondria in some of these divisions does not depend on the concurrent fate inducing FGF-MAPK signaling. In the notochord, which is an evolutionary novelty of chordates, the convergence of cell fate from two disparate lineages revealed modular structure in the gene regulatory network beyond the known master regulator T/Brachyury. Comparison to single cell transcriptomes of the early mouse embryo showed a clear match of cell types at the tissue level and supported the hypothesis of developmental-genetic toolkit. This study provides a high-resolution single cell dataset to understand chordate embryogenesis and the relationship between fate trajectories and the cell lineage. HighlightsO_LITranscriptome profiles of 47 cell types across 8 stages in early chordate embryo C_LIO_LIBipotent mother in asymmetric division shows the default daughter fate C_LIO_LIModular structure of the notochord GRN beyond the known function of T C_LIO_LIInvariant lineage and manual cell isolation provide truth to trajectory analysis C_LI

developmental biology↗

bioRxiv2019

Hallmarks of slow translation initiation revealed in mitochondrially localizing mRNA sequences

The mRNA of some, but not all, nuclear encoded mitochondrial proteins localize to the periphery of mitochondria. Previous studies have shown that both the nascent polypeptide chain and an mRNA binding protein play a role in this phenomenon, and have noted a positive correlation between mRNA length and mitochondrial localization. Here, we report the first investigation into the relationship between mRNA translation initiation rate and mRNA mitochondrial localization. Our results indicate that translation initiation promoting factors such as Kozak sequences are associated with cytosolic localization, while inhibiting factors such as 5' UTR secondary structure correlate with mitochondrial localization. Moreover, the frequencies of nucleotides in various positions of the 5' UTR show higher correlation with localization than the 3' UTR. These results indicate that mitochondrial localization is associated with slow translation initiation. Interestingly this may help explain why short mRNAs, which are thought to initiate translation rapidly, seldom localize to mitochondria. We propose a model in which translating mRNA has reduced mobility and tends not to reach mitochondria. Finally, we explore this model with a simulation of mRNA diffusion using previously estimated translation initiation probabilities, confirming that our model can produce localization values similar to those measured in experimental studies.

molecular biology↗