Biology subjects

Higashiyama, T.

Publications and source records attributed to Higashiyama, T..

31 records · Page 2Linked to original sources

bioRxiv2022

Chemical screen of Arabidopsis zygote and proteomics in tobacco BY-2 cells identify general plant cell division inhibitors

Cell division is essential for growth and development and involves events such as spindle assembly, chromosome separation, and cell plate formation. In plants, the tools used to control these events at the desired time are still poor because the genetic approach is ineffective owing to a high redundancy and lethality, as well as harmful side effects. Accordingly, we screened cell division-affecting compounds, with a focus on Arabidopsis thaliana zygotes, which individually develop in maternal ovules; the cell division was reliably traceable without time-lapse observations. We then identified the target events of the identified compounds using tobacco BY-2 cells for live-cell imaging and proteomics. As a result, we isolated two compounds, PD-180970 and PP2. PD-180970 disrupts microtubule (MT) organization and, thus, nuclear separation, presumably by inhibiting MT-associated proteins (MAP70). PP2 affected class II Kinesin-12 localization at the phragmoplast emerging site and impaired cytokinesis. Moreover, neither chemical caused irreversible damage to viability but they were effective in multiple plant species such as cucumber (Cucumis sativus) and moss (Physcomitrium patens). We propose that the combination of chemical screening based on Arabidopsis zygotes and target event specification focusing on tobacco BY-2 cells can be used to effectively identify novel tools and transiently control specific cell division events that are conserved in diverse plant species.

plant biology↗

bioRxiv2022

IZUMO1 is a sperm fusogen

Mammalian sperm-egg adhesion depends on the trans-interaction between the sperm-specific type I glycoprotein IZUMO1 and its oocyte-specific GPI-anchored receptor JUNO. However, the mechanisms and proteins (fusogens) which mediate the following step of gamete fusion remain unknown. Using live imaging and content mixing assays in a heterologous system and structure-guided mutagenesis, we unveil an unexpected function for IZUMO1 in cell-to-cell fusion. We show that IZUMO1 alone is sufficient to induce fusion, and that this ability is retained in a mutant unable to bind JUNO. On the other hand, a triple mutation in exposed aromatic residues prevents this fusogenic activity without impairing JUNO interaction. Our findings suggest a second, crucial function for IZUMO1 as a unilateral mouse gamete fusogen. HighlightsO_LIIZUMO1 expression in somatic cells in culture induces cell-to-cell fusion C_LIO_LIThe fusogenic activity of IZUMO1 is unilateral C_LIO_LICell fusion is independent of the binding of IZUMO1 to JUNO C_LIO_LIIZUMO1-mediated cell merger depends on its transmembrane domain, and three solvent-exposed aromatic residues C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/478669v2_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@1f06f47org.highwire.dtl.DTLVardef@19d5adorg.highwire.dtl.DTLVardef@1b8d6eorg.highwire.dtl.DTLVardef@e23e26_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

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↗

bioRxiv2021

Plasma membrane damage limits replicative lifespan in yeast and human fibroblasts

Plasma membrane damage (PMD) occurs in all cell types due to environmental perturbation and cell-autonomous activities. However, cellular outcomes of PMD remain largely unknown except for recovery or death. Here, using budding yeast and normal human fibroblasts, we show that cellular senescence, irreversible cell cycle arrest contributing to organismal aging, is the long-term outcome of PMD. To identify the genes essential for PMD response, we developed a simple PMD-damaging assay using a detergent and performed a systematic yeast genome-wide screen. The screen identified 48 genes. The top hits in the screen are the endosomal sorting complexes required for transport (ESCRT) genes, encoding the well-described plasma membrane repair proteins in eukaryotes. Unexpectedly, the replicative lifespan regulator genes are enriched in our 48 hits. This finding suggests a close genetic association between the PMD response and the replicative lifespan regulations. Indeed, we show that PMD limits the replicative lifespan in budding yeast; the ESCRT activator AAA-ATPase VPS4-overexpression extends it. These results suggest that PMD limits replicative lifespan in budding yeast. Moreover, in normal human fibroblasts, we find that PMD induces premature senescence via the Ca2+-p53 axis but not the major senescence pathway, ATM/ATR pathway. Consistent with the results in yeast, transient overexpression of ESCRT-III, CHMP4B, suppressed the PMD-dependent senescence in normal human fibroblasts. Our study proposes that PMD limits cellular lifespan in two different eukaryotic cell types and highlights an underappreciated but ubiquitous senescent cell subtype, namely PMD-dependent senescent cells.

cell biology↗

bioRxiv2021

Detection of a biolistic delivery of fluorescent markers and CRISPR/Cas9 to the pollen tube

In recent years, genome-editing techniques, such as the CRISPR/Cas9 system, have been highlighted as a new approach to plant breeding. Agrobacterium-mediated transformation has been widely utilized to generate transgenic plants by introducing plasmid DNA containing CRISPR/Cas9 into plant cells. However, this method is generally applicable to a limited range of plants, such as model species. To overcome this limitation, we developed a method to genetically modify male germ cells without the need for Agrobacterium-mediated transfection and tissue culture, by using tobacco as a model. In this study, plasmid DNA containing sequences of Cas9, guide RNA, and fluorescent reporter was introduced into pollen using a biolistic delivery system. Based on the transient expression of fluorescent reporters, the Arabidopsis UBQ10 promoter was found to be the most suitable for driving expression of the delivered gene in pollen tubes. We also evaluated delivery efficiency in male germ cells in the pollen by expression of the introduced fluorescent marker. Mutations were detected in the target gene in the genomic DNA extracted from CRISPR/Cas9 introduced pollen tubes but were not detected in the negative control. Bombarded pollen germinated pollen tubes on the stigma and produced two sperm cells within the pistil. We also observed ovules showing fluorescence derived from bombarded pollen. The findings of this study provide important insights into the editing of pollen tube genomes and the delivery of genome-modified male germ cells for seed production.

plant biology↗

bioRxiv2021

Pilot study: Trehalose-induced remodelling of the human microbiota affects Clostridioides difficile infection outcome in an in vitro colonic model

Within the human intestinal tract, dietary, microbial- and host-derived compounds are used as signals by many pathogenic organisms, including Clostridioides difficile. Trehalose has been reported to enhance virulence of certain C. difficile ribotypes; however, such variants are widespread and not correlated with clinical outcomes for patients suffering from C. difficile infection (CDI). Here, we make preliminary observations to how to trehalose supplementation affects the microbiota in an in vitro model and show that trehalose can reduce the outgrowth of C. difficile, preventing simulated CDI. Three clinically reflective human gut models simulated the effects of sugar (trehalose or glucose) or saline ingestion on the microbiota. Models were instilled with sugar or saline and further exposed to C. difficile spores. The recovery of the microbiota following antibiotic treatment and CDI induction was monitored in each model. The human microbiota remodeled to utilise the bioavailable trehalose. Clindamycin induction caused simulated CDI in models supplemented with either glucose or saline; however, trehalose supplementation did not result in CDI, although limited spore germination did occur. The absence of CDI in trehalose model was associated with enhanced abundances of Finegoldia, Faecalibacterium and Oscillospira, and reduced abundances of Klebsiella and Clostridium spp., compared with the other models. Functional analysis of the microbiota in the trehalose model revealed differences in the metabolic pathways, such as amino acid metabolism, which could be attributed to prevention of CDI. Our data show that trehalose supplementation remodelled the microbiota, which prevented simulated CDI, potentially due to enhanced recovery of nutritionally competitive microbiota against C. difficile.

microbiology↗

bioRxiv2020

Defective Repression of OLE3::LUC 1 (DROL1) is specifically required for the splicing of AT-AC-type introns in Arabidopsis

An Arabidopsis mutant named defective repression of OLE3::LUC 1 (drol1) was originally isolated as a mutant with defects in the repression of OLEOSIN3 (OLE3) after seed germination. In this study, we show that DROL1 is an Arabidopsis homolog of yeast DIB1, a subunit of U5 snRNP in the spliceosome. It is also part of a new subfamily that is specific to a certain class of eukaryotes. Comprehensive analysis of the intron splicing using RNA-Seq analysis of the drol1 mutants revealed that most of the minor introns with AT-AC dinucleotide termini had reduced levels of splicing. Only two nucleotide substitutions from AT-AC to GT-AG enabled AT-AC-type introns to be spliced in drol1 mutants. Forty-eight genes, including those having important roles in abiotic stress responses and cell proliferation, exhibited reduced splicing of AT-AC-type introns in the drol1 mutants. Additionally, drol1 mutant seedlings showed retarded growth, similar to that caused by the activation of abscisic acid signaling, possibly as a result of reduced AT-AC-type intron splicing in the endosomal Na+/H+ antiporters and plant-specific histone deacetylases. These results indicate that DROL1 is specifically involved in the splicing of minor introns with AT-AC termini, and that this plays an important role in plant growth and development. Significance statementDefective Repression of OLE3::LUC 1 (DROL1) is a homolog of yeast DIB1, which is a subunit of the U5 snRNP in the spliceosome, but is also part of a new subfamily that is specific to a certain class of eukaryotes. Using RNA-Seq we show that introns with AT-AC dinucleotide termini were specifically retained in the transcriptome of drol1 mutants and that their splicing plays an important role in plant growth and development.

molecular biology↗

bioRxiv2020

Dynamics of the cell fate specifications during female gametophyte development in Arabidopsis

The female gametophytes of angiosperms contain cells with distinct functions, such as those that enable reproduction via pollen tube attraction and fertilization. Although the female gametophyte undergoes unique developmental processes, such as several rounds of nuclear division without cell plate formation, and the final cellularization, it remains unknown when and how the cell fate is determined during their development. Here, we visualized the living dynamics of female gametophyte development and performed transcriptome analysis of its individual cell types, to assess the cell fate specifications in Arabidopsis thaliana. We recorded time lapses of the nuclear dynamics and cell plate formation from the one-nucleate stage to the seven-cell stage after cellularization, using the in vitro ovule culture system. The movies showed that the nuclear division occurred along the micropylar-chalazal axis. During cellularization, the polar nuclei migrated while associating with forming edge of the cell plate. Then, each polar nucleus migrated to fuse linearly towards each other. We also tracked the gene expression dynamics and identified that the expression of the MYB98pro::GFP, a synergid-specific marker, was initiated before cellularization, and then restricted to the synergid cells after cellularization. This indicated that cell fates are determined immediately after cellularization. Transcriptome analysis of the female gametophyte cells of the wild type and myb98 mutant, revealed that the myb98 synergid cells had the egg cell-like gene expression profile. Although in the myb98, the egg cell-specific gene expressions were properly initiated only in the egg cells after cellularization, but subsequently expressed ectopically in one of the two synergid cells. These results, together with the various initiation timings of the egg cell-specific genes suggest the complex regulation of the individual gametophyte cells, such as cellularization-triggered fate initiation, MYB98-dependent fate maintenance, cell morphogenesis, and organelle positioning. Our system of live-cell imaging and cell-type-specific gene expression analysis provides insights into the dynamics and mechanisms of cell fate specifications in the development of female gametophytes in plants.

plant biology↗

bioRxiv2020

Cell-cell adhesion in plant grafting is facilitated by β-1,4-glucanases

Plant grafting is conducted for vegetative propagation in plants, whereby a piece of living tissue is attached to another tissue through establishment of cell-cell adhesion. Plant grafting has a long history in agriculture and has been applied to improve crop traits for thousands of years1. Plant grafting has mostly relied on the natural ability of a plant for wound healing. However, the compatibility of cell-cell adhesion typically limits graft combinations to closely related species2-4, and the mechanism by which cell-cell adhesion of injured tissues is established is largely unknown. Here, we show that a subclade of {beta}-1,4-glucanases secreted into the extracellular region facilitates cell-cell adhesion near the graft interface. Nicotiana shows a propensity for cell-cell adhesion with a diverse range of angiosperms, including vegetables, fruit trees, and monocots, in which cell wall reconstruction was promoted in a similar manner to conventional intrafamily grafting5-7. Using transcriptomic approaches, we identified a specific clade of {beta}-1,4-glucanases that is upregulated during grafting in successful graft combinations but not in incompatible grafts and precedes graft adhesion in inter- and intrafamily grafts. Grafting was facilitated with an overexpressor of the {beta}-1,4-glucanase and, using Nicotiana stem as an interscion, we produced tomato fruits on rootstocks from other plant families. Our results demonstrate that the mechanism of cell-cell adhesion is partly conserved in plants and is a potential target to enhance plant grafting techniques.

plant biology↗

bioRxiv2019

Micrografting device for testing environmental conditions for grafting and systemic signaling in Arabidopsis

SummaryGrafting techniques have been applied in studies of systemic, long-distance signaling in several model plants. Seedling grafting in Arabidopsis, known as micrografting, enables investigation of the molecular mechanisms of systemic signaling between shoots and roots. However, conventional micrografting requires a high level of skill, limiting its use. Thus, an easier user-friendly method is needed. Here, we developed a silicone microscaled device, the micrografting chip, to obviate the need for training and to generate less stressed and more uniformly grafted seedlings. The chip has tandemly arrayed units, each of which consists of a seed pocket for seed germination and a micro-path with pairs of pillars for hypocotyl holding. Grafting, including seed germination, micrografting manipulation, and establishment of tissue reunion, is performed on the chip. Using the micrografting chip, we evaluated the effect of temperature and the carbon source on grafting and showed that a temperature of 27{degrees}C and a sucrose concentration of 0.5% were optimal. We also used the chip to investigate the mechanism of systemic signaling of iron status using a quadruple nicotianamine synthase (nas) mutant. The constitutive iron-deficiency response in the nas mutant because of aberrant partitioning was significantly rescued by grafting of wild-type shoots or roots, suggesting that shoot-and root-ward translocation of nicotianamine-iron complexes is essential for iron mobilization. Thus, our micrografting chip will promote studies of long-distance signaling in plants. Significance StatementA number of micrografting studies on systemic, long-distance signaling have been performed, but the technique is not yet used widely. Here, we developed a silicone-based micrografting chip to improve the ease-of-use, efficiency, and success rate of micrografting, even for untrained users.

plant biology↗

bioRxiv2019

KAKU4-mediated deformation of the vegetative nucleus controls its precedent migration over sperm cells in pollen tubes

A putative nuclear lamina protein, KAKU4, modulates nuclear morphology in Arabidopsis thaliana seedlings but its physiological significance is unknown. KAKU4 was strongly expressed in mature pollen grains, each of which has a vegetative cell and two sperm cells. KAKU4 protein was highly abundant on the envelopes of vegetative nuclei (VNs) and less abundant on the envelopes of sperm cell nuclei (SCNs) in pollen grains and elongating pollen tubes. VN is irregularly shaped in wild-type pollen. However, KAKU4 deficiency caused it to become more spherical. These results suggest that the dense accumulation of KAKU4 is responsible for the irregular shape of the VNs. After a pollen grain germinates, the VN and SCNs migrate to the tip of the pollen tube. In the wild type, the VN preceded the SCNs in 91-93% of the pollen tubes, whereas in kaku4 mutants, the VN trailed the SCNs in 39-58% of the pollen tubes. kaku4 pollen was less competitive than wild-type pollen after pollination, although it had an ability to fertilize. Taken together, our results suggest that controlling the nuclear shape in vegetative cells of pollen grains by KAKU4 ensures the orderly migration of the VN and sperm cells in pollen tubes.\n\nHighlightThe nuclear envelope protein KAKU4 is involved in controlling the migration order of vegetative nuclei and sperm cells in pollen tubes, affecting the competitive ability of pollen for fertilization.

plant biology↗

bioRxiv2019

A peptide pair coordinates regular ovule initiation patterns with seed number and fruit size

Ovule development in Arabidopsis thaliana involves pattern formation which ensures that ovules are regularly arranged in the pistils to reduce competition for nutrients and space. Mechanisms underlying pattern formation in plants, such as phyllotaxis, flower morphogenesis or lateral root initiation, have been extensively studied, and genes controlling the initiation of ovules have been identified. However, how a regular spacing of ovules is achieved is not known. Using natural variation analysis combined with quantitative trait locus analysis, we found that the spacing of ovules in the developing fruits is controlled by two secreted peptides, EPFL2 and EPFL9 (also known as Stomagen), and their receptors from the ERECTA (ER) family that act from the carpel wall and the placental tissue. We found that a signalling pathway controlled by EPFL9 acting from the carpel wall through the LRR-receptor kinases ER, ERL1 and ERL2 promotes fruit growth. Regular spacing of ovules depends on EPFL2 expression in the carpel wall and in the inter-ovule spaces, where it acts through ERL1 and ERL2. Loss of EPFL2 signalling results in shorter fruits and irregular spacing of ovules or even ovule twinning. The EPFL2 expression pattern between ovules is under negative-feedback regulation by auxin, which accumulates in the arising ovule primordia. We propose that the auxin-EPFL2 signalling module evolved to control the initiation and regular, equidistant spacing of ovule primordia, which serves to minimise competition between developing seeds. Together, EPFL2 and EPFL9 coordinate ovule patterning and thereby seed number with fruit growth through a set of shared receptors.

plant biology↗

bioRxiv2019

Presynaptic MAST Kinase Controls Bidirectional Post-SynapticResponses to Convey Stimulus Valence in C. elegans

Presynaptic plasticity is known to modulate the strength of synaptic transmission. However, it remains unknown whether regulation in presynaptic neurons alters the directionality -positive or negative-of postsynaptic responses. We report here that the C. elegans homologs of MAST kinase, Stomatin and Diacylglycerol kinase act in a thermosensory neuron to elicit in its postsynaptic neuron an excitatory or inhibitory response that correlates with the valence of thermal stimuli. By monitoring neural activity of the valence-coding interneuron in freely behaving animals, we show that the alteration between excitatory and inhibitory responses of the interneuron is mediated by controlling the balance of two opposing signals released from the presynaptic neuron. These alternative transmissions further generate opposing behavioral outputs necessary for the navigation on thermal gradients. Our findings reveal the previously unrecognized capability of presynaptic regulation to evoke bidirectional postsynaptic responses and suggest a molecular mechanism of determining stimulus valence.

neuroscience↗