Biology subjects

Fujita, Y.

Publications and source records attributed to Fujita, Y..

49 records · Page 3Linked to original sources

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↗

bioRxiv2021

Tumor-Cell Invasion Initiates at Invasion Hotspots, an Epithelial Tissue-Intrinsic Microenvironment

Malignant cancers emerge in epithelial tissues through a progressive process in which a single transformed mutant cell becomes tumorigenic and invasive. Although numerous genes involved in the malignant transformation of cancer cells have been described, how tumor cells launch an invasion into the basal side of epithelial tissues remains elusive. Here, using a Drosophila wing imaginal disc epithelia, we show that genetically mosaic clones of cells mutant for a neoplastic-tumor-suppressor gene (nTSG) in combination with the oncogenic Ras (RasV12) expression initiate invasion into the basal side of the epithelial layer at specific spots in the epithelial tissue. In this "invasion hotspot", the oncogenic double-mutant cells activate c-Jun N-terminal kinase (JNK) signaling, which causes basal extrusion of the double-mutant cells and destruction of basement membrane through upregulation of a matrix metalloprotease, MMP1. Conversely, in other regions of the epithelial tissue, the double-mutant cells do not strongly activate JNK, deviate from the apical side of the epithelial layer, and show benign tumor growth in the lumen. These data indicate that the onset of tumor-cell invasion is highly dependent on the tissue-intrinsic local microenvironment. Given the conservation of genetic signaling pathways involved in this process, initiation of tumor-cell invasion from invasion hotspots in Drosophila wing imaginal epithelia could help us to understand the developmental mechanisms of invasive cancers.

cancer biology↗

bioRxiv2021

Phosphate starvation response precedes drought response in field-grown plants

Drought severely damages crop production, even under conditions so mild that the leaves show no signs of wilting. However, it is unclear how field-grown plants respond to mild drought. Here, we show through 6 years of field trials that ridges are a useful experimental tool to mimic mild drought stress in the field. Mild drought reduces inorganic phosphate levels in the leaves to activate the phosphate starvation response in soybean plants in the field. Using Arabidopsis thaliana and its mutant plants grown in pots under controlled environments, we demonstrate that phosphate starvation response (PSR) occurs before abscisic acid response under progressive mild drought and that PSR plays a crucial role in plant growth under mild drought. Our observations in the field and laboratory using model crop and experimental plants provide insight into the molecular response to mild drought in field-grown plants and the relationship between nutrition and drought stress response.

plant biology↗

bioRxiv2021

Src activation in lipid rafts confers epithelial cells with invasive potential to escape from apical extrusion during cell competition

Abnormal/cancerous cells within healthy epithelial tissues undergo apical extrusion to protect against carcinogenesis, while they acquire invasive capacity once carcinogenesis progresses. However, the molecular mechanisms by which cancer cells escape from apical extrusion and invade surrounding tissues remain elusive. We found that during competition within epithelial cell layers, Src-transformed cells underwent basal delamination by Src activation within lipid rafts, whereas they were apically extruded when Src was outside of lipid rafts. Comparative analysis of contrasting phenotypes revealed that activation of the Src-STAT3-MMP axis through lipid rafts was required for basal delamination. CUB domain-containing protein 1 (CDCP1) was identified as an Src activating scaffold in lipid rafts, and its overexpression induced basal delamination. In renal cancer spheroids, CDCP1 promoted HGF-dependent invasion by activating the Src-STAT3-MMP axis. Overall, these results suggest that Src activation in lipid raft confers resistance to apical extrusion and invasive potential on epithelial cells to promote carcinogenesis.

cell biology↗

bioRxiv2021

Influenza virus ribonucleoprotein complex formation occurs in the nucleolus

Influenza A virus double-helical ribonucleoprotein complex (vRNP) performs transcription and replication of viral genomic RNA (vRNA). Unlike most RNA viruses, vRNP formation accompanied by vRNA replication is carried out in the nucleus of virus-infected cell. However, the precise subnuclear site remains unknown. Here, we report the subnuclear site of vRNP formation in influenza virus. We found that all vRNP components were colocalized in the nucleolus of virus-infected cells at early stage of infection. Mutational analysis showed that nucleolar localization of viral nucleoprotein, a major vRNP component, is critical for functional double-helical vRNP formation. Furthermore, nucleolar disruption of virus-infected cells inhibited vRNP component assembly into double-helical vRNPs, resulting in decreased vRNA transcription and replication. Collectively, our findings demonstrate that the vRNA replication-coupled vRNP formation occurs in the nucleolus, demonstrating the importance of the nucleolus for influenza virus life cycle.

microbiology↗

bioRxiv2020

RNF5 Regulation of RBBP4 Defines Acute Myeloid Leukemia Growth and Susceptibility to Histone Deacetylase Inhibitors

Acute myeloid leukemia (AML) remains incurable, largely due to its resistance to conventional treatments. Here, we found that increased expression and abundance of the ubiquitin ligase RNF5 contributes to AML development and survival. High RNF5 expression in AML patients correlated with poor prognosis. RNF5 inhibition decreased AML cell growth in culture and in vivo, and blocked development of MLL-AF9-driven leukemogenesis in mice, prolonging their survival. RNF5 inhibition led to transcriptional changes that overlapped with those seen upon HDAC1 inhibition. RNF5 induced the formation of K29 ubiquitin chains on the histone-binding protein RBBP4, promoting its recruitment and subsequent epigenetic regulation of genes involved in AML development and maintenance. Correspondingly, RNF5 or RBBP4 knockdown enhanced the sensitivity of AML cells to histone deacetylase (HDAC) inhibitors. Notably, low expression of RNF5 and HDAC coincided with a favorable prognosis. Our studies identified ERAD-independent role for RNF5, demonstrating that its control of RBBP4 constitutes an epigenetic pathway that drives AML while highlighting RNF5/RBBP4 as markers to stratify patients for treatment with HDAC inhibitors.

cancer biology↗

bioRxiv2020

Human bronchial epithelial cell-derived extracellular vesicle therapy for pulmonary fibrosis via inhibition of TGF-β-WNT crosstalk

Idiopathic pulmonary fibrosis (IPF) is characterized by devastating and progressive lung parenchymal fibrosis, resulting in poor patient prognosis. An aberrant recapitulation of developmental lung gene expression, including genes for transforming growth factor (TGF)-{beta} and WNT, has been widely implicated in the pathogenic IPF wound healing process that results from repetitive alveolar epithelial injury. Extracellular vesicles (EVs) have been shown to carry bioactive molecules and to be involved in various physiological and pathological processes. Here, we demonstrate that, by attenuating WNT signaling, human bronchial epithelial cell-derived EVs (HBEC EVs) inhibit TGF-{beta} mediated induction of both myofibroblast differentiation and lung epithelial cellular senescence. This effect of HBEC EVs is more pronounced than that observed with mesenchymal stem cell-derived EVs. Mechanistically, the HBEC EV microRNA (miRNA) cargo is primarily responsible for attenuating both myofibroblast differentiation and cellular senescence. This attenuation occurs via inhibition of canonical and non-canonical WNT signaling pathways. Among enriched miRNA species present in HBEC EVs, miR-16, miR-26a, miR-26b, miR-141, miR-148a, and miR-200a are mechanistically involved in reducing WNT5A and WNT10B expression in LFs, and in reducing WNT3A, WNT5A, and WNT10B expression in HBECs. Mouse models utilizing intratracheal administration of EVs demonstrate efficient attenuation of bleomycin-induced lung fibrosis development accompanied by reduced expression of both {beta}-catenin and markers of cellular senescence. These findings indicate that EVs derived from normal resident lung HBECs may possess anti-fibrotic properties. They further suggest that, via miRNA-mediated inhibition of TGF-{beta}-WNT crosstalk, HBEC EVs administration can be a promising anti-fibrotic modality of treatment for IPF.

molecular biology↗

bioRxiv2020

Curing genetic skin disease through altered replication stress response

Revertant mosaicism, or natural gene therapy, refers to the spontaneous in vivo reversion of an inherited mutation in a somatic cell1. Only [~]50 human genetic disorders exhibit revertant mosaicism, implicating a distinctive role played by mutant proteins in somatic correction of a pathogenic germline mutation2. However, the process by which mutant proteins induce somatic genetic reversion in these diseases remains unknown. Here we show that heterozygous pathogenic CARD14 mutations causing autoinflammatory skin diseases, including psoriasis and pityriasis rubra pilaris, are repaired mainly via homologous recombination. Rather than altering the DNA damage response to exogenous stimuli such as X-irradiation or etoposide treatment, mutant CARD14 increased DNA double-strand breaks under conditions of replication stress. Furthermore, mutant CARD14 suppressed new origin firings without promoting crossover events in the replication stress state. Together, these results suggest that mutant CARD14 alters the replication stress response and preferentially drives break-induced replication (BIR), which is generally suppressed in eukaryotes3. Our results highlight the involvement of BIR in reversion events, thus revealing a previously undescribed role of BIR that could potentially be exploited to develop therapeutics for currently intractable genetic diseases.

molecular biology↗

bioRxiv2020

Isolation and characterization of Streptomyces bacteriophages and Streptomyces strains encoding biosynthetic arsenals

The threat to public health posed by drug-resistant bacteria is rapidly increasing, as some of healthcares most potent antibiotics are becoming obsolete. Approximately two-thirds of the worlds antibiotics are derived from natural products produced by Streptomyces encoded biosynthetic gene clusters. Thus, in order to identify novel gene clusters, we sequenced the genomes of four bioactive Streptomyces strains isolated from the soil in San Diego County and used Bacterial Cytological Profiling adapted for agar plate culturing in order to examine the mechanisms of bacterial inhibition exhibited by these strains. In the four strains, we identified 101 biosynthetic gene clusters. Some of these clusters were predicted to produce previously studied antibiotics; however, the known mechanisms of these molecules could not fully account for the antibacterial activity exhibited by the strains, suggesting that novel clusters might encode antibiotics. When assessed for their ability to inhibit the growth of clinically isolated pathogens, three Streptomyces strains demonstrated activity against methicillin-resistant Staphylococcus aureus. Additionally, due to the utility of bacteriophages for genetically manipulating bacterial strains via transduction, we also isolated four new phages (BartholomewSD, IceWarrior, Shawty, and TrvxScott) against S. platensis. A genomic analysis of our phages revealed nearly 200 uncharacterized proteins, including a new site-specific serine integrase that could prove to be a useful genetic tool. Sequence analysis of the Streptomyces strains identified CRISPR-Cas systems and specific spacer sequences that allowed us to predict phage host ranges. Ultimately, this study identified Streptomyces strains with the potential to produce novel chemical matter as well as integrase-encoding phages that could potentially be used to manipulate these strains.

microbiology↗

bioRxiv2020

vRNA-vRNA interactions in influenza A virus HA vRNA packaging

The genome of the influenza A virus is composed of eight single-stranded negative-sense RNA segments (vRNAs). The eight different vRNAs are selectively packaged into progeny virions. This process likely involves specific interactions among vRNAs via segment-specific packaging signals located in the 3 and 5 terminal coding regions of vRNAs. To identify vRNA(s) that interact with hemagglutinin (HA) vRNA during genome packaging, we generated a mutant virus, HA 5m2, which possessed five silent mutations in the 5 packaging signal region of HA vRNA. The HA 5m2 virus had a specific defect in HA vRNA incorporation, which reduced the viral replication efficiency. After serial passaging in cells, the virus acquired additional mutations in the 5 terminal packaging signal regions of both HA and PB2 vRNAs. These mutations contributed to recovery of viral growth and packaging efficiency of HA vRNA. A direct RNA-RNA interaction between the 5 ends of HA and PB2 vRNAs was confirmed in vitro. Our results indicate that direct interactions of HA vRNA with PB2 vRNA via their packaging signal regions are important for selective genome packaging and enhance our knowledge on the emergence of pandemic influenza viruses through genetic reassortment.

microbiology↗

bioRxiv2019

Computational characteristics of the striatal dopamine system described by reinforcement learning with fast generalization

Generalization enables applying past experience to similar but nonidentical situations. Therefore, it may be essential for adaptive behaviors. Recent neurobiological observation indicates that the striatal dopamine system achieves generalization and subsequent discrimination by updating corticostriatal synaptic connections in differential response to reward and punishment. To analyze how the computational characteristics in this system affect behaviors, we proposed a novel reinforcement learning model with multilayer neural networks in which the synaptic weights of only the last layer are updated according to the prediction error. We set fixed connections between the input and hidden layers so as to maintain the similarity of inputs in the hidden-layer representation. This network enabled fast generalization, and thereby facilitated safe and efficient exploration in reinforcement learning tasks, compared to algorithms which do not show generalization. However, disturbance in the network induced aberrant valuation. In conclusion, the unique computation suggested by corticostriatal plasticity has the advantage of providing safe and quick adaptations to unknown environments, but on the other hand has the potential defect which can induce maladaptive behaviors like delusional symptoms of psychiatric disorders. Author summaryThe brain has an ability to generalize knowledge obtained from reward- and punishment-related learning. Animals that have been trained to associate a stimulus with subsequent reward or punishment respond not only to the same stimulus but also to resembling stimuli. How does generalization affect behaviors in situations where individuals are required to adapt to unknown environments? It may enable efficient learning and promote adaptive behaviors, but inappropriate generalization may disrupt behaviors by associating reward or punishment with irrelevant stimuli. The effect of generalization here should depend on computational characteristics of underlying biological basis in the brain, namely, the striatal dopamine system. In this research, we made a novel computational model based on the characteristics of the striatal dopamine system. Our model enabled fast generalization and showed its advantage of providing safe and quick adaptation to unknown environments. By contrast, disturbance of our model induced abnormal behaviors. The results suggested the advantage and the shortcoming of generalization by the striatal dopamine system.

neuroscience↗

bioRxiv2018

Stress relaxation in epithelial monolayers is controlled by actomyosin

Epithelial monolayers are one-cell thick tissue sheets that separate internal and external environments. As part of their function, they withstand extrinsic mechanical stresses applied at high strain rate. However, little is known about how monolayers respond to mechanical deformations. In stress relaxation tests, monolayers respond in a biphasic manner and stress dissipation is accompanied by an increase in monolayer resting length, pointing to active remodelling of cell architecture during relaxation. Consistent with this, actomyosin remodels at a rate commensurate with mechanical relaxation and governs the rate of monolayer stress relaxation - as in single cells. By contrast, junctional complexes and intermediate filaments form stable connections between cells, enabling monolayers to behave rheologically as single cells. Together, these data show actomyosin cytoskeletal dynamics govern the rheological properties of monolayers by enabling active, ATP-dependent changes in the resting length. These findings have far-reaching consequences for our understanding of developmental morphogenesis and tissue response to mechanical stress.

biophysics↗

bioRxiv2018

Src-transformed cells hijack mitosis to extrude from the epithelium

At the initial stage of carcinogenesis single mutated cells appear within an epithelium. Mammalian in vitro experiments show that potentially cancerous cells undergo live apical extrusion from normal monolayers. However, the mechanism underlying this process in vivo remains poorly understood. Mosaic expression of the oncogene vSrc in a simple epithelium of the early zebrafish embryo results in apical extrusion of transformed cells. Here we find that during extrusion components of the cytokinetic ring are recruited to adherens junctions of transformed cells, stimulating formation of a misoriented pseudo cytokinetic ring. During extrusion, the ring constricts and separates the basal from the apical part of the cell releasing both from the epithelium. This process requires cell cycle progression and occurs immediately after vSrc-transformed cell enters mitosis. To achieve extrusion, vSrc coordinates cell cycle progression, junctional integrity, cell survival and apicobasal polarity. Without vSrc, modulating these cellular processes reconstitutes vSrc-like extrusion, confirming their sufficiency for this process.

cell biology↗