Biology subjects

Wada, H.

Publications and source records attributed to Wada, H..

16 recordsLinked to original sources

bioRxiv2026

Reconstitution in a minimal synthetic bacterium reveals distinct Mollicute MreB-based motility systems driven by orthogonal force directions

MreB, a bacterial actin homolog, is widely conserved in bacteria where it functions as a scaffold for a peptidoglycan synthesis. Intriguingly, members of the cell wall-lacking Mollicutes retain multiple mreB genes despite the absence of peptidoglycan. Previous work demonstrated that two MreB isoforms from Spiroplasma can reconstitute swimming motility in the minimal synthetic bacterium syn3B, indicating that MreB was repurposed for motility. Here, we expressed seven MreBs from Haloplasma contractile, an early-diverging Mollicute, in syn3B. Approximately 50% of cells elongated, and 15% exhibited curving and coiling movements that resembled Haloplasma but differed from Spiroplasma motility. Systematic analysis of isoform combinations revealed that movement requires specific MreB pairs, and that additional isoforms enhance motility efficiency. Cryo-electron tomography showed membrane-associated ribbons composed of MreB filaments. Mathematical modeling demonstrated that Haloplasma- and Spiroplasma-type movements can be reproduced by altering only the direction of force generation. Thus, diversification of force orientation within an actin homolog enabled independent evolution of distinct motility systems in Mollicutes.

Synthetic Biology↗

bioRxiv2025

Mimicking the night shift: Hypothalamic-pituitary-adrenal (HPA) axis regulation, energy homeostasis, and oxidative stress in a model diurnal songbird

Everchanging socioeconomic demands, coupled with modern day technological advancements have lifted constraints on human activity patterns, enabling us to take advantage of the 24-h period. However, varying patterns of light at night and sleep/wake may come at the cost of disrupting endogenous rhythms that govern biological processes and maintain homeostasis. To this end, we exposed zebra finches (Taeniopygia guttata castanotis), a diurnal songbird, to a consistent light/dark cycle of 12L:12D or alternating lighting patterns of 12D:12L and 12L:12D every three days for 66 days to mimic night shift work conditions. Surprisingly, we found that corticosterone profiles were inverted in birds exposed to simulated night shift work, with a slight trend indicating the inversion of melatonin profiles. Moreover, they had poor HPA axis reactivity in response to a standardized capture and restraint challenge and a significant reduction in corticosterone levels at ZT0 after simulated night shift work. Yet, we did not detect changes in body mass, subcutaneous fat deposits, or 4-hydroxynonaneal damage in the brain or liver. Our study underscores the need to capitalize on unique model organisms to evaluate consequences and resilience of simulated night shift work to inform public health decisions and the role of circadian plasticity.

physiology↗

bioRxiv2025

Bacteria break through one-micrometer-square passages by flagellar wrapping

Confined spaces are omnipresent in the micro-environments, including soil aggregates and intestinal crypts, yet little is known about how bacteria behave under such conditions where movement is challenging due to limited diffusion. Stinkbug symbiont Caballeronia insecticola navigates a narrow gut passage about one micrometer in diameter to reach the stinkbugs symbiotic organ. Here, we developed a microfluidic device mimicking the hosts sorting organ, wherein bacterial cells are confined in a quasi-one-dimensional fashion, and revealed that this bacterium wraps flagellar filaments around its cell body like a screw thread to control fluid flow and generate propulsion for smooth and directional movement in narrow passages. Physical simulations and genetic experiments revealed that hook flexibility is essential for this wrapping; increasing hook rigidity impaired both wrapping motility and infectivity. Thus, flagellar wrapping likely represents an evolutionary innovation, enabling bacteria to break through confined environments using their motility machinery.

microbiology↗

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

Early development of the mineralized external skeleton of the polyplacophoran mollusk, with insight into the evolutionary history of shell plates and spicules.

Recent molecular phylogenetic studies have raised two questions about the evolutionary history of the calcified exoskeleton of mollusks. The first question concerns the homology of the two types of skeleton; whether spicules and shell plates share an evolutionary origin. The second question is the homology of the shell plates between chitons and other mollusks, including gastropods and bivalves. To gain insight into these questions, we examined the early development of shell plates and spicules in chitons. We identified several developmental genes that are involved in both shell plates and spicules, suggesting that spicules and shell plates share a common evolutionary origin. We also found that subpopulations of the dorsal shell field (the ridge and the plate field) have specific gene expression profiles. The differential gene expression of the ridge and plate field is not identical to the profiles of the zones of the gastropod shell field. This observation may suggest an independent evolutionary origin of the shell plates in chitons and gastropods.

evolutionary biology↗

bioRxiv2024

Development of shell field populations in gastropods

The embryonic shell field of mollusks first appears during gastrulation of the dorsal ectoderm and subsequently develops into the shell-secreting mantle in adult animals. Although several lines of evidence have revealed that this shell field lineage is exclusively derived from the second quartet (2q) of the 16-cell embryos, it is generally believed that the establishment of the shell field fate would be accomplished only after receiving inductive signals from the invaginated endoderm. Despite being accepted as a comprehensive model for molluskan shell field specification, the validity of this induction hypothesis remains questionable owing to the lack of clear experimental evidence and contradictory results. Here, we attempted to re-investigate the inductive role of the endoderm in shell field fate establishment in the limpet Nipponacmea fuscoviridis by experimentally disrupting cell-cell contacts between cell lineages after the 16-cell stage. First, we characterized the shell field cell population by performing two-color in situ hybridization. We characterized at least three cell populations in the developing shell field. Using single-cell transcriptome analysis, we identified several specific effector genes for each population, as well as transcription factor genes. Differentiation of each shell field population was inspected in 2q blastomeres isolated from other cells of the 16-cell embryos. Despite the absence of any interlineage interactions (including ectoderm-endoderm contacts), the expression of marker genes for each shell field population was observed in the isolated 2q fragments. In addition, the expression of several shell field genes was detected in embryos in which cytokinesis was blocked at the 16-cell stage. We concluded that the early process of shell field differentiation in the 2q lineage occurs mostly independently of the interactions with other lineages.

developmental biology↗

bioRxiv2023

Plastid anionic lipids are required for membrane development and protochlorophyllide synthesis in etioplasts

Dark-germinated angiosperms develop the chloroplast precursors called etioplasts in cotyledon cells. Etioplasts develop lattice membrane structures called prolamellar bodies (PLBs), where the chlorophyll intermediate protochlorophyllide (Pchlide) forms a ternary complex with NADPH and light-dependent NADPH-Pchlide oxidoreductase (LPOR). The lipid bilayers of etioplast membranes are mainly composed of galactolipids, which play important roles in membrane-associated processes in etioplasts. Although etioplast membranes also contain two anionic lipids, phosphatidylglycerol (PG) and sulfoquinovosyldiacylglycerol (SQDG), the roles of these anionic lipids are unknown. To reveal the importance of PG and SQDG for the development of etioplasts, we characterized etiolated Arabidopsis mutants deficient in the biosynthesis of PG and SQDG. A partial deficiency in PG biosynthesis loosened the lattice structure of PLBs and impaired the insertion of Mg2+ into protoporphyrin IX, leading to a significant decrease in Pchlide content. Although a complete lack of SQDG biosynthesis did not notably affect both PLB formation and Pchlide biosynthesis, the lack of SQDG in addition to the partial deficiency of PG caused strong impairments of these processes. The results suggested that PG is required for PLB formation and Pchlide biosynthesis, whereas SQDG plays an auxiliary role in these processes. Notably, the PG deficiency and the lack of SQDG oppositely affected the dynamics of LPOR complexes after photoconversion, suggesting different involvements of PG and SQDG in the organization of LPOR complexes. Our data demonstrate pleiotropic roles of anionic lipids in etioplast development.

plant biology↗

bioRxiv2022

Novel Escherichia coli genetic markers for tracking domestic wastewater contamination in environmental waters

Escherichia coli has been used as an indicator of fecal pollution in environmental waters. However, its presence in environmental waters does not provide information on the source of water pollution. Identifying the source of water pollution is paramount to be able to effectively reduce contamination. The present study aimed to identify E. coli microbial source tracking (MST) markers that can be used to identify domestic wastewater contamination in environmental waters. We first analyzed wastewater E. coli genomes sequenced by us (n = 50) and RefSeq animal E. coli genomes of fecal origin (n = 82), and identified 144 candidate wastewater-associated marker genes. The sensitivity and specificity of the candidate marker genes were then assessed by screening the genes in 335 RefSeq wastewater E. coli genomes and 3,318 RefSeq animal E. coli genomes. We finally identified two MST markers, namely W_nqrC and W_clsA_2, which could be used for detection of wastewater-associated E. coli isolates. These two markers showed higher performance than the previously developed human wastewater-associated E. coli markers H8 and H12. When used in combination, W_nqrC and W_clsA_2 showed specificity of 98.9% and sensitivity of 25.7%. PCR assays to detect W_nqrC and W_clsA_2 were also developed and validated. The developed PCR assays are potentially useful for detecting E. coli isolates of wastewater origin in environmental waters, though users should keep in mind that the sensitivity of these markers is not high. Further studies are needed to assess the applicability of the developed markers to a culture-independent approach.

microbiology↗

bioRxiv2021

Phosphatidylglycerol synthesis facilitates plastid gene expression and light induction of nuclear photosynthetic genes

Phosphatidylglycerol (PG) is the only major phospholipid in the thylakoid membrane of chloroplasts. PG is essential for photosynthesis and loss of PG in Arabidopsis thaliana results in severe defects of growth and chloroplast development with decreased chlorophyll accumulation, impaired thylakoid formation, and downregulation of photosynthesis-associated genes encoded in nuclear and plastid genomes. However, how the absence of PG affects the gene expression and plant growth remains unclear. To elucidate this mechanism, we investigated the growth and transcriptional profiles of a PG-deficient Arabidopsis mutant pgp1-2 under various light conditions. Microarray analysis demonstrated that reactive oxygen species-responsive genes were upregulated in pgp1-2. Decreased growth light did not alleviated the impaired leaf development and the downregulation of photosynthesis-associated genes in pgp1-2, indicating limited impacts of photooxidative stress on the defects of pgp1-2. Illumination to dark-adapted pgp1-2 triggered downregulation of photosynthesis-associated nuclear-encoded genes (PhANGs), while plastid-encoded genes were constantly suppressed. Overexpression of GOLDEN2-LIKE1 (GLK1), a transcription factor regulating chloroplast development, in pgp1-2 upregulated PhANGs but not plastid-encoded genes along with chlorophyll accumulation. Our data suggest a broad impact of PG biosynthesis on nuclear-encoded genes partially via GLK1 and a specific involvement of this lipid in the plastid gene expression and plant development.

plant biology↗

bioRxiv2019

Minimally invasive, pressure probe based sampling allows for in-situ gene expression analyses in plant cells.

BackgroundGene expression analyses are conducted using multiple approaches and increasingly research has been focused on assessing gene expression at the level of a tissue or even single-cells. To date, methods to assess gene expression at the single-cell in plant tissues have been semi-quantitative, require tissue disruption, and/or involve laborious, possibly artifact-inducing manipulation. In this work, we used grape berries (Vitis vinifera L. Zinfandel) as a model in order to examine the validity and reproducibility of an in-situ gene expression analysis method combining a cell pressure probe (CPP) with quantitative PCR (qPCR).\n\nResultsWe developed a method to directly assess gene expression levels via qPCR from cellular fluids sampled in-situ with a CPP. Cellular fluids, with volumes in the picoliter range, were collected from intact berries with a CPP at various depths across skin and mesocarp tissues. The expression of a key anthocyanin biosynthetic gene, UDP-glucose: flavonoid 3-O-glucosyltransferase (VviUFGT), was analyzed as a test case since its expression is restricted to cells producing anthocyanins in grape berry skins during ripening. The method identifies samples contaminated with significant levels of genomic DNA by amplifying a region of VviUFGT that spans an intron. Therefore false positives were discarded which occurred in 28% of the samples tested. Shallow probing of skin cells showed high VviUFGT expression as expected while deeper probing of mesocarp cells resulted in no VviUFGT expression.\n\nConclusionsThe clear correspondence of VviUFGT expression to the targeted cell samples suggests that the in-situ gene expression analysis using a CPP is reliable and does not result in contamination as the probe moves through tissues. This method can be paired to single-cell transcriptomic analyses in the future. We conclude that this technique represents a minimally invasive method of sampling plant cells in-situ which creates an opportunity for the analysis of cellular level, spatiotemporal responses in heterogeneous plant tissues.

plant biology↗

bioRxiv2019

Comprehensive epigenome characterization reveals diverse transcriptional regulation across human vascular endothelial cells

BackgroundEndothelial cells (ECs) make up the innermost layer throughout the entire vasculature. Their phenotypes and physiological functions are initially regulated by developmental signals and extracellular stimuli. The underlying molecular mechanisms responsible for the diverse phenotypes of ECs from different organs are not well understood.\n\nResultsTo characterize the transcriptomic and epigenomic landscape in the vascular system, we cataloged gene expression and active histone marks in nine types of human ECs (generating 148 genome-wide datasets) and carried out a comprehensive analysis with chromatin interaction data. We identified 3,765 EC-specific enhancers, some of which were associated with disease-associated genetic variations. We also identified various candidate marker genes for each EC type. Notably, reflecting the developmental origins of ECs and their roles in angiogenesis, vasculogenesis and wound healing.\n\nConclusionsWhile the importance of several HOX genes for early vascular development and adult angiogenesis in pathological conditions has been reported, a systematic analysis of the regulation and roles of HOX genes in mature tissue cells has been lacking. These datasets provide a valuable resource for understanding the vascular system and associated diseases.

genomics↗

bioRxiv2018

Trynity controls epidermal barrier function and respiratory tube maturation in Drosophila by modulating apical extracellular matrix nano-patterning

The outer surface of insects is covered by the cuticle, which is derived from the apical extracellular matrix (aECM). The aECM is secreted by epidermal cells during embryogenesis. The aECM exhibits large variations in structure, function, and constituent molecules, reflecting the enormous diversity in insect appearances. To investigate the molecular principles of aECM organization and function, here we studied the role of a conserved aECM protein, the ZP domain protein Trynity, in Drosophila melanogaster. We first identified trynity as an essential gene for epidermal barrier function. trynity mutation caused disintegration of the outermost envelope layer of the cuticle, resulting in small- molecule leakage and in growth and molting defects. In addition, the tracheal tubules of trynity mutants showed defects in pore-like structures of the cuticle, and the mutant tracheal cells failed to absorb luminal proteins and liquid. Our findings indicated that trynity plays essential roles in organizing nano-level structures in the envelope layer of the cuticle that both restrict molecular trafficking through the epidermis and promote the massive absorption pulse in the trachea.\n\nSummary StatementThe zona pellucida domain protein Trynity controls the structural organization and function of the apical extracellular matrix in the epidermis and trachea of Drosophila.

developmental biology↗

bioRxiv2018

De Novo assembly of the goldfish (Carassius auratus) genome and the evolution of genes after whole genome duplication

For over a thousand years throughout Asia, the common goldfish (Carassius auratus) was raised for both food and as an ornamental pet. Selective breeding over more than 500 years has created a wide array of body and pigmentation variation particularly valued by ornamental fish enthusiasts. As a very close relative of the common carp (Cyprinus carpio), goldfish shares the recent genome duplication that occurred approximately 14-16 million years ago (mya) in their common ancestor. The combination of centuries of breeding and a wide array of interesting body morphologies is an exciting opportunity to link genotype to phenotype as well as understanding the dynamics of genome evolution and speciation. Here we generated a high-quality draft sequence of a \"Wakin\" goldfish using 71X PacBio long-reads. We identified 70,324 coding genes and more than 11,000 non-coding transcripts. We found that the two sub-genomes in goldfish retained extensive synteny and collinearity between goldfish and zebrafish. However, \"ohnologous\" genes were lost quickly after the carp whole-genome duplication, and the expression of 30% of the retained duplicated gene diverged significantly across seven tissues sampled. Loss of sequence identity and/or exons determined the divergence of the expression across all tissues, while loss of conserved, non-coding elements determined expression variance between different tissues. This draft assembly also provides an important resource for comparative genomics with the very commonly used zebrafish model (Danio rerio), and for understanding the underlying genetic causes of goldfish variants.

genomics↗

bioRxiv2018

Endoderm Morphogenesis Reveals Integration of Distinct Processes in the Development and Evolution of Pharyngeal Arches

The vertebrate pharyngeal arches (PAs) are established by a combination of two styles of segmentation; the most anterior 2 PAs are simultaneously but the others are sequentially formed. However, the mechanism underlying their coexistence is unclear. Here, we show that the simultaneous and sequential segmentation discretely proceeded, respectively, but were finally integrated at the second PP (PP2), by dynamic morphogenesis of pharyngeal endoderm in the zebrafish. The coordination of these 2 distinct processes appears to be common in the PA development of many vertebrates, in which specific developmental defects posterior to the PP2 are caused by mutations of particular genes or perturbation of retinoic acid signaling. Surprisingly, comparative analysis of PA segmentation showed that the combinatorial styles of PA development is present in shark but not in lamprey, suggesting that PA segmentation was modified in the stem gnathostomes corresponding to the drastic pharyngeal innovations, such as PA2-derived opercular.

developmental biology↗

bioRxiv2017

Multiple strategies for heat adaptation in rice endosperms revealed by on-site cell-specific analysis

Plant cells have multiple strategies to adapt to environmental stresses. Rice endosperms form chalkiness in a part of the tissue under heat conditions during the grain-filling stage, although nitrogen supply reduces chalky rice. Air spaces formed in the cells cause an irregular light reflection and create chalkiness, yet what exactly occurs remains unclear at cell level. Through on-site cell-specific analysis, we show that heat-treated cells adjust osmotically and retard protein synthesis to preserve protein storage vacuoles in the cytosol, resulting in air space formation. Application of nitrogen enhances heat tolerance to sustain protein body and amyloplast development during strong osmotic adjustment, which diminishes air spaces to avoid chalkiness. Hence, we conclude that rice endosperm cells could alter organelle compartments spatially during the heat adaptation, depending on the available nitrogen level. Our findings provide new insight into the cellular mechanism of rice chalky formation as a strategy for heat acclimation.

plant biology↗