Biology subjects

Tanaka, K.

Publications and source records attributed to Tanaka, K..

At least 73 records · Page 4Linked to original sources

bioRxiv2022

Structural and biochemical insights into heterotetramer formation between human oncogenic K-Ras4BG12V and Rgl2, a RalA/B activator

About a quarter of total human cancers carry mutations in Ras isoforms. Accumulating evidence suggests that small GTPases, RalA and RalB, and their activators, Ral guanine nucleotide exchange factors (RalGEFs), play an essential role in oncogenic Ras-induced signalling. We studied the interaction between human KRas4B and the Ras association (RA) domain of Rgl2 (Rgl2RA), one of the RA-containing RalGEFs. We show that the G12V oncogenic KRas4B mutation changes the interaction kinetics with Rgl2RA. The crystal structure of the KRas4BG12V: Rgl2RA complex shows a 2:2 heterotetramer where the Switch I and Switch II regions of each KRasG12V interact with both Rgl2RA molecules. This structural arrangement is highly similar to the HRasE31K:RALGDSRA crystal structure and is distinct from the well-characterised Ras:Raf complex. Interestingly, the G12V mutation was found at the dimer interface of KRas4BG12V with its partner. Our study reveals a potentially distinct mode of Ras:effector complex formation by RalGEFs, and offers a possible mechanistic explanation for how the oncogenic KRas4BG12V hyperactivates the RalA/B pathway.

biochemistry↗

bioRxiv2022

Remarkable improvement in detection of readthrough downstream-of-gene transcripts by semi-extractable RNA-sequencing

The mammalian cell nucleus contains dozens of membrane-less nuclear bodies that play significant roles in various aspects of gene expression. Several nuclear bodies are nucleated by specific architectural noncoding RNAs (arcRNAs) acting as structural scaffolds. We have reported that a minor population of cellular RNAs exhibits an unusual semi-extractable feature upon using the conventional procedure of RNA preparation and that needle shearing or heating of cell lysates remarkably improves extraction of dozens of RNAs. Because semi-extractable RNAs, including known arcRNAs, commonly localize in nuclear bodies, this feature may be a hallmark of arcRNAs. Using the semi-extractability of RNA, we performed genome-wide screening of semi-extractable long non-coding RNAs to identify new candidate arcRNAs for arcRNA under hyperosmotic and heat stress conditions. After screening stress-inducible and semi-extractable RNAs, hundreds of readthrough downstream-of-gene (DoG) transcripts over several hundreds of kilobases, many of which were not detected among RNAs prepared by the conventional extraction procedure, were found to be stress-inducible and semi-extractable. We further characterized some of the abundant DoGs and found that stress-inducible transient extension of the 3-UTR made DoGs semi-extractable. Furthermore, they were localized in distinct nuclear foci that were sensitive to 1,6-hexanediol. These data suggest that semi-extractable DoGs exhibit arcRNA-like features and our semi-extractable RNA-seq is a powerful tool to extensively monitor DoGs that are induced under specific physiological conditions.

molecular biology↗

bioRxiv2022

Characterisation of an Escherichia coli line that completely lacks ribonucleotide reduction yields insights into the evolution of obligate intracellularity.

All life requires ribonucleotide reduction for de novo synthesis of deoxyribonucleotides. A handful of obligate intracellular species are known to lack ribonucleotide reduction and are instead dependent on their host for deoxyribonucleotide synthesis. As ribonucleotide reduction has on occasion been lost in obligate intracellular parasites and endosymbionts, we reasoned that it should in principle be possible to knock this process out entirely under conditions where deoxyribonucleotides are present in the growth media. We report here the creation of a strain of E. coli where all three ribonucleotide reductase operons have been fully deleted. Our strain is able to grow in the presence of deoxyribonucleosides and shows slowed but substantial growth. Under limiting deoxyribonucleoside levels, we observe a distinctive filamentous cell morphology, where cells grow but do not appear to divide regularly. Finally, we examined whether our lines are able to adapt to limited supplies of deoxyribonucleosides, as might occur in the evolutionary switch from de novo synthesis to dependence on host production during the evolution of parasitism or endosymbiosis. Over the course of an evolution experiment, we observe a 25-fold reduction in the minimum concentration of exogenous deoxyribonucleosides necessary for growth. Genome analysis of replicate lines reveals that several lines carry mutations in deoB and cdd. deoB codes for phosphopentomutase, a key part of the deoxyriboaldolase pathway, which has been hypothesised as an alternative to ribonucleotide reduction for deoxyribonucleotide synthesis. Rather than synthesis via this pathway complementing the loss of ribonucleotide reduction, our experiments reveal that mutations appear that reduce or eliminate the capacity for this pathway to catabolise deoxyribonucleotides, thus preventing their loss via central metabolism. Mutational inactivation of both deoB and cdd is also observed in a number of obligate intracellular bacteria that have lost ribonucleotide reduction. We conclude that our experiments recapitulate key evolutionary steps in the adaptation to intracellular life without ribonucleotide reduction.

microbiology↗

bioRxiv2022

Fibronectin-integrin α5 signaling promotes thoracic aortic aneurysm in a mouse model of Marfan syndrome

BackgroundMarfan syndrome, caused by mutations in the gene for the extracellular matrix (ECM) glycoprotein fibrillin-1, leads to thoracic aortic aneurysms (TAAs). Phenotypic modulation of vascular smooth muscle cells (SMCs) and ECM remodeling are characteristics of both non-syndromic and Marfan aneurysms. The ECM protein fibronectin (FN) is elevated in the tunica media of TAAs and amplifies inflammatory signaling in endothelial and SMCs through its main receptor, integrin 5{beta}1. We investigated the role of integrin 5-specific signals in Marfan mice in which the cytoplasmic domain of integrin 5 was replaced with that of integrin 2 (denoted 5/2 chimera). MethodsWe used 5/2 chimera mouse crossed with Fbn1mgR/mgR genetic background (mgR, a mouse model of Marfan syndrome) to compare the survival rate and pathogenesis of TAAs among wild type, 5/2, mgR and 5/2; mgR mice. Further biochemical and microscopic analysis of porcine and mouse aortic SMCs allowed us to identify the molecular mechanisms by which FN affects SMCs and subsequent development of TAAs. ResultsFN was elevated in the thoracic aortas from Marfan patients, in non-syndromic aneurysms and in the mgR mouse model of Marfan syndrome. The 5/2 mutation greatly prolonged survival of Marfan mice, with improved elastic fiber integrity, mechanical properties, SMC density, and SMC contractile gene expression. Furthermore, in vitro, plating of wild-type, but not 5/2, SMCs on FN decreased contractile gene expression and activated inflammatory pathways. These effects correlated with increased NF-kB activation and immune cell infiltration in the mgR aortas, which was rescued in the 5/2 mgR aortas. ConclusionsFN-integrin 5 signaling is a significant driver of TAA in the mgR mouse model. This pathway warrants further investigation as a therapeutic target.

pathology↗

bioRxiv2022

Glutamine synthetase mRNA releases sRNA from its 3'UTR to regulate carbon/nitrogen metabolic balance

Glutamine synthetase is the key enzyme of nitrogen assimilation, which is encoded in the first cistron of glnALG operon and is induced under nitrogen limiting conditions through transcriptional activation by NtrBC in Salmonella and E. coli. 2-oxoglutarate serves as the carbon skeleton of glutamate and glutamine, but how 2-oxoglutarate fluctuation is controlled in response to nitrogen availability remained unknown. We show that the glnA mRNA produces an Hfq-dependent GlnZ sRNA from its 3 sUTR through RNase E-mediated cleavage. Through a base-pairing mechanism, GlnZ primarily regulates sucA, encoding the E1o component of 2-oxoglutarate dehydrogenase. In the cells grown on glutamine as the nitrogen source, the endogenous GlnZ represses the expression of SucA to redirect the carbon flow from the TCA cycle to the nitrogen assimilation pathway. This study also clarifies that the release of GlnZ sRNA from the glnA mRNA by RNase E is essential for the post-transcriptional regulation of sucA, and thus the mRNA coordinates the two independent functions to balance the supply and demand of the fundamental metabolites.

microbiology↗

bioRxiv2022

Secondary reversion to sexual monomorphism associated with tissue-specific loss of doublesex expression

Animal evolution is characterized by frequent turnover of sexually dimorphic traits - new sex- specific characters are gained, and some ancestral sex-specific characters are lost, in many lineages. In insects, sexual differentiation is predominantly cell-autonomous and depends on the expression of the doublesex (dsx) transcription factor. In most cases, cells that transcribe dsx have the potential to undergo sex-specific differentiation, while those that lack dsx expression do not. Consistent with this mode of development, comparative research has shown that the origin of new sex-specific traits can be associated with the origin of new spatial domains of dsx expression. In this report, we examine the opposite situation - a secondary loss of the sex comb, a male-specific grasping structure that develops on the front legs of some drosophilid species. We show that, while the origin of the sex comb is linked to an evolutionary gain of dsx expression in the leg, sex comb loss in a newly identified species of Lordiphosa (Drosophilidae) is associated with a secondary loss of dsx expression. We discuss how the developmental control of sexual dimorphism affects the mechanisms by which sex-specific traits can evolve.

evolutionary biology↗

bioRxiv2022

Accumulated 3-phosphoglycerate in darkness is a primary starting substrate for stepwise photosynthetic induction process in cyanobacteria

Photosynthesis must maintain stability and robustness throughout fluctuating natural environments. In cyanobacteria, dark-to-light transition leads to drastic metabolic changes from dark respiratory metabolism to CO2 fixation through the Calvin-Benson-Bassham (CBB) cycle using energy and redox equivalents provided by photosynthetic electron transfer. Previous studies showed that catabolic metabolism supports the smooth transition into CBB cycle metabolism. However, metabolic mechanisms for robust initiation of photosynthesis are poorly understood due to lack of dynamic metabolic characterizations of dark-to-light transitions. Here, we show rapid (on a time scale of seconds) dynamic changes in absolute metabolite concentrations and 13C tracer incorporation after strong or weak light irradiation in the cyanobacterium Synechocystis sp. PCC 6803. Integration of this data enables estimation of time-resolved nonstationary metabolic flux underlying CBB cycle activation. This dynamic metabolic analysis indicates that downstream glycolytic intermediates including phosphoglycerate and phosphoenolpyruvate accumulate under dark conditions as major substrates for initial CO2 fixation. Compared with wild-type Synechocystis, significant delays in the initiation of oxygen evolution are observed in 12 h dark preincubated mutants deficient in glycogen degradation or oxidative pentose phosphate pathway ({Delta}zwf, {Delta}gnd, and{Delta} glgP). Accordingly, the degree of delay in the oxygen evolution initiation is proportional to the accumulated pool size of the glycolytic intermediates. These observations indicate that the accumulation of glycolytic intermediates is essential for efficient metabolism switching under fluctuating light environments.

plant biology↗

bioRxiv2022

Improving the Safety of Cultured Meat Using Basal Medium Prepared using Food Ingredients

Currently, meat production involves environmental issues and high costs; one solution to address this may be to produce meat via cell culture. Therefore, there is an urgent demand for the development of cell culture methods that replace raw materials with food materials to produce cultured meat as food. In this study, we designed food-grade-Dulbeccos Modified Eagles Medium (FG-DMEM); when the cells cultured in FG-DMEM and regular DMEM were compared, there was no significant difference in cell growth or differentiation. Therefore, FG-DMEM can be used for research and safe production of cultured meat.

cell biology↗

bioRxiv2022

An actin-like filament from Clostridium botulinum exhibits a novel mechanism of filament dynamics

Here, we report the discovery of a ParM protein from Clostridium botulinum (CBgs-ParM), which forms a double-stranded polar filament. CBgs-ParM shares many similarities in its basic filament architecture with actin, however, Pi release after nucleotide hydrolysis induces a large lateral strand shift of ~2.5 nm. We identified the ParR (CBgs-ParR) that acts as a nucleation factor in the initial stage of polymerization, similar to ParR from Escherichia coli plasmid R1. CBgs-ParR also functions as a depolymerization factor, probably by recognizing the structural change in the CBgs-ParM filament after Pi release. Comparison with CBH-ParM, another ParM from Clostridum botulinum, showed that subunit-subunit interacting regions largely differ, preventing co-polymerization, implying a selection pressure in evolution to prevent interference between different ParMRC systems.

biophysics↗

bioRxiv2022

Simultaneous overexpression of multiple herbicide-metabolizing genes for broad-spectrum resistance in an agricultural weed Echinochloa phyllopogon

O_LIPrevious research unveiled that the overexpression of catalytically promiscuous CYP81A cytochrome P450s underlies the multiple-herbicide resistance (MHR) in a Californian population of Echinochloa phyllopogon. However, it does not fully accommodate the resistance to diverse herbicides in MHR E. phyllopogon although the genetic inheritance of MHR was suggested as under a single gene control. C_LIO_LIWe investigated the high-level resistance to diclofop-methyl in MHR E. phyllopogon. Detailed diclofop-methyl metabolism was analyzed, followed by gene expression study and functional characterization of P450 genes. The generality of the MHR mechanism was investigated using another MHR line. C_LIO_LIThe MHR line rapidly produced two distinct hydroxylated-diclofop-acid, only one of which was the major metabolite produced by CYP81A12/21. Gene expression study identified the genetically linked overexpression of a novel gene CYP709C69 with CYP81A12/21 in the MHR line. The gene conferred diclofop-methyl resistance in plants and produced another hydroxylated-diclofop-acid in yeast. The activity was observed in some CYP709C in plants. Unlike the broad substrate-specificity in CYP81As, CYP709C69 showed narrow substrate-specificity. The overexpression of the CYP81A and CYP709C69 was also observed in another MHR line. C_LIO_LIThe present findings establish a novel concept that genetically-linked simultaneous overexpression of herbicide-metabolizing genes enhances and broadens the profile of metabolic resistance in weeds. C_LI

plant biology↗

bioRxiv2021

Nuclear activation in dual-durotaxing cells on a matrix with cell-scale stiffness-heterogeneity

Living organisms are typically composed of various tissues with microscopic cell-scale stiffness-heterogeneity, in which some cells receive dynamically fluctuating mechanical stimuli from the heterogeneous extracellular milieu during long-term movement. Although intracellular stress dynamics (ISD), which are closely related to the regulation of cell functions such as proliferation and differentiation, can be characteristically modulated in cells migrating on a matrix with stiffness-heterogeneity, it has been unclear how the mode of fluctuation of ISD affects cell functions. In the present study, we demonstrate that mesenchymal stem cells (MSCs) dual-durotaxing (i.e., both forward and reverse durotaxis) on microelastically-patterned gels with stiff triangular domains markedly amplify the fluctuation of ISD, nuclear shape, and the spatial distribution of chromatins, which makes the cells remain far from tensional equilibrium. We provide evidence that amplified chromatin fluctuation in the dual-durotaxing MSCs can cause activation of cellular vigor and maintenance of the stemness.

biophysics↗

bioRxiv2021

Verification of our empirical understanding of the physiology and ecology of two contrasting plantation species using a trait database

The effects of climate change on forest ecosystems take on increasing importance more than ever. Information on plant traits is a powerful predictor of ecosystem dynamics and functioning. We reviewed the major ecological traits, such as foliar gas exchange and nutrients, xylem morphology and drought tolerance, of Cryptomeria japonica and Chamaecyparis obtusa, which are major timber species in East Asia, especially in Japan, by using a recently developed functional trait database for both species (SugiHinokiDB). Empirically, C. obtusa has been planted under drier conditions, whereas C. japonica has been planted under wetter conditions. Our analyses revealed followings: The maximum photosynthetic rate, stomatal conductance, foliar nutrient content and soil-to-foliage hydraulic conductance were higher in C. japonica than in C. obtusa and were consistent with the higher growth rate of C. japonica. In contrast, the foliar turgor loss point and xylem pressure corresponding to 50% conductivity, which indicate drought tolerance, were lower in C. obtusa than in C. japonica and are consistent with the drier habitat of C. obtusa. Ontogenetic shifts were also observed; as the age and height of the trees increased, many foliar nutrient concentrations decreased, and the foliar minimum midday water potential and specific leaf area also decreased. This suggests that an ontogenetic reduction in photosynthesis occurred due to an increase in drought stress with tree height and age. However, among the Cupressaceae worldwide, the drought tolerance of C. japonica and C. obtusa is not as high. This may be related to the fact that the Japanese archipelago has historically not been subjected to strong dryness. The maximum photosynthetic rate showed intermediate values within the family, indicating that C. japonica and C. obtusa exhibit relatively high growth rates in the Cupressaceae family, and this is thought to be the reason why they have been selected as economically suitable timber species in Japanese forestry. This study clearly demonstrated that the plant trait database provides us a promising opportunity to verify out empirical knowledge of plantation management and helps us to understand effect of climate change on plantation forests by using trait-based modelling.

ecology↗

bioRxiv2021

Genes responsible for avoiding attack of a beetle, relating to the duration of death feigning

Predator avoidance is an important behavior that affects the degree of adaptation of organisms. We compared the DNA variation of one of the predator-avoidance behaviors, the recently extensively studied "death-feigning behavior," between the long strain bred for feigning death for a long time and the short strain bred for feigning death for a short time. To clarify how the difference in DNA sequences between the long and short strains corresponds to the physiological characteristics of the death-feigning duration at the transcriptome level, we performed comprehensive and comparative analyses of gene variants in Tribolium castaneum strains using DNA-re-sequence. The duration of death feigning involves many gene pathways, including caffeine metabolism, tyrosine metabolism, tryptophan metabolism, metabolism of xenobiotics by cytochrome P450, longevity regulating pathways, and circadian rhythm. Artificial selection based on the duration of death feigning results in the preservation of variants of genes in these pathways in the long strain. When an animal wake up from a near-death experience is closely related to its success in avoiding predation. This study suggests that many metabolic pathways and related genes may be involved in the decision-making process of anti-predator animal behavior by forming a network in addition to the tyrosine metabolic system, including dopamine, revealed in previous studies.

animal behavior and cognition↗

bioRxiv2021

Genome-wide chromosomal association of Upf1 is linked to Pol II transcription in Schizosaccharomyces pombe

Although the RNA helicase Upf1 has hitherto been examined mostly in relation to its cytoplasmic role in nonsense mediated mRNA decay (NMD), here we report high-throughput ChIP data indicating genome-wide association of Upf1 with active genes in Schizosaccharomyces pombe. This association is RNase sensitive and it correlates with Pol II transcription and mRNA expression levels. While changes in Pol II occupancy were detected at only some genes in a Upf1-deficient (upf1{Delta}) strain, there is an increased Ser2 Pol II signal at all highly transcribed genes examined by ChIP-qPCR. Furthermore, upf1{Delta} cells are hypersensitive to the transcription elongation inhibitor 6-azauracil and display Pol II abnormalities suggestive of Pol II hyperphosphorylation. A significant proportion of the genes associated with Upf1 in wild-type conditions are also mis-regulated in upf1{Delta}. These data envisage that by operating on the nascent transcript Upf1 might influence Pol II phosphorylation and transcription.

molecular biology↗

bioRxiv2021

Coral symbionts exhibit a polycistronic flavodiiron gene leading to functional proteins in photosynthesis

Photosynthesis in cyanobacteria, green algae, and basal land plants is protected against excess reducing pressure on the photosynthetic chain by flavodiiron proteins (FLV) that dissipate photosynthetic electrons by reducing O2. In these organisms, the genes encoding FLV are always conserved in the form of a pair of two-type isozymes (FLVA and FLVB) that are believed to function in O2 photo-reduction as a heterodimer. While coral symbionts (dinoflagellates of the family Symbiodiniaceae) are the only algae to harbor FLV in photosynthetic red plastid lineage, only one gene is found in transcriptomes and its role and activity remain unknown. Here, we characterized the FLV genes in Symbiodiniaceae and found that its coding region is composed of tandemly repeated FLV sequences. By measuring the O2-dependent electron flow and P700 oxidation, we suggest that this atypical FLV is active in vivo. Based on the amino-acid sequence alignment and the phylogenetic analysis, we conclude that in coral symbionts, the gene pair for FLVA and FLVB have been fused to construct one coding region for a hybrid enzyme, which presumably occurred when or after both genes were inherited from basal green algae to the dinoflagellate. Immunodetection suggested the FLV polypeptide to be cleaved by a post-translational mechanism, adding it to the rare cases of polycistronic genes in eukaryotes. Our results demonstrate that FLV are active in coral symbionts with genomic arrangement that is unique to these species. The implication of these unique features on their symbiotic living environment is discussed.

plant biology↗

bioRxiv2020

Novel multicellular prokaryote discovered next to an underground stream

The emergence of multicellularity is a key event in the evolution of life and is an attractive challenge among researchers, including those investigating the artificial design of cellular behavior1. Multicellular organisms are widely distributed on Earth, and retracing the specific conditions conducive for the initial transition from unicellularity to multicellularity is difficult. However, by examining organisms that inhabit unique (e.g., isolated) environmental niches, we may be able to get a glimpse into primitive multicellularity in the context of a given environment. Here we report the discovery of a new bacterium that displayed multicellular-like characteristics and behavior. The bacterium, which was isolated adjacent to an underground stream in a limestone cave, is to be named Jeongeupia sacculi sp. nov. HS-3. On a solid surface, HS-3 self-organizes its filamentous cells to form an appearance similar to the nematic phase of a liquid crystal2. Mature colonies produce and accommodate clusters of coccobacillus progeny, and release them upon contact with water. HS-3 demonstrated novel, spatiotemporally regulated multicellularity that can resolve the so-called competition-dispersal trade-off problem3. This study illustrates a hypothetical missing link on the emergence of multicellularity.

evolutionary biology↗

bioRxiv2020

A quantitative demonstration of NADP+/NADPH redox homeostasis in cyanobacterial cells

In photosynthetic organisms, it is recognized that the intracellular NADP+/NADPH ratio is regulated within an appropriate range for the cooperative function of a wide variety of physiological processes. However, despite its importance, there is large variability in the values of the NADP+/NADPH ratio quantitatively estimated to date. In the present study, the light-response of the NADP+/NADPH ratio was investigated by applying a novel NADP(H) extraction method using phenol / chloroform / isoamyl alcohol (PCI) in the cyanobacterium Synechocystis sp. PCC 6803. The light-response of NADP(H) observed using PCI extraction was qualitatively consistent with the NADPH fluorescence time course measured in vivo. Moreover, the results obtained by PCI extraction and the fluorescence-based methods were also consistent in a mutant lacking the ability to oxidize NAD(P)H in the respiratory chain, and exhibiting a unique NADPH light-response. These observations indicate that the PCI extraction method allowed quantitative determination of NADP(H) redox. Notably, the PCI extraction method showed that not all NADP(H) was oxidized or reduced by light-dark transition, indicating that some NADP(H) is not light-responsive. Specifically, 64% of total NADP(H) was observed as non-light-responsive in the wild-type cells. The variation of the intracellular NADP+/NADPH ratio is limited to a narrow range due to the presence of non-light-responsive NADP(H).

plant biology↗

bioRxiv2020

Modeling population size independent tissue epigenomes by ChIL-seq with single-thin sections

Recent advances in omics studies have enabled analysis at the single-cell level; however, methods for analyzing the whole cell of large organs and tissues remain challenging. Here, we developed a method named tsChIL to understand the diverse cellular dynamics at the tissue level using high-depth epigenomic data. tsChIL allowed the analysis of a single tissue section and could reproducibly acquire epigenomic profiles from several types of tissues, based on the distribution of target epigenomic states, tissue morphology, and number of cells. The proposed method enabled the independent evaluation of changes in cell populations and gene activation of cells in regenerating skeletal muscle tissues, using a statistical model of RNA polymerase II distribution on gene loci. Thus, the integrative analysis by tsChIL can elucidate in vivo cell-type dynamics of tissues.

molecular biology↗