Biology subjects

Fujiwara, T.

Publications and source records attributed to Fujiwara, T..

At least 37 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

Cure of congenital purpura fulminans via expression of engineered protein C through neonatal genome editing in mice

Protein C (PC) is a plasma anticoagulant encoded by PROC; mutation in both PROC alleles results in neonatal purpura fulminans--a fatal systemic thrombotic disorder. In the present study, we aimed to develop a genome editing treatment to cure congenital PC deficiency. First, we generated an engineered activated PC to insert a self-cleaving peptide sequence between light and heavy chains. The engineered PC could be released in its activated form and significantly prolonged the plasma coagulation time independent of the cofactor activity of protein S in vitro. The adeno-associated virus (AAV) vector-mediated expression of the engineered PC, but not wild-type PC, prolonged coagulation time owing to the inhibition of activated coagulation factor V in a dose-dependent manner and abolished pathological thrombus formation in vivo in C57BL/6 mice. The insertion of EGFP sequence conjugated with self-cleaving peptide sequence at Alb locus via neonatal in vivo genome editing using AAV vector resulted in the expression of EGFP in 7% of liver cells, mainly via homology-directed repair, in mice. Finally, we succeeded in improving the survival of PC-deficient mice by expressing the engineered PC via neonatal genome editing in vivo. These results suggest that the expression of the engineered PC via neonatal genome editing is a potential cure for severe congenital PC deficiency. One Sentence SummaryEctopic expression of an engineered protein C via genome editing cures protein C deficiency in mice.

bioengineering↗

bioRxiv2022

SEN1 is responsible for molybdate transport into nodule symbiosomes for nitrogen fixation in Lotus japonicus

Symbiotic nitrogen fixation (SNF) in legume root nodules requires a steady supply of molybdenum (Mo) for synthesis of the iron-Mo cofactor for nitrogenase in bacteroids. For this nutrient to be exported by the host plant it must cross the peribacteroid membrane (PBM), however, the molybdate transporter responsible has not yet been identified. A Lotus japonicus symbiotic mutant, sen1, forms nodules that do not fix nitrogen; it has nodule defects and bacteroid degradation. The biochemical function and subcellular localization of SEN1 protein remains to be elucidated. Here, we found a new phenotype in which the SEN1 mutation resulted in increased Mo accumulation in the nodule host fractions but decreased Mo accumulation in the bacteroids at 10 days post inoculation. We identified the molybdate efflux transport activity of SEN1 via heterologous expression in yeast. SEN1 was expressed exclusively in nodules, and its expression was stable in response to varying Mo supply in nutrient solution. In situ immunostaining verified that the SEN1 protein is localized, in part, to the PBM in the rhizobium-infected cells. Taken together, these results confirmed that SEN1 is responsible for mediating molybdate efflux from the cytosol of nodule host cells to the symbiosomes for SNF. Furthermore, SEN1 mutation reduced the expression of nifD and nifK, suggesting that SEN1 may be pertinent to iron-Mo-cofactor assembly. This work fills the knowledge gap regarding how molybdate is allocated from the host plant to the bacteroids; such knowledge is critical for developing new SNF biological systems in non-legume plants. One-sentence summarySEN1 is localized partly in the peribacteroid membrane of nodule cells and mediates the molybdate exportation from the host plant cytosol to the symbiosomes for symbiotic nitrogen fixation.

plant biology↗

bioRxiv2022

FUS regulates RAN translation through modulating the G-quadruplex structure of GGGGCC repeat RNA in C9orf72-linked ALS/FTD

Abnormal expansions of GGGGCC repeat sequence in the noncoding region of the C9orf72 gene is the most common cause of familial amyotrophic lateral sclerosis and frontotemporal dementia (C9-ALS/FTD). The expanded repeat sequence is translated into dipeptide repeat proteins (DPRs) by noncanonical repeat-associated non-AUG (RAN) translation. Since DPRs play central roles in the pathogenesis of C9-ALS/FTD, we here investigate the regulatory mechanisms of RAN translation, focusing on the effects of RNA-binding proteins (RBPs) targeting GGGGCC repeat RNAs. Using C9-ALS/FTD model flies, we demonstrated that the ALS/FTD-linked RBP FUS suppresses RAN translation and neurodegeneration in an RNA-binding activity-dependent manner. Moreover, we found that FUS directly binds to and modulates the G-quadruplex structure of GGGGCC repeat RNA as an RNA chaperone, resulting in the suppression of RAN translation in vitro. These results reveal a previously unrecognized regulatory mechanism of RAN translation by G-quadruplex-targeting RBPs, providing therapeutic insights for C9-ALS/FTD and other repeat expansion diseases.

neuroscience↗

bioRxiv2022

Transposons repressed by H3K27me3 were co-opted as cis-regulatory elements of H3K27me3 controlled protein coding genes during evolution of plants

The mobility of transposable elements (TEs) contributes to evolution of genomes 1,2. Meanwhile, their uncontrolled activity causes genomic instability and therefore expression of TEs is silenced by host genomes 3,4. TEs are marked with DNA and H3K9 methylation that are associated with silencing in flowering plants 5, animals, and fungi 6. Yet, in distantly related eukaryotes TEs are instead marked by H3K27me3 deposited by the Polycomb Repressive Complex 2 (PRC2) 7-11, an epigenetic mark associated with gene silencing in multicellular eukaryotes 12-15. It was therefore proposed that the ancestral activity of PRC2 was the deposition of H3K27me3 to silence TEs 16. To test this hypothesis we obtained mutants deprived of PRC2 activity and used genomics to analyze the role of PRC2 in extant species along the lineage of Archaeplastida. While in the red alga Cyanidioschyzon merolae more TEs than genes were repressed by PRC2, an opposite trend was observed in bryophytes Marchantia polymorpha and Anthoceros agrestis. In the red alga, TEs silenced by H3K27me3 are in subtelomeres but in bryophytes, TEs and genes marked by H3K27me3 form coregulated transcriptional units. The latter trend was also observed in the flowering plant Arabidopsis thaliana, and we identified cis-elements recognised by transcription factors in TEs flanking genes repressed by PRC2. Together with the silencing of TEs by PRC2 in ciliates that diverged early from an ancestor common with Archaeplastida, our findings support the hypothesis that PRC2 deposited H3K27me3 to silence TEs in early lineages of eukaryotes. During evolution, TE fragments marked with H3K27me3 were selected to shape transcriptional regulation that control networks of genes regulated by PRC2. HighlightsH3K27me3 marks a decreasing proportion of TEs during evolution of plants The polycomb repressive complex 2 represses TEs in red algae and bryophytes H3K27me3-marked TEs in flowering plants contain transcription factor binding sites Transcription factors bind TEs and regulate networks of genes controlled by PRC2

evolutionary biology↗

bioRxiv2022

Energy transfer in ubiquitous rhodopsin pumps with xanthophyll antennas

Energy transfer from light-harvesting ketocarotenoids to light-driven proton pumps xanthorhodopsins has been previously demonstrated in two unique cases: an extreme halophilic bacterium1 and a terrestrial cyanobacterium2. Attempts to find carotenoids that bind and transfer energy to rhodopsin proton pumps from the abundant marine and freshwater photoheterotrophs have thus far failed3-5. Here, using functional metagenomics combined with chromophore extraction from the environment, we detected light energy transfer from the widespread hydroxylated carotenoids zeaxanthin and lutein to the retinal moiety of xanthorhodopsins and proteorhodopsins. The light-harvesting carotenoids transfer up to 42% of the harvested energy in the violet/blue-light range to the green-light absorbing retinal chromophore. Our data suggest that these antennas have a significant impact on rhodopsin phototrophy in the worlds lakes, seas and oceans.

microbiology↗

bioRxiv2021

iTRVZ: liquid nano-platform for signal integration on the plasma membrane

Cell survival in immune-competent organisms relies on both growth and immune evasion, yet these processes have been studied as independent cellular programs. Here, using super-resolution single-molecule imaging, we identify GEM (Growth and Evasion Metastable hub), a {approx}34-nm, metastable, nano-liquid, plasma membrane hub that physically integrates receptor-type tyrosine kinase (RTK) inputs with CD59-mediated immune-evasion signaling within the same assembly. GEM is formed by LLPS-like protein clustering on PI(4,5)P2-containing raft-nanodomains. Upon concurrent RTK and CD59 stimulations, GEM recruits both receptors and downstream kinases, enabling reciprocal activation via nano-liquid confinement that supralinearly amplifies PLC{gamma}-IP3-Ca2+ and PI3K-Akt survival signaling outputs. In mice, disrupting GEM suppresses tumor growth in vivo, establishing nano-liquid GEM as an integrator that physically couples growth and CD59-mediated immune-evasion signaling for cell survival.

cell biology↗

bioRxiv2021

Oscillatory movement of a dynein-microtubule complex crosslinked with DNA origami

During repetitive bending of cilia and flagella, axonemal dynein molecules move in an oscillatory manner along a microtubule (MT), but how the minus-end-directed motor dynein can oscillate back and forth is unknown. There are various factors that may regulate the dynein activities, e.g., the nexin-dynein regulatory complex, radial spokes, and central apparatus. In order to understand the basic mechanism of the oscillatory movement, we constructed a simple model system composed of MTs, outer-arm dyneins, and DNA origami that crosslinks the MTs. Electron microscopy (EM) showed patches of dynein molecules crossbridging two MTs in two opposite orientations; the oppositely oriented dyneins are expected to produce opposing forces. The optical trapping experiments showed that the dynein-MT-DNA-origami complex actually oscillate back and forth after photolysis of caged ATP. Intriguingly, the complex, when held at one end, showed repetitive bending motions. The results show that a simple system composed of ensembles of oppositely oriented dyneins, MTs, and inter-MT crosslinkers, without the additional regulatory structures, has an intrinsic ability to cause oscillation and repetitive bending motions.

biophysics↗

bioRxiv2021

Biochemical propensity mapping for structural and functional anatomy of importin α IBB domain

Importin has been described as a nuclear protein transport receptor that enables proteins synthesized in the cytoplasm to translocate into the nucleus. Besides its function in nuclear transport, an increasing number of studies have examined its non-nuclear transport functions. In both nuclear transport and non-nuclear transport, a functional domain called the IBB domain (importin {beta} binding domain) plays a key role in regulating importin behavior, and is a common interacting domain for multiple binding partners. However, it is not yet fully understood how the IBB domain interacts with multiple binding partners, which leads to the switching of importin function. In this study, we have distinguished the location and propensities of amino acids important for each function of the importin IBB domain by mapping the biochemical/physicochemical propensities of evolutionarily conserved amino acids of the IBB domain onto the structure associated with each function. We found important residues that are universally conserved for IBB functions across organisms and families, in addition to those previously known, as well as residues that are presumed to be responsible for the differences in complex-forming ability between families and for functional switching.

cell biology↗

bioRxiv2021

Walking strides direct rapid and flexible recruitment of visual circuits for course control in

Flexible mapping between activity in sensory systems and movement parameters is a hallmark of successful motor control. This flexibility depends on continuous comparison of short-term postural dynamics and the longer-term goals of an animal, thereby necessitating neural mechanisms that can operate across multiple timescales. To understand how such body-brain interactions emerge to control movement across timescales, we performed whole-cell patch recordings from visual neurons involved in course control in Drosophila. We demonstrate that the activity of leg mechanosensory cells, propagating via specific ascending neurons, is critical to provide a clock signal to the visual circuit for stride-by-stride steering adjustments and, at longer timescales, information on speed-associated motor context to flexibly recruit visual circuits for course control. Thus, our data reveal a stride-based mechanism for the control of high-performance walking operating at multiple timescales. We propose that this mechanism functions as a general basis for adaptive control of locomotion.

neuroscience↗

bioRxiv2021

Time-series Multi-spectral Imaging in Soybean for Improving Biomass and Genomic Prediction Accuracy

Multi-spectral (MS) imaging enables the measurement of characteristics important for increasing the prediction accuracy of genotypic and phenotypic values for yield-related traits. In this study, we evaluated the potential application of temporal MS imaging for the prediction of above-ground biomass (AGB) and determined which developmental stages should be used for accurate prediction in soybean. Field experiments with 198 accessions of soybean were conducted with four different irrigation levels. Five vegetation indices (VIs) were calculated using MS images from soybean canopies from early to late growth stages. To predict the genotypic values of AGB, VIs at the different growth stages were used as secondary traits in a multi-trait genomic prediction. The accuracy of the prediction model increased starting at an early stage of growth (31 days after sowing). To predict phenotypic values of AGB, we employed multi-kernel genomic prediction. Consequently, the prediction accuracy of phenotypic values reached a maximum at a relatively early growth stage (38 days after sowing). Hence, the optimal timing for MS imaging may depend on the irrigation levels.

plant biology↗

bioRxiv2021

Transferrin receptor 1-mediated iron uptake regulates bone mass in mice via osteoclast mitochondria and cytoskeleton

Increased intracellular iron spurs mitochondrial biogenesis and respiration to satisfy high-energy demand during osteoclast differentiation and bone-resorbing activities. Transferrin receptor 1 (TFR1) mediates cellular iron uptake through endocytosis of iron-loaded transferrin and its expression increases during osteoclast differentiation. Nonetheless, the precise functions of TFR1 and TFR1-mediated iron uptake in osteoclast biology and skeletal homeostasis remain incompletely understood. To investigate the role of TFR1 in osteoclast lineage cells, we conditionally deleted Tfr1 gene in myeloid precursors or mature osteoclasts by crossing Tfr1- floxed mice with LysM-Cre and Ctsk-Cre mice, respectively. Skeletal phenotyping by {micro}CT and histology unveiled that loss of Tfr1 in osteoclast progenitor cells resulted in a three-fold increase in trabecular bone mass in the long bones of 10-week old female but not male mice. Although high trabecular bone volume in long bones was seen in both male and female mice with deletion of Tfr1 in mature osteoclasts, this phenotype was more pronounced in female knockout mice. Mechanistically, disruption of Tfr1 expression attenuated mitochondrial metabolism and cytoskeletal organization in mature osteoclasts, leading to decreased bone resorption with no impact on osteoclastogenesis. These results indicate that Tfr1-mediated iron uptake is specifically required for osteoclast function and is indispensable for bone remodeling.

cell biology↗

bioRxiv2021

Liquid-liquid phase separation of florigen activation complex induces flowering

Floral transition, regulated by the systemic action of the mobile florigen protein FLOWERING LOCUS T (FT), is essential for successful plant reproduction1. How FT controls downstream gene expression remains incompletely understood, although it relies on the florigen activation complex (FAC), a core component of FT function2-4. The FAC is a nucleus-localized transcriptional activator of genes encoding MADS-box transcription factors critical to reproductive development and consists of florigen FT; a scaffold 14-3-3 protein that is a key component for complex assembly; and FD, a basic leucine-zipper protein that recruits the FAC to DNA. Here we report that the FAC exhibits phase separation. In rice shoot apical meristem cells, rice florigen Heading date 3a (Hd3a) fused to the green fluorescent protein formed speckles in the nucleus. The FAC speckle is formed in a FAC-dependent manner in tobacco cells. Recombinant Hd3a, but not OsFD1, phase-separated in vitro, and this effect was enhanced in the presence of 14-3-3 protein. Furthermore, mutations affecting functionally important residues in the pocket region or C-terminal disordered region of Hd3a affected FAC phase separation, providing a biochemical framework for the proteins effect on flowering. The ability to form condensates via phase transition represents a previously unknown mechanism for gene activation by the FAC.

plant biology↗

bioRxiv2021

Exploration of novel αβ-protein folds through de novo design

Most naturally occurring protein folds have likely been discovered1-3. The question is whether natural evolution has exhaustively sampled almost all possible protein folds4, or whether a large fraction of the possible folds remains unexplored5-7. To address this question, we introduce a set of rules for {beta}-sheet topology to predict novel folds, and carry out the systematic de novo protein design for the novel folds predicted by the rules. The rules predicted eight novel {beta}-folds with a four-stranded {beta}-sheet, including a knot-forming one. We designed proteins for all the predicted {beta}-folds and found that all the designs are monomeric with high thermal stability and fold into the structures close to the design models, demonstrating the ability of the set of rules to predict novel {beta}-folds. The rules also predicted about twelve thousand novel {beta}-folds with five- to eight-stranded {beta}-sheets; the number is far exceeding the number of {beta}-folds observed so far. This result suggests that the enormous number of {beta}-folds are possible but have not emerged or become extinct due to evolutionary bias. The predicted novel folds should open up the possibility of designing functional proteins of our interests.

biophysics↗

bioRxiv2021

Design of complicated all-α protein structures

A wide range of de novo protein structure designs have been achieved, but the complexity of naturally occurring protein structures is still far beyond these designs. To expand the diversity and complexity of de novo designed protein structures, we sought to develop a method for designing "difficult-to-describe"-helical protein structures composed of irregularly aligned -helices like globins. Backbone structure libraries consisting of a myriad of -helical structures with 5- or 6-helices were generated by combining 18 helix-loop-helix motifs and canonical -helices, and five distinct topologies were selected for de novo design. The designs were found to be monomeric with high thermal stability in solution and fold into the target topologies with atomic accuracy. This study demonstrated that complicated -helical proteins are created using typical building blocks. The method we developed would enable us to explore the universe of protein structures for designing novel functional proteins.

biophysics↗

bioRxiv2021

CZON-cutter: a CRISPR-Cas9 system with multiplexed organelle imaging in a simple unicellular alga

The simple cellular structure of the unicellular alga Cyanidioschyzon merolae consists of one nucleus, one mitochondrion, one chloroplast, and one peroxisome per cell and offers unique advantages to investigate mechanisms of organellar proliferation and the cell cycle. Here, we describe an engineered clustered, regularly interspaced, short palindromic repeats (CRISPR)-associated protein 9 (Cas9) system, CZON-cutter, for simultaneous genome editing and organellar visualization. We engineered a C. merolae strain expressing a nuclear-localized Cas9-Venus nuclease to target editing at a locus defined by a single-guide RNA (sgRNA). We then successfully edited the algal genome and visualized the mitochondrion and peroxisome in transformants by fluorescent protein reporters with different excitation wavelengths. Fluorescent protein labeling of organelles in living transformants allows validation of phenotypes associated with organellar proliferation and the cell cycle, even when the edited gene is essential. Combined with the exceptional biological features of C. merolae, CZON-cutter will be instrumental for investigating cellular and organellar division in a high-throughput manner. SummaryAn engineered CRISPR-Cas9 system, named CZON-cutter, for simultaneous genome editing and fluorescent protein labeling of organelles in Cyanidioschyzon merolae can be used to validate intracellular function of a particular gene, even if it is essential.

molecular biology↗

bioRxiv2020

Pattern recognition receptors confer plant salt tolerance via WRKY18/WRKY40 transcription factors

Pattern recognition receptors (PRRs) bind microbe- and damage-associated molecular patterns (MAMPs/DAMPs, respectively) to enhance host immunity in animals and plants. Here, we report that PRRs also confer salt tolerance in the model plant Arabidopsis thaliana following recognition of cognate ligands, such as bacterial flagellin and EF-Tu and the endogenous Pep peptides. Pattern-triggered salt tolerance (PTST) requires the PRR-associated kinases BAK1 and BIK1, and the NADPH oxidase RBOHD. Transcriptome profiling reveals an inventory of PTST target genes, which increase or acquire salt responsiveness following an exposure to immunogenic patterns. In their regulatory DNA sequences, specific binding sites for a subset of WRKY transcription factors are over-represented. Accordingly, PTST requires WRKY40 and WRKY18, which activate salt tolerance-related genes but attenuate pathogen defense-related genes, including the EDS1 immunity activator. PRR signaling leads to sustained WRKY40/WRKY18 accumulation under salt stress and utilizes both WRKYs for salt tolerance. The PRR-WRKY40/WRKY18 module also confers salt tolerance after challenge with non-pathogenic bacteria. Our findings give molecular insight into signaling plasticity underlying biotic-abiotic stress cross-tolerance in plants conferred by PRRs.

plant biology↗

bioRxiv2020

OsNLP4 is required for nitrate assimilation gene expressions and nitrate-dependent growth in rice

In plants, nitrate is important nutrient and signaling molecule that modulates the expression of many genes and regulates growth. In paddy grown rice, nitrogen is mostly supplied in the form of ammonium, but nitrate also shares substantial portion of available nitrogen. A number of nitrogen transporters and nitrate assimilation enzymes have been identified and functionally characterized. However, little is known about the nitrate sensor system and regulatory mechanisms of these nitrate related genes. In recent years, NIN-like proteins (NLPs) have been described as key transcription factors of nitrogen responses in Arabidopsis thaliana. But the functions of OsNLPs in rice are still elusive. Here we report the characterization of OsNLP4 to reveal its function in rice. Growths of OsNLP4 knockout mutants were reduced under the nitrate supply, but not under ammonium supply. The mRNA accumulation of genes involved in nitrate assimilation were declined significantly and nitrate uptake rate and nitrate reductase activity were also impaired in the mutants. Using rice protoplast transient expression system, OsNLP4-GFP fusion was localized to nucleus irrespective of nitrate conditions. OsNLP4 was also required for normal yield under paddy field conditions. We propose the OsNLP4 is essential for regulation of genes involved in nitrate assimilation and nitrate-dependent growth in rice. One sentence summaryThe osnlp4 mutants exhibit abnormal nitrate response and poor growth under nitrate supply and in paddy field conditions.

plant biology↗