Biology subjects

Shibata, Y.

Publications and source records attributed to Shibata, Y..

33 records · Page 2Linked to original sources

bioRxiv2023

Ribosomal protein mutation suppresses gonadal leader cell migration defects in mig-17/ADAMTS mutants in Caenorhabditis elegans

The migration of the gonadal distal tip cells (DTCs) in Caenorhabditis elegans provides an excellent model for studying the migration of epithelial tubes during organogenesis. Mutations in the mig-17/ADAMTS gene cause misdirected migration of DTCs during gonad formation, resulting in deformed gonad arms. An amino-acid substitution in RPL- 20 corresponding to the mammalian RPL18a/eL20, a component of the 60S ribosomal large subunit, showed a slow growth phenotype and strongly suppressed the mig-17 gonadal defects. Slow-growing mutants clk-1 and clk-2 also suppressed mig-17, although weaker than rlp-20 mutants. MIG-17 recruits FBL-1C/fibulin-1C to the gonadal basement membrane to regulate DTC migration. Reducing the gene dosage of fbl-1 by half partially compromised the suppressor activity of the mutant rpl-20 gene on mig-17. Analysis using the mNeonGreen-FBL-1 reporter revealed that its localization to the gonadal basement membrane was significantly reduced in mig-17, whereas it was recovered to the wild-type levels in mig-17; rpl-20 double mutants. These results indicate that the rpl-20 mutation suppresses mig-17 gonadal defects through dual mechanisms: deceleration of growth rate and enhancement of FBL-1C recruitment to the gonadal basement membrane.

developmental biology↗

bioRxiv2023

Length estimation of fish detected as non-occluded using a smartphone application and deep learning techniques

Uncertainty in stock assessment can be reduced if accurate and precise length composition of catch is available. Length data are usually manually collected, although this method is costly and time-consuming. Recently, some studies have estimated fish species and length from images using deep learning by installing camera systems in fishing vessels or a fish auction center. Once the deep learning model is properly trained, it does not require expensive and time-consuming manual labor. However, several previous studies have focused on monitoring fishing practices using an electronic monitoring system (EMS); therefore, it is necessary to solve many challenges, such as counting the total number of fish in the catch. In this study, we proposed a new deep learning-based method to estimate fish length using images. Species identification was not performed by the model, and images were taken manually by the measurers; however, length composition was obtained only for non-occluded fish detected by the model. A smartphone application was developed to calculate scale information (cm/pixel) from a known size fish box in fish images, and the Mask R-CNN (Region-based convolutional neural networks) model was trained using 76,161 fish to predict non-occluded fish. Two experiments were conducted to confirm whether the proposed method resulted in errors in the length composition. First, we manually measured the total length (TL) for each of the five fish categories and estimated the TL using deep learning and calculated the bias. Second, multiple fish in a fish box were photographed simultaneously, and the difference between the mean TL estimated from the non-occluded fish and the true TL from all fish was calculated. The results indicated that the biases of all five species categories were within {+/-} 3%. Moreover, the difference was within {+/-} 1.5% regardless of the number of fish in the fish box. In the proposed method, deep learning was used not to replace the measurer but to increase their measurement efficiency. The proposed method is expected to increase opportunities for the application of deep learning-based fish length estimation in areas of research that are different from the scope of conventional EMS.

ecology↗

bioRxiv2022

Membrane curvature-generating proteins crucial for autophagosome formation

Autophagy is essential for cellular homeostasis and begins with the formation of a phagophore, a cup-like membrane sheet consisting of two closely apposed lipid bilayers connected by a highly curved rim. How the membrane sheet forms, bends, and eventually generates an autophagosome that enwraps cargo remains enigmatic. Specifically, it is unclear how the high membrane curvature of the phagophore rim, an energetically unfavorable state, is stabilized. Here, we demonstrate that phagophore formation requires the conserved, membrane curvature-generating REEP1 proteins. The REEP1 family proteins (REEP1-4 in vertebrates) differ from the related endoplasmic reticulum-shaping REEPs in abundance and membrane topology. In fission yeast, the single REEP1 ortholog is involved in both bulk and selective autophagy. It is recruited at early stages of phagophore formation and required for their maturation into autophagosomes. The function of REEP1 relies on its ability to generate high membrane curvature and its localization to the phagophore rim. Mammalian REEP1 proteins also associate with phagophores upon induction of autophagy and colocalize with early autophagic markers. We propose that REEP1 proteins stabilize the phagophores highly curved rim so that the two membrane sheets are kept in close proximity to form the autophagosome. Defective autophagy may underlie the effect of curvature-compromising mutations in human REEP1 proteins linked to neurological disease.

cell biology↗

bioRxiv2022

Identification, expression, and purification of DNA cytosine 5-methyltransferases with short recognition sequences

BackgroundDNA methyltransferases (MTases) are enzymes that induce methylation, one of the representative epigenetic modifications of DNA, and are also useful tools for analyzing epigenomes. However, regarding DNA cytosine 5-methylation, MTases identified so far have drawbacks in that their recognition sequences overlap with those for intrinsic DNA methylation in mammalian cells and/or that the recognition sequence is too long for fine epigenetic mapping. To identify MTases with short recognition sequences that never overlap with the CG dinucleotide, we systematically investigated the 25 candidate enzymes identified using a database search, which showed high similarity to known cytosine 5-MTases recognizing short sequences. ResultsWe identified MTases with six new recognition sequences, including TCTG, CC, CNG, TCG, GCY, and GGCA. Because the recognition sequence never overlapped with the CG dinucleotide, MTases recognizing the CC dinucleotide were promising. ConclusionsIn the current study, we established a procedure for producing active CC-methylating MTases and applied it to nucleosome occupancy and methylome sequencing to prove the usefulness of the enzyme for fine epigenetic mapping. MTases that never overlap with CG dinucleotides would allow us to profile multiple epigenomes simultaneously.

biochemistry↗

bioRxiv2021

Chlamydomonas State Transition Is Quiet Around Pyrenoid and Independent from Thylakoid Deformation

Photosynthetic organisms have developed a rapid regulation mechanism called state transition (ST) to rapidly adjust the excitation balance between two photosystems by light-harvesting complex II (LHCII) movement. Though many researchers have assumed coupling of the ultrastructural dynamics of the thylakoid membrane to the ST mechanism, how ST is related to the ultrastructural dynamic of the thylakoid in Chlamydomonas remains elusive. To clarify the above-mentioned relation, here we used two specialized microscope techniques, observation via the excitation-spectral microscope (ESM) developed recently by us and the super-resolution imaging based on structured illumination microscopy (SIM). The ESM observation revealed a highly reversible rearrangement of LHCII-related fluorescence. More importantly, it clarified lower ST activity in the region surrounding the pyrenoid, which is the specific subcellular compartment associated with the carbon-fixation reaction. On the other hand, the SIM observation resolved partially irreversible fine thylakoid transformations induced by the ST-inducing illumination. Fine irreversible thylakoid transformation was also observed for the Stt7-kinase-lacking mutant. This result, together with the nearly equal structural changes in the less active ST regions around the pyrenoid, suggested the independence of the observed fine structural changes from the LHCII phosphorylation.

biophysics↗

bioRxiv2020

KDM2B promotes cell viability by enhancing DNA damage response in canine hemangiosarcoma

Epigenetic regulators have been implicated in tumorigenesis of many types of cancer; however, their roles in endothelial cell cancers such as canine hemangiosarcoma (HSA) have not been studied. In this study, we found that lysine-specific demethylase 2B (Kdm2b) was highly expressed in HSA cell lines compared to normal canine endothelial cells. Silencing of Kdm2b in HSA cells resulted to increased cell death in vitro compared to the scramble control by inducing apoptosis through the inactivation of the DNA repair pathways and accumulation of DNA damage. Similarly, doxycycline-induced Kdm2b silencing in tumor xenografts resulted to decreased tumor sizes compared to the scramble control. Furthermore, Kdm2b was also highly expressed in clinical cases of HSA, and its expression levels was higher than in hemangioma, a benign counterpart of HSA. Based on these results, we hypothesized that pharmacological Kdm2b inhibition can also induce HSA cell death and can be used as an alternative treatment for HSA. We treated HSA cells with GSK-J4, a histone demethylase inhibitor, and found that GSK-J4 treatment also induced apoptosis and cell death. On top of that, GSK-J4 treatment in HSA tumor-bearing mice decreased tumor sizes without any obvious side-effects. In this study, we demonstrated that Kdm2b acts as an oncogene in HSA by enhancing DNA damage response and can be used as a biomarker to differentiate HSA from hemangioma. Moreover, we indicated that histone demethylase inhibitor GSK-J4 can be used as a therapeutic alternative to doxorubicin for HSA treatment.

cancer biology↗

bioRxiv2020

Genetic interactions among ADAMTS metalloproteases and basement membrane molecules in cell migration in Caenorhabditis elegans

During development of the Caenorhabditis elegans gonad, the gonadal leader cells, called distal tip cells (DTCs), migrate in a U-shaped pattern to form the U-shaped gonad arms. The ADAMTS (a disintegrin and metalloprotease with thrombospondin motifs) family metalloproteases MIG-17 and GON-1 are required for correct DTC migration. Mutations in mig-17 result in misshapen gonads due to the misdirected DTC migration, and mutations in gon-1 result in shortened and swollen gonads due to the premature termination of DTC migration. Although the phenotypes shown by mig-17 and gon-1 mutants are very different from one another, mutations that result in amino acid substitutions in the same basement membrane protein genes, emb-9/collagen IV a1, let-2/collagen IV a2 and fbl-1/fibulin-1, were identified as genetic suppressors of mig-17 and gon-1 mutants. To understand the roles shared by these two proteases, we examined the effects of the mig-17 suppressors on gon-1 and the effects of the gon-1 suppressors and enhancers on mig-17 gonadal defects. Some of the emb-9, let-2 and fbl-1 mutations suppressed both mig-17 and gon-1, whereas others acted only on mig-17 or gon-1. These results suggest that mig-17 and gon-1 have their specific functions as well as functions commonly shared between them for gonad formation. The levels of collagen IV accumulation in the DTC basement membrane were significantly higher in the gon-1 mutants as compared with wild type and were reduced to the wild-type levels when combined with suppressor mutations, but not with enhancer mutations, suggesting that the ability to reduce collagen IV levels is important for gon-1 suppression.

developmental biology↗

bioRxiv2020

Identifications of novel avian and mammalian deltaviruses filled the large evolutionally gaps and revealed inter-family transmission of deltaviruses

Hepatitis delta virus (HDV) is a satellite virus that requires hepadnavirus envelope proteins for its transmission. Although recent studies identified HDV-related deltaviruses in certain animals, the evolution of deltaviruses, such as the origin of HDV and the mechanism of its coevolution with its helper viruses, is unknown, mainly because of the phylogenetic gaps among deltaviruses. Here we identified novel deltaviruses of passerine birds, woodchucks, and white-tailed deer by extensive database searches and molecular surveillance. Phylogenetic and molecular epidemiological analyses suggest that HDV originated from mammalian deltaviruses and the past interspecies transmission of mammalian and passerine deltaviruses. Further, metaviromic and experimental analyses suggest that the satellite-helper relationship between HDV and hepadnavirus was established after the divergence of the HDV lineage from non-HDV mammalian deltaviruses. Our findings enhance our understanding of deltavirus evolution, diversity, and transmission, indicating the importance of further surveillance for deltaviruses.

microbiology↗

bioRxiv2020

Mechanism and inhibition of SARS-CoV-2 PLpro

Coronaviruses, including SARS-CoV-2, encode multifunctional proteases that are essential for viral replication and evasion of host innate immune mechanisms. The papain-like protease PLpro cleaves the viral polyprotein, and reverses inflammatory ubiquitin and anti-viral ubiquitin-like ISG15 protein modifications1,2. Drugs that target SARS-CoV-2 PLpro (hereafter, SARS2 PLpro) may hence be effective as treatments or prophylaxis for COVID-19, reducing viral load and reinstating innate immune responses3. We here characterise SARS2 PLpro in molecular and biochemical detail. SARS2 PLpro cleaves Lys48-linked polyubiquitin and ISG15 modifications with high activity. Structures of PLpro bound to ubiquitin and ISG15 reveal that the S1 ubiquitin binding site is responsible for high ISG15 activity, while the S2 binding site provides Lys48 chain specificity and cleavage efficiency. We further exploit two strategies to target PLpro. A repurposing approach, screening 3727 unique approved drugs and clinical compounds against SARS2 PLpro, identified no compounds that inhibited PLpro consistently or that could be validated in counterscreens. More promisingly, non-covalent small molecule SARS PLpro inhibitors were able to inhibit SARS2 PLpro with high potency and excellent antiviral activity in SARS-CoV-2 infection models.

biochemistry↗

bioRxiv2020

Estimating maximum sustainable yield of snow crab (Chionoecetes opilio) off Tohoku Japan via a state-space assessment model with time-varying natural mortality

Yield from fisheries is a tangible benefit of ecosystem services and sustaining or restoring a fish stock level to achieve a maximum sustainable yield (MSY). Snow crab (Chionoecetes opilio) off Tohoku has been managed by a total allowable catch since 1996, although their abundance has not increased even after 2011, when fishing pressure rapidly decreased because of the Great East Japan Earthquake. This implies that their biological characteristics, such as recruits, natural mortality coefficient (M), and terminal molting probabilities (p), might have changed. We developed "just another state-space stock assessment model (JASAM)" to estimate the MSY of the snow crab off Tohoku, Japan, considering interannual variations in M and p. The multi-model inference revealed that M increased from 0.2 in 1997 to 0.59 in 2018, although it was not different among the instars, sex, nor terminal molt of crabs. The parameter p also increased by 1.34-2.46 times depending on the instar growth stages from 1997 to 2018. We estimated the MSYs in three scenarios, which drastically changed if M and p were set as they were in the past or at the current values estimated from this study. This result indicated that the MSY of snow crab would also be time-varying based on their time-varying biological characteristics.

ecology↗

bioRxiv2019

Scalable method for groupwise comparisons and its application to chromatin accessibility among primates

Changes in transcriptional regulation are thought to be a major contributor to the evolution of phenotypic traits, but the contribution of changes in chromatin accessibility to the evolution of gene expression remains almost entirely unknown. To address this important gap in knowledge, we developed a new method to identify DNase I Hypersensitive (DHS) sites with differential chromatin accessibility between species using a joint modeling approach. Our method overcomes several limitations inherent to conventional threshold-based pairwise comparisons that become increasingly apparent as the number of species analyzed rises. Our approach employs a single quantitative test which is more sensitive than existing pairwise methods. To illustrate, we applied our joint approach to DHS sites in fibroblast cells from five primates (human, chimpanzee, gorilla, orangutan, and rhesus macaque). We identified 89,744 DHS sites, of which 41% are identified as differential between species using the joint model compared with 33% using the conventional pairwise approach. The joint model provides a principled approach to distinguishing single from multiple chromatin accessibility changes among species. We found that non differential DHS sites are enriched for nucleotide conservation. Differential DHS sites with decreased chromatin accessibility relative to rhesus macaque occur more commonly near transcription start sites (TSS), while those with increased chromatin accessibility occur more commonly distal to TSS. Further, differential DHS sites near TSS are less cell type-specific than more distal regulatory elements. Taken together, these results point to distinct classes of DHS sites, each with distinct characteristics of selection, genomic location, and cell type specificity.

evolutionary biology↗

bioRxiv2019

Extrachromosomal circular DNA, microDNA, without canonical promoters produce short regulatory RNAs that suppress gene expression

Interest in extrachromosomal circular DNA (eccDNA) molecules has increased recently because of their widespread presence in normal cells across every species ranging from yeast to humans, their increased levels in cancer cells, and their overlap with oncogenic and drug-resistant genes. However, the majority of eccDNA (microDNA) are too small to carry protein coding genes. We have tested functional capabilities of microDNA, by creating artificial microDNA molecules mimicking known microDNA sequences and have discovered that they express functional small regulatory RNA including microRNA and novel si-like RNA. MicroDNA is transcribed in vitro and in vivo independent of a canonical promoter sequence. MicroDNA which carry miRNA genes form transcripts which are processed into mature miRNA molecules, through the endogenous RNA-interference pathway, which repress a luciferase reporter gene as well as endogenous mRNA targets of the miRNA. Further, microDNA containing sequences of exons repress the endogenous gene from which the microDNA was derived through the formation of novel si-like RNA. We also show that endogenous microDNA associate with RNA polymerases subunits POLR2H and POLR3F. Together, these results suggest that microDNA may modulate gene expression through the production of both known and novel regulatory small RNA.

molecular biology↗

bioRxiv2018

Mammalian SWI/SNF collaborates with a polycomb-associated protein to regulate male germ line transcription in the mouse

A deficiency in BRG1, the catalytic subunit of the SWI/SNF chromatin remodeling complex, results in a meiotic arrest during spermatogenesis. Here, we explore the causative mechanisms. BRG1 is preferentially enriched at active promoters of genes essential for spermatogonial pluripotency and meiosis. In contrast, BRG1 is also associated with the repression of somatic genes. Chromatin accessibility at these target promoters is dependent upon BRG1. These results favor a model where BRG1 coordinates spermatogenic transcription to ensure meiotic progression. In spermatocytes, BRG1 interacts with SCML2, a testes specific PRC1 factor that is associated with the repression of somatic genes. We present evidence to suggest that BRG1 and SCML2 concordantly regulate genes during meiosis. Furthermore, BRG1 is required for the proper localization of SCML2 and its associated deubiquitinase, USP7, to the sex chromosomes during pachynema. SCML2 associated, mono ubiquitination of histone H2A lysine 119 (H2AK119ub1) and acetylation of histone lysine 27 (H3K27ac) are elevated in Brg1cKO testes. Coincidentally, the PRC1 ubiquitin ligase, RNF2 is activated while a histone H2A/H2B deubiquitinase, USP3 is repressed. Thus, BRG1 impacts the male epigenome by influencing the localization and expression of epigenetic modifiers. This mechanism highlights a novel paradigm of co-operativity between SWI/SNF and PRC1.\n\nSummary statementBRG1, a catalytic subunit of SWI/SNF chromatin remodeling complex, interacts with SCML2 (Sex comb on midleg-like 2), a polycomb repressive 1 (PRC1) factor, to regulate transcription during spermatogenesis. This represents a novel paradigm of SWI/SNF-PRC1 co-operation during gametogenesis.

developmental biology↗

bioRxiv2018

Repulsive Guidance Molecule Acts in Axon Branching in Caenorhabditis elegans

Repulsive guidance molecules (RGMs) are evolutionarily conserved proteins implicated in repulsive axon guidance. Here we report the function of the Caenorhabditis elegans ortholog DRAG-1 in axon branching. The axons of hermaphrodite-specific neurons (HSNs) branch at the region abutting the vulval muscles and innervate these muscles to control egg laying. The drag-1 mutants exhibited defects in HSN axon branching in addition to a small body size and egg laying-defective phenotype. DRAG-1 expression in the hypodermal cells was required for the branching of these axons. The C-terminal glycosylphosphatidylinositol anchor of DRAG-1 was important for its function. Genetic analyses suggested that the membrane receptor UNC-40, but neither SMA-1/{beta}H-spectrin nor SMA-5/MAP kinase 7, acts in the same pathway with DRAG-1 in HSN branching. We propose that DRAG-1 expressed in the hypodermis signals via the UNC-40 receptor expressed in HSNs to elicit branching activity of HSN axons.

developmental biology↗

bioRxiv2018

Maintenance of Cell Fates by Regulation of the Histone Variant H3.3 in Caenorhabditis elegans

HighlightsTLK-1 maintains cell fates by repression of selector genes\n\nTLK-1 and downstream H3 chaperone CAF1 inhibit H3.3 deposition\n\nLoss of sin-3 suppresses the defect in cell-fate maintenance of tlk-1 mutants\n\nAcH4-binding protein BET-1 is necessary for sin-3 suppression\n\nSummaryCell-fate maintenance is important to preserve the variety of cell types that are essential for the formation and function of tissues. We previously showed that the acetylated histone H4-binding protein BET-1 maintains cell fate by recruiting the histone variant H2A.z. Here, we report that Caenorhabditis elegans tousled-like kinase TLK-1 and the histone H3 chaperone CAF1 maintain cell fate by preventing the incorporation of histone variant H3.3 into nucleosomes, thereby repressing ectopic expression of transcription factors that induce cell-fate specification. Genetic analyses suggested that TLK-1 and BET-1 act in parallel pathways. In tlk-1 mutants, the loss of SIN-3, which promotes histone acetylation, suppressed a defect in cell-fate maintenance in a manner dependent on MYST family histone acetyltransferase MYS-2 and BET-1. sin-3 mutation also suppressed abnormal H3.3 incorporation. Thus, we propose that the regulation and interaction of histone variants play crucial roles in cell-fate maintenance through the regulation of selector genes.

developmental biology↗