Biology subjects

Inagaki, S.

Publications and source records attributed to Inagaki, S..

At least 19 recordsLinked to original sources

bioRxiv2026

Direct observation of feeding behavior in the kleptoplastic sea slug Plakobranchus ocellatus type black (Gastropoda, Sacoglossa)

The feeding ecology of sacoglossans, an exceptionally stenophagous group among marine animals, has been extensively studied because these organisms exhibit kleptoplasty--a phenomenon in which chloroplasts derived from ingested algae are sequestered and functionally maintained within animal cells. The genus Plakobranchus is notable for its exceptional capacity to retain photosynthetically active chloroplasts for prolonged periods. Nevertheless, direct observations of its feeding behavior have remained limited, largely due to subsequent taxonomic revisions and its cryptic, benthic lifestyle. In the present study, we focus on Plakobranchus ocellatus type black (PoB). Using high-resolution macro videography, we obtained, for the first time, video recordings that directly document the feeding behavior of this lineage. Under illuminated conditions, we demonstrate active feeding on the fan-shaped fronds of udoteacean algae, clarify which algal life-history stages serve as food, and identify Ventalia sp. 4 as a previously unrecognized dietary component. We further describe a characteristic inverted "headstand" posture adopted during feeding and discuss this behavior in relation to the species benthic and semi-burrowing lifestyle, proposing that it may partly explain the historical rarity of feeding observations in the field. The confirmation of feeding behavior represents a critical step toward establishing a stable laboratory culture system essential for experimental studies of kleptoplasty.

animal behavior and cognition↗

bioRxiv2026

The circadian clock gates lateral root development

Lateral root (LR) formation remodels root architecture, yet how temporal information integrates with hormonal cues remains unclear. We show that the circadian clock component EARLY FLOWERING 3 (ELF3) acts as a temporal gatekeeper of root organogenesis in Arabidopsis thaliana. Time-resolved transcriptomics, imaging, and genetics reveal that ELF3 restrains hormone-induced pericycle proliferation by maintaining rhythmic expression of key regulators under callus-inducing conditions. Loss of ELF3 disrupts rhythmic gene expression, enhancing callus growth and accelerating LR development. ELF3 functions through its target LNK1 to regulate the MADS-box genes AGL14 and AGL20. Notably, callus-inducing signals partially restore rhythmicity in elf3 mutants, revealing feedback from hormonal signaling to the circadian system. These findings identify ELF3 as an integrator of circadian and hormonal inputs that temporally constrains root developmental plasticity.

plant biology↗

bioRxiv2025

Sexual life cycle establishes the unicellular red algae Cyanidiophyceae as a genetically tractable model lineage for eukaryotic evolution

The thermo-acidophilic unicellular algal class Cyanidiophyceae is the earliest-branching lineage in red algae, which diverged from Viridiplantae lineage (green algae and land plants) soon after chloroplast establishment in the common ancestor of Archaeplastida. Cyanidiophyceae possess extremely simple genomes (8.7-17.8 Mb; approximately 4,800- 7,800 genes), and the cell-wall-less, genetically tractable strain Cyanidioschyzon merolae 10D has served as a model organism. However, its unknown sexual life cycle has limited its utility in studies of evolution and genetics. Inspired by the recent discovery of sexual reproduction in the cyanidiophycean genus Galdieria, we identified similar life cycles in the other cyanidiophycean genera Cyanidioschyzon, Cyanidiococcus, and Cyanidium. In these genera, the cell-walled diploid form, exclusively observed in nature, produces a cell-wall-less haploid form when the culture pH is lowered, and both proliferate asexually. In addition, the cell-wall-less Cyanidioschyzon merolae 10D strain has been shown to be a haploid clone that forms a cell-walled diploid through mating with other haploid clones. Building on these findings, we generated high-quality genomic resources with phase-specific transcriptomes and developed genetic manipulation systems using the cell-wall-less haploids of these genera. We further uncovered phase-specific distribution of histone H3 lysine 27 trimethylation linked to haploid- and diploid-specific gene expression, including transcription factors involved in differentiation associated with sexual reproduction in plants. Additionally, biparental inheritance of organelle DNA occurs following isogamous mating of haploid cells but resolves into uniparental inheritance during diploid proliferation. These advances position Cyanidiophyceae as a powerful model lineage for studying early Archaeplastida evolution, the shared mechanisms of photosynthetic eukaryotes, and their environmental adaptation.

plant biology↗

bioRxiv2025

MBD8 is required for LDL2-mediated transcriptional repression downstream of H3K9me2 in Arabidopsis

Histone H3 lysine 9 methylation (H3K9me) is a conserved epigenetic mark for transcriptional gene repression. In Arabidopsis, H3K9 dimethylation (H3K9me2) promotes the removal of a transcription-associated mark, H3K4 monomethylation (H3K4me1), and this process within genes requires the histone demethylase LDL2. However, how LDL2 specifically functions in genes that accumulate H3K9me2 remains unknown. Here, we show that methyl-CpG-binding domain protein 8 (MBD8) binds to LDL2 and plays a role in H3K4me1 removal and transcriptional gene repression downstream of H3K9me2. MBD8 preferentially binds GC-poor DNA, regardless of its cytosine methylation status. Interestingly, LDL2 protein levels were significantly reduced in the mbd8 mutant background, suggesting that MBD8 stabilizes the LDL2 protein, facilitating H3K4me1 removal and the transcriptional repression of H3K9me-marked genes. The domain necessary for the MBD8-LDL2 interaction is conserved among plants. We propose that MBD proteins have diverse functions beyond their methylated DNA-binding capabilities.

plant biology↗

bioRxiv2025

WIND1 controls cell fate transition through histone acetylation and deacetylation during somatic embryogenesis

Regeneration involves large-scale transcriptional reprogramming to drive cell identity transitions. These transcriptional changes are tightly coupled with chromatin remodelling but molecular mechanisms that coordinate these changes remain unclear. Here we show that WOUND INDUCED DEDIFFERENTIATION 1 (WIND1) transcription factor promotes somatic embryogenesis by repressing pre-existing cell fate and activating new cell identity programmes. WIND1 interacts with histone deacetylase HISTONE DEACETYLASE 9 (HDA9) and histone acetyltransferase complex component HOMOLOG OF YEAST ADA1 2a (ADA2a) via conserved N-terminal domain. These interactions enable WIND1 to mediate both H3K27 deacetylation and acetylation at distinct target loci, leading to repression of shoot identity genes such as AINTEGUMENTA (ANT) and activation of embryogenesis regulators including LEAFY COTYLEDON 2 (LEC2). Our findings identify WIND1 as a bifunctional chromatin regulator that integrates opposing histone acetylation dynamics to coordinate transcriptional reprogramming. This mechanism provides a molecular framework for how a transcription factor directs complex cell fate transitions during regeneration

plant biology↗

bioRxiv2025

H3K4me2 orchestrates H2A.Z and Polycomb repressive marks in Arabidopsis

The dimethylation of histone H3 lysine 4 (H3K4me2) plays an important role in developmental phase transitions in plants, such as regeneration from the callus and the initiation of flowering. H3K4me2 in plants is correlated with transcriptionally repressed states, which can be accounted for by transcription-coupled active H3K4me2 demethylation, but the converse molecular pathway by which H3K4me2 represses transcription remains largely unexplored. Here, we show that H3K4me2 colocalizes with the facultatively repressive marks H2A.Z, H2A ubiquitination (H2Aub), and H3K27me3 in the gene body. Our genetic analyses reveal that H3K4me2 functions upstream but not downstream of these repressive marks. Interestingly, in genes with diurnal H3K4me2 oscillation, H3K4me2 oscillates in antiphase with transcription but in phase with H2A.Z. In addition, the genetic manipulation of H3K4me2 affects the oscillating profiles of H2A.Z, suggesting the efficient relay from H3K4me2 to H2A.Z. Notably, the diurnal oscillation of H2Aub is much weaker than that of H2A.Z despite the overall similarity in their distributions. These results suggest that H3K4me2 orchestrates H2A.Z and H2Aub with distinct dynamics. Our findings reveal that plants employ H3K4me2 to transmit transcriptional cessation into repressive chromatin states.

genetics↗

bioRxiv2024

Tartrazine cannot make live tissues transparent

Withdrawal StatementThe authors have withdrawn their manuscript because one of the conclusions was wrong. The authors sincerely apologize to Ou et al. and the research community for the confusion. In this preprint, the authors performed an abdominal skin clearing experiment following the methods section in Ou et al. (ref. 1) and observed only a marginal clearing effect. In this condition (Figure 1), the authors detected tartrazine only in the stratum corneum (SC) of the skin, where only dead cells are present (Figure 1E, F). However, following a more detailed protocol with additional tips kindly provided by Ou et al. (ref. 1), the abdominal skin became more transparent. It seems that mechanical damage to the SC barrier is critical for successful clearing. In this condition, the authors detected strong tartrazine signals under the SC, including the dermis where live cells are located. We are still concerned about the issue of osmolality and toxicity to live cells; cleared cells must be under hypertonic conditions (Figure 3 and 4). However, since Figure 1 (especially Figure 1E, F) no longer stands, and the title can be misinterpreted as if the original results (ref. 1) were irreproducible, the authors believe it is best to withdraw this preprint. Therefore, the authors do not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding author.

bioengineering↗

bioRxiv2024

Isotonic and minimally invasive optical clearing media for live cell imaging ex vivo and in vivo

Tissue clearing has been widely used for fluorescence imaging of fixed tissues, but not for live tissues due to its toxicity. Here we develop minimally invasive optical clearing media for fluorescence imaging of live mammalian tissues. Light scattering is minimized by adding spherical polymers with low osmolarity to the extracellular medium. A clearing medium containing bovine serum albumin (SeeDB-Live) is minimally invasive to live cells, allowing for structural and functional imaging of live tissues, such as spheroids, organoids, acute brain slices, and the mouse brain in vivo. SeeDB-Live minimally affects the electrophysiological properties and sensory responses of neurons. We demonstrate its utility for widefield imaging of subcellular voltage dynamics, such as backpropagating action potentials, in acute brain slices. We also utilize SeeDB-Live for widefield voltage imaging of dozens of dendrites in vivo, demonstrating population dynamics. Thus, SeeDB-Live expands the scale and modalities of fluorescence imaging of live mammalian tissues.

neuroscience↗

bioRxiv2024

Endoplasmic reticulum patterns insect cuticle nanostructure

Insect cuticles with nano-level structures exhibit functional surface properties such as structural color and superhydrophobicity. Despite the enormous influence the cuticle has had on biomimetic industrial applications, molecular and cellular mechanisms of cuticular extracellular matrix (ECM) assembly into nanoscale structures remain poorly understood in insects and other taxa. Ghiradella (1989) described how a crystallin-like lattice of endoplasmic reticulum (ER) prefigures the patterning of the porous cuticle of the butterfly wing scale with structural color1. Building on that insight, we show that the nanopore structure of the olfactory (olf) organs, which serve as molecular filters in Drosophila, is built through a novel process in which ER material is trafficked to the plasma membrane mediated by the autophagy pathway. The process is controlled by the insect-specific protein Gore-tex/Osiris23 (Gox)2, which is localized to the tubular ER of olf hair cells. Gox recruits Ref(2)P, the fly counterpart of mammalian p62/SQSTM13, to initiate ER scission. The excised ER fragments are processed by autophagy to gain access to the plasma membrane and trigger membrane invagination, which plays a role in remodeling the cuticular envelope layer to the nanopore formation. This repurposing of the ER phagy for machinery to support the fabrication of nanoscale ECM by the Gox protein sheds light on the nanopatterning of insect cuticles and their genetic control.

developmental biology↗

bioRxiv2024

Mechanical control of the insect extracellular matrix nanostructure

Nanoscale modifications of apical extracellular matrix (ECM) have created a variety of functional surfaces with distinct physical properties, as exemplified by structural coloration and superhydrophobicity in animals and plants1. While the surface nanostructures of various organisms have inspired numerous biomimetic applications, the biological mechanisms underlying ECM patterning at the nanoscale remain largely unknown. Here, we investigated the morphogenesis of cuticular pores in Drosophila olfactory organs2,3. Hundreds of uniform-sized nanopores of [~]50 nm permit selective access of odorant molecules to olfactory neurons. We showed that matrices composed of the zona pellucida domain (ZPD) protein4-6 cover sensory organs in cell type-specific patterns and combinations. The ZPD proteins Dyl, Tyn, Mey, and Nyo form matrices with specific mixing and sorting properties, restricting cell growth and movement. The disruption of ZPD matrices leads to detachment of the envelope layer of the cuticle from the plasma membrane, and reduced numbers and irregularly sized nanopores. Our results suggest that compressive force from the ECM is essential for robust nanopore morphogenesis. This work reveals a previously unrecognized role for ZPD proteins as modular units that establish the mechanical environment required to modulate the nanoscale assembly of cuticle materials, opening a new biological context to these biomimetically important structures.

developmental biology↗

bioRxiv2024

Osiris gene family defines the cuticle nano-patterns of Drosophila

Nanostructures of pores and protrusions in the insect cuticle modify molecular permeability and surface wetting and help insects sense a variety of environmental cues. The cellular mechanism specifying cuticle nanostructures is poorly understood. Here, we show that insect-specific Osiris family genes are expressed in various cuticle-secreting cells in the Drosophila head in the early stage of cuticle secretion and collectively cover nearly the entire surface of the head epidermis. We show that each sense organ cell with various cuticular nanostructures expresses a unique combination of Osiris genes. Osiris gene mutations caused various cuticle defects in the corneal nipples of the eye and pores of the chemosensory sensilla. Osiris genes provide an entry point for investigating cuticle nanopatterning in insects.

developmental biology↗

bioRxiv2023

H3K9me regulates heterochromatin silencing through incoherent feedforward loops

Histone H3 lysine-9 methylation (H3K9me) is associated with condensed and transcriptionally inactive heterochromatin1. Although it has long been known that H3K9me silences transcription to control a wide variety of biological phenomena in many eukaryotic species2,3, how the silencing is regulated under the control of H3K9me is still largely unclear. Moreover, how cells delimit regions with H3K9me to avoid silencing essential genes remains unexplored. Here, using Arabidopsis genetic systems that induce H3K9me2 and its associated non-CG DNA methylation (mCH) in the transcribed region of genes de novo, we show that the accumulation of H3K9me2/mCH paradoxically also leads to the deposition of the euchromatic mark H3K36me3. This induction of H3K36me3 depends on a SET domain methyltransferase, ASHH3, and brings about anti-silencing by preventing the demethylation of H3K4me1 by LDL2, which mediates transcriptional silencing downstream of H3K9me2/mCH4. H3K9me2/mCH-driven antagonistic actions of ASHH3-H3K36me3 and LDL2-H3K4me1-loss also regulate the de novo silencing of reactivated transposable elements (TEs). These results demonstrate that H3K9me2 both facilitates and impedes silencing, and the incoherent feedforward loops fine-tune the fate of genes and TEs. Our results illuminate a novel elaborate mechanism for partitioning chromatin domains and provide insights into the molecular basis underlying natural epigenetic variation.

genetics↗

bioRxiv2023

Cotranscriptional demethylation induces global loss of H3K4me2 from active genes in Arabidopsis

Based on studies of animals and yeasts, methylation of histone H3 lysine 4 (H3K4me1/2/3, for mono-, di-, and tri-methylation, respectively) is regarded as the key epigenetic modification of transcriptionally active genes. In plants, however, H3K4me2 correlates negatively with transcription, and the regulatory mechanisms of this counterintuitive H3K4me2 distribution in plants remain largely unexplored. A previous genetic screen for factors regulating plant regeneration identified Arabidopsis LYSINE-SPECIFIC DEMETHYLASE 1-LIKE 3 (LDL3), which is a major H3K4me2 demethylase. Here, we show that LDL3-mediated H3K4me2 demethylation depends on the transcription elongation factor Paf1C and phosphorylation of the C-terminal domain (CTD) of RNA polymerase II (RNAPII). In addition, LDL3 binds to phosphorylated RNAPII. These results suggest that LDL3 is recruited to transcribed genes by binding to elongating RNAPII and demethylates H3K4me2 cotranscriptionally. Importantly, the negative correlation between H3K4me2 and transcription is disrupted in the ldl3 mutant, demonstrating the genome-wide impacts of the transcription-driven LDL3 pathway to control H3K4me2 in plants. Our findings implicate H3K4me2 in plants as chromatin memory for transcriptionally repressive states, which ensures robust gene control.

plant biology↗

bioRxiv2022

Automated neuronal reconstruction with super-multicolour fluorescence imaging

Fluorescence imaging is widely used for the mesoscopic mapping of neuronal connectivity. However, neurite reconstruction is challenging, especially when neurons are densely labelled. Here we report a strategy for the fully automated reconstruction of densely labelled neuronal circuits. Firstly, we established stochastic "super-multicolour" labelling with up to seven different fluorescent proteins using the Tetbow method. With this method, each neuron was labelled with a unique combination of fluorescent proteins, which were then imaged and separated by linear unmixing. We also established an automated neurite reconstruction pipeline based on the quantitative analysis of multiple dyes (QDyeFinder). To classify colour combinations, we used a newly developed unsupervised clustering algorithm, dCrawler, in which data points in multi-dimensional space were clustered based on a given threshold distance. Our new strategy allows for the reconstruction of neurites for up to hundreds of neurons at a millimetre scale without manual tracing.

neuroscience↗

bioRxiv2022

Local induction of IAA5 and IAA29 promotes DNA damage-triggered stem cell death in Arabidopsis roots

Plants generate organs continuously during post-embryonic development, thus how to preserve stem cells in changing environments is crucial for their survival. Genotoxic stress threatens genome stability in all somatic cells, whereas, in the meristem, only stem cells undergo cell death in response to DNA damage, followed by stem cell replenishment. It was previously shown that inhibition of downward auxin flow in roots participates in DNA damage-induced stem cell death; however, how cell death is confined to stem cells in tissues with reduced auxin content remains elusive. Here we show that, in response to DNA double-strand breaks, the Aux/IAA family members IAA5 and IAA29, which encode the negative regulators of auxin signaling, were locally induced in vascular stem cells and their daughters in Arabidopsis roots. This is an active response to DNA damage in which the transcription factor SUPPRESSOR OF GAMMA RESPONSE 1 directly induces their expression. In the iaa5 iaa29 double mutant, DNA damage-induced stem cell death was significantly suppressed, while it was fully restored by the expression of a stable form of IAA5 in vascular stem cells. Our genetic data revealed that both the suppression of auxin signaling and reduced auxin content are prerequisite for cell death induction, suggesting the central role in maintaining genome integrity in stem cells. One-sentence summaryThe induction of IAA5 and IAA29 promotes stem cell death in Arabidopsis roots under DNA stress.

plant biology↗

bioRxiv2022

Repetitive DNA promotes RNAi-mediated heterochromatin formation via an anti-silencing factor in fission yeast.

In most eukaryotes, constitutive heterochromatin, defined by histone H3 lysine 9 methylation (H3K9me), is enriched on repetitive DNA, such as pericentromeric repeats and transposons. Furthermore, repetitive transgenes also induce heterochromatin formation in diverse model organisms. However, the mechanisms that promote heterochromatin formation at repetitive DNA elements are still not clear. Here, using fission yeast, we show that tandemly repeated mRNA genes promote RNA interference (RNAi)-mediated heterochromatin formation in co-operation with an anti-silencing factor, Epe1. Although the presence of tandemly repeated genes itself does not cause heterochromatin formation, once complementary small RNAs are artificially supplied in trans, the RNAi machinery assembled on the repeated genes starts producing cognate small RNAs in cis to autonomously maintain heterochromatin at these sites. The establishment of this "repeat-induced RNAi" depends on the copy number of repeated genes and also requires Epe1, which is known to remove H3K9me and derepress the transcription of genes underlying heterochromatin. Analogous to repeated genes, the DNA sequence underlying constitutive heterochromatin encodes widespread transcription start sites (TSSs), from which Epe1 activates ncRNA transcription to promote RNAi-mediated heterochromatin formation. Our results suggest that, when repetitive transcription units underlie heterochromatin, Epe1 generates sufficient transcripts for the activation of RNAi without disruption of heterochromatin.

biochemistry↗

bioRxiv2021

The mechanochemical origins of the microtubule sliding motility within the kinesin-5 domain organization

The conserved kinesin-5 bipolar tetrameric motors slide apart microtubules during mitotic spindle assembly and elongation. Kinesin-5 bipolar organization originates from its conserved tetrameric helical minifilament, which position the C-terminal tail domains of two subunits near the N-terminal motor domains of two anti-parallel subunits (Scholey et al, 2014). This unique tetrameric structure enables kinesin-5 to simultaneously engage two microtubules and transmit forces between them, and for multiple kinesin-5 motors to organize via tail to motor interactions during microtubule sliding (Bodrug et al, 2020). Here, we show how these two structural adaptations, the kinesin-5 tail-motor domain interactions and the length of the tetrameric minifilament, determine critical aspects of kinesin-5 motility and sliding mechanisms. An x-ray structure of the 34-nm kinesin-5 minifilament reveals how the dual dimeric N-terminal coiled-coils emerge from the tetrameric central bundle. Using this structure, we generated active bipolar mini-tetrameric motors from Drosophila and human orthologs, which are half the length of native kinesin-5. Using single-molecule motility assays, we show that kinesin-5 tail domains promote mini-tetramers static pauses that punctuate processive motility. During such pauses, kinesin-5 mini-tetramers form multi-motor clusters mediated via tail to motor domain cross-interactions. These clusters undergo slow and highly processive motility and accumulate at microtubule plus-ends. In contrast to native kinesin-5, mini-tetramers require tail domains to initiate microtubule crosslinking. Although mini-tetramers are highly strained in initially aligning microtubules, they slide microtubules more efficiently than native kinesin-5, due to their decreased minifilament flexibility. Our studies reveal that the conserved kinesin-5 motor-tail mediated clustering and the length of the tetrameric minifilament are key features for sliding motility and are critical in organizing microtubules during mitotic spindle assembly and elongation.

cell biology↗

bioRxiv2021

Transcription-coupled and epigenome-encoded mechanisms direct H3K4 methylation

Mono-, di-, and trimethylation of histone H3 lysine 4 (H3K4me1/2/3) are associated with transcription, yet it remains controversial whether H3K4me1/2/3 promote or result from transcription. Our previous characterizations of Arabidopsis H3K4 demethylases suggest roles for H3K4me1 in transcription. However, the control of H3K4me1 remains unexplored in Arabidopsis, in which no methylase for H3K4me1 has been identified. Here, we identified three Arabidopsis methylases that direct H3K4me1. Analyses of their genome-wide localization using ChIP-seq and machine learning revealed that one of the enzymes cooperates with the transcription machinery, while the other two are associated with specific histone modifications and DNA sequences. Importantly, these two types of localization patterns are also found for the other H3K4 methylases in Arabidopsis and mice. These results suggest that H3K4me1/2/3 are established and maintained via interplay with transcription as well as inputs from other chromatin features, presumably enabling elaborate gene control.

molecular biology↗