Biology subjects

Ueda, M.

Publications and source records attributed to Ueda, M..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

Deep learning-based cytoskeleton segmentation for accurate high-throughput measurement of cytoskeleton density

Microscopic analyses of cytoskeleton organization are crucial for understanding various cellular activities, including cell proliferation and environmental responses in plants. Traditionally, assessments of cytoskeleton dynamics have been qualitative, relying on microscopy-assisted visual inspection. However, the transition to quantitative digital microscopy has introduced new technical challenges, with segmentation of cytoskeleton structures proving particularly demanding. In this study, we examined the utility of a deep learning-based segmentation method for accurate quantitative evaluation of cytoskeleton organization using confocal microscopic images of the cortical microtubules in tobacco BY-2 cells. The results showed that, although conventional methods sufficed for measurement of cytoskeleton angles and parallelness, the deep learning-based method significantly improved the accuracy of density measurements. To assess the versatility of the method, we extended our analysis to physiologically significant models in the context of changes in cytoskeleton density, namely Arabidopsis thaliana guard cells and zygotes. The deep learning-based method successfully improved the accuracy of cytoskeleton density measurements for quantitative evaluations of physiological changes in both stomatal movement in guard cells and intracellular polarization in elongating zygotes, confirming its utility in these applications. The results demonstrate the effectiveness of deep learning-based segmentation in providing precise and high-throughput measurements of cytoskeleton density, and has the potential to automate and expedite analyses of large-scale image datasets.

cell biology↗

bioRxiv2024

Brassinosteroids function as the plant male and female reproductive hormone coordinating gene expression.

Brassinosteroids (BRs) are steroid hormones identified in plants. Besides promoting cell elongation and division, BRs facilitate the development of both male and female reproductive tissues. In animals, reproductive steroid hormones play an essential role in reproductive tissue development by regulating gene expression. Here, we focused on the function of BRs during fertilization. We measured the content of biologically active BRs, brassinolide (BL) and castasterone (CS), in the reproductive tissues of Arabidopsis thaliana. Both BL and CS accumulated abundantly in pollen grains and in larger amounts in pistils than in leaves. To evaluate BL function during fertilization, we used an in vitro guidance assay with exogenously applied BL. Although pollen tubes need to be elongated through the pistils for efficient capacitation, BL treatment promoted pollen tube capacitation and improved attraction to ovules in vitro. Transcriptome analysis demonstrated that BL treatment induced the expression of half of the genes expressed in pollen tubes that elongated through the pistils. These results indicated that BL supplied from pistils is a key factor for pollen tube capacitation. However, using the bri1 mutant for the guidance assay resulted in reduced pollen tube capacitation, suggesting that BRI1-signaling in pistils is also important. Furthermore, BRs act on ovules. Exogenous BL application to ovules maintained guidance capacity by promoting the expression of small secreted proteins involved in pollen tube attraction and gamete fusion. Overall, BRs play a significant role as male and female reproductive hormones throughout the plant fertilization process.

plant biology↗

bioRxiv2024

Downstream metabolites of (+)-cis-12-oxo-phytodienoic acid function as noncanonical bioactive jasmonates in Arabidopsis thaliana.

(+)-cis-12-oxo-phytodienoic acid (cis-OPDA) is a biosynthetic precursor of the plant hormone (+)-7-iso-jasmonoyl-L-isoleucine (JA-Ile). It functions as an endogenous chemical signal independent of the JA-Ile receptor COI1-JAZ in Arabidopsis thaliana. The bioactive form of cis-OPDA that induces COI1-JAZ-independent gene expression remains unknown. In this study, we hypothesized that the genuine bioactive forms of cis-OPDA are the downstream metabolites, which upregulate the expression of the OPDA marker genes such as ZAT10/ERF5 in a JA-Ile-independent manner. These downstream metabolites function independently of the JA-Ile-COI1-JAZ-MYCs canonical jasmonate signaling module, and its electrophilic nature is essential for its bioactivity.

plant biology↗

bioRxiv2024

HD-ZIP IV genes are essential for embryo initial cell polarization in the radial axis initiation in Arabidopsis

Plants develop along apical-basal and radial axes. In Arabidopsis thaliana, the radial axis becomes evident when the cells of the eight-cell proembryo divide periclinally, forming inner and outer cell layers. Although changes in cell polarity or morphology likely precede this oriented cell division, the initial events and the factors regulating radial axis formation remain elusive. Here, we report that three transcription factors belonging to class IV homeodomain-leucine zipper (HD-ZIP IV) family redundantly regulate radial pattern formation: HOMEODOMAIN GLABROUS11 (HDG11), HDG12, and PROTODERMAL FACTOR2 (PDF2). The hdg11 hdg12 pdf2 triple mutant failed to undergo periclinal division at the eight-cell stage and cell differentiation along the radial axis. Live-cell imaging revealed that this failure in radial axis formation can be traced back to the behavior of the embryo initial cell (apical cell), which is generated by zygote division. In the wild type, the apical cell grows longitudinally and then radially and its nucleus remains at the bottom of the cell, where the vertical cell plate emerges. By contrast, the mutant apical cell elongates longitudinally and its nucleus releases from its basal position, resulting in a transverse division. Computer simulations based on the live-cell imaging data confirmed the importance of the geometric rule (the minimal plane principle and nucleus-passing principle) in determining the cell division plane. We propose that HDG11, HDG12, and PDF2 promote apical cell polarization, i.e., radial cell growth and basal nuclear retention, as the initial event of radial axis formation during embryogenesis.

plant biology↗

bioRxiv2024

Viral antigen mismatch affects antiviral T-cell response and may impair immunotherapeutic efficacy against ATL

Human T-cell leukemia virus type 1 (HTLV-1) has the potential to transform primary CD4+ T cells in vitro within a short time; however, the majority of infected individuals maintain an asymptomatic and disease-free condition, suggesting the existence of an equilibrium between the proliferation of infected cells and host immunity. The decline in anti-viral immunity contributes to the transformation of the infected cells, leading to the development of adult T-cell leukemia/lymphoma (ATL). This study identified a variation in a major viral antigen, HTLV-1 Tax, in human leukocyte antigen-A24 (HLA-A24) positive individuals. Two variants of Tax301-309 peptides, SFHNLHLLF (Tax301-309 A) and SFHSLHLLF (Tax301-309 B) were found to induce distinct T-cell immune responses in HLA-A24 positive individuals. There was a disparity between two Tax301-309 peptides in the detection of anti-Tax301-309 cytotoxic T-lymphocytes (CTLs) binding to A24/peptide multimers by flow cytometry analysis. More importantly, over half of the anti-Tax TCRs of anti-Tax CTLs from infected individuals did not recognize mismatched Tax301-309 peptides by Enzyme-Linked Immunospot (ELISpot) assay using Jurkat T cells expressing the anti-Tax301-309 specific TCR. These findings underscore the importance of matching the viral antigen epitope type in T-cell-based immunotherapy against ATL by using viral antigen Tax. Key pointsO_LIEpitope heterogeneity in the major viral antigen in HTLV-1 infection causes different T-cell responses in infected individuals. C_LIO_LIRecommended guideline; performing virus typing to obtain optimal efficacy in T-cell-mediated immunotherapy against the viral antigen Tax C_LI

immunology↗

bioRxiv2023

Single-molecule tracking-based drug screening

The single-molecule tracking of transmembrane receptors in living cells has provided significant insights into signaling mechanisms, such as mobility and clustering upon their activation/inactivation, making it a potential screening method for drug discovery. Here we show that single-molecule tracking-based screening can be used to explore compounds both detectable and undetectable by conventional methods for disease-related receptors. Using an automated system for a fast large-scale single-molecule analysis, we screened for epidermal growth factor receptor (EGFR) from 1,134 of FDA approved drugs. The 18 hit compounds included all EGFR-targeted tyrosine kinase inhibitors (TKIs) in the library that suppressed any phosphorylation-dependent mobility shift of EGFR, proving the concept of this approach. The remaining hit compounds are not reported as EGFR-targeted drugs and did not inhibit EGF-induced EGFR phosphorylation. These non-TKI compounds affected the mobility and/or clustering of EGFR without EGF and induced EGFR internalization, to impede EGFR-dependent cell growth. Thus, single-molecule tracking provides a new modality for discovering novel therapeutics on various receptor functions with previously untargeted mechanisms.

pharmacology and toxicology↗

bioRxiv2023

Surface-engineered extracellular vesicles to modulate antigen-specific T cell expansion for cancer immunotherapy

Extracellular vesicles (EVs), including exosomes, are emerging as novel mediators of cell-cell communications, involved in various processes such as immune activation and immunosuppression. Despite the recent development of several EVs-based cancer immunotherapies, their clinical efficacy remained limited. Here, using fusion with tetraspanin as one of the EV engineering techniques, we created antigen-presenting extracellular vesicles (AP-EVs) to reproduce the functional characteristics of professional antigen-presenting cells (APCs). AP-EVs were also equipped with surface-bound IL-2, a feature not inherent to APCs, which facilitated selective delivery of IL-2 to antigen-specific CD8+ T cells. AP-EVs were engineered to express a peptide-major histocompatibility class I (pMHCI) complex, a costimulatory CD80 molecule, and IL-2, allowing the simultaneous presentation of multiple immune modulators to antigen-specific CD8+ T cells. This promoted the clonal expansion and differentiation of antigen-specific cytotoxic T lymphocytes, leading to potent anticancer immune responses. Combination therapy with AP-EVs and anti-PD-1 demonstrated enhanced anticancer immunity against established tumors compared with anti-PD-1 monotherapy. Our engineered EVs represent a novel effective strategy for cancer immunotherapy.

bioengineering↗

bioRxiv2023

Comprehensive and quantitative analysis of intracellular structure polarization at the apical-basal axis in elongating Arabidopsis zygotes

A comprehensive and quantitative evaluation of multiple intracellular structures or proteins is a promising approach to provide a deeper understanding of and new insights into cellular polarity. In this study, we developed an image analysis pipeline to obtain intensity profiles of fluorescent probes along the apical-basal axis in elongating Arabidopsis thaliana zygotes based on two-photon live-cell imaging data. This technique showed the intracellular distribution of actin filaments, mitochondria, microtubules, and vacuolar membranes along the apical-basal axis in elongating zygotes from the onset of cell elongation to just before asymmetric cell division. Hierarchical cluster analysis of the quantitative data on intracellular distribution revealed that the zygote may be compartmentalized into two parts, with a boundary located 43.6% from the cell tip, immediately after fertilization. To explore the biological significance of this compartmentalization, we examined the positions of the asymmetric cell divisions from the dataset used in this distribution analysis. We found that the cell division plane was reproducibly inserted 20.5% from the cell tip. This position corresponded well with the midpoint of the compartmentalized apical region, suggesting a potential relationship between the zygote compartmentalization, which begins with cell elongation, and the position of the asymmetric cell division.

cell biology↗

bioRxiv2023

RasGEFX triggers spontaneous Ras excitation with RasGEFB/M/U for random cell migration

Excitable systems of eukaryotic chemotaxis can generate asymmetric signals of Ras-GTP-enriched domains spontaneously to drive random cell migration without guidance cues. The molecules responsible for the spontaneous signal generation remain elusive. Here, we characterized RasGEFs encoded in Dictyostelium discoideum by the live-cell imaging of the Ras dynamics and hierarchical clustering, finding that RasGEFX triggers symmetry breaking to generate a Ras-GTP-enriched domain and is essential for random migration in combination with RasGEFB/M/U but dispensable for chemotaxis. Among RasGEFs, RasGEFX and RasGEFB co-localize with Ras-GTP on oscillatory waves propagating along the membrane and regulate the temporal periods and spatial sizes, respectively, of the waves and thus the cytoskeletal dynamics differently. RasGEFU localized uniformly on the membrane and regulated cell adhesion. These findings provide mechanistic insights into the internal signal generation that operates independently of external chemotaxis signaling and suggest a coordinated control of the cytoskeletal dynamics by multiple RasGEFs for spontaneous motility.

molecular biology↗

bioRxiv2023

Upregulated Fcrl5 disrupts B cell anergy and causes autoimmunity

B cell anergy plays a critical role in maintaining self-tolerance by inhibiting autoreactive B cell activation to prevent autoimmune diseases. Here, we demonstrated that Fc receptor-like 5 (Fcrl5) upregulation contributes to autoimmune disease pathogenesis by disrupting B cell anergy. Fcrl5--a gene whose homologs are associated with human autoimmune diseases--is highly expressed in age/autoimmunity-associated B cells (ABCs), an autoreactive B cell subset. By generating B cell-specific Fcrl5 transgenic mice, we demonstrated that Fcrl5 overexpression in B cells caused systemic autoimmunity with age. Furthermore, Fcrl5 upregulation in B cells exacerbated the systemic lupus erythematosus-like disease model and increased the levels of ABCs and activated T cells. Mechanistically, an increase in Fcrl5 expression broke B cell anergy, activating autoreactive B cells in the presence of a self-antigen. Fcrl5 facilitated toll-like receptor signaling, reactivating anergic B cells. Thus, Fcrl5 is a potential regulator of B cell-mediated autoimmunity by regulating B cell anergy. This study provides important insights into the role of Fcrl5 in breaking B cell anergy and its effect on the pathogenesis of autoimmune diseases.

immunology↗

bioRxiv2023

Conjugation of cis-OPDA with amino acids is a conserved pathway affecting cis-OPDA homeostasis upon stress responses

Jasmonates (JAs) are a family of oxylipin phytohormones regulating plant development and growth and mediating defense versus growth responses. The upstream JA biosynthetic precursor cis-(+)-12-oxo-phytodienoic acid (cis-OPDA) has been reported to act independently of the COI1-mediated JA signaling in several stress-induced and developmental processes. However, its means of perception and metabolism are only partially understood. Furthermore, cis-OPDA, but not JA, occurs in non-vascular plant species, such as bryophytes, exhibiting specific functions in defense and development. A few years ago, a low abundant isoleucine analog of the biologically active JA-Ile, OPDA-Ile, was detected in wounded leaves of flowering plants, opening up to the possibility that conjugation of cis-OPDA to amino acids might be a relevant mechanism for cis-OPDA regulation. Here, we extended the analysis of amino acid conjugates of cis-OPDA and identified naturally occurring OPDA-Val, OPDA-Phe, OPDA-Ala, OPDA-Glu, and OPDA-Asp in response to biotic and abiotic stress in Arabidopsis. The newly identified OPDA-amino acid conjugates show cis-OPDA-related plant responses in a JAR1-dependent manner. We also discovered that the synthesis and hydrolysis of cis-OPDA amino acid conjugates are regulated by members of the amidosynthetase GH3 and the amidohydrolase ILR1/ILL families. Finally, we found that the cis-OPDA conjugative pathway already functions in non-vascular plants and gymnosperms. Thus, one level of regulation by which plants modulate cis-OPDA homeostasis is the synthesis and hydrolysis of OPDA-amino acid conjugates, which temporarily store cis-OPDA in stress responses.

plant biology↗

bioRxiv2023

Origami-based growing tube model for reproducing shell shapes

Mathematical models for shell morphology have been well-studied in theoretical morphology. The growing tube model proposed by Takashi Okamoto was developed to reproduce heteromorph ammonoids and can generate various shells by changing three parameters: curvature, torsion, and enlarging ratio. Previous studies in theoretical morphology have employed computers to visualize mathematical models. However, computers are not accessible to everyone, and merely observing the graphics of the reproduced shells does not deepen understanding of the mathematical model. Therefore, in this study, we considered using origami, which is inexpensive and readily available, to make users understand mathematical models through hands-on folding. First, we simplified the crease pattern of seashells, originally devised by an origami artist Tomoko Fuse, to create a basic crease pattern. We then identified the crease pattern elements corresponding to the three parameters in the growing tube model and the aperture-apex position determiner. Based on them, we reproduced various shell shapes, including a heteromorph-ammonoid-like shape, with origami. Finally, we investigated the constraints between parameters in the origami-growing tube model. The origami-growing tube model is expected to help disseminate mathematical models and promote theoretical biology.

scientific communication and education↗

bioRxiv2023

A dynamic partitioning mechanism polarizes membrane protein distribution

The plasma membrane is widely regarded as the hub of the signal transduction network activities that drives numerous physiological responses, including cell polarity and migration. Yet, the symmetry breaking process in the membrane, that leads to dynamic compartmentalization of different proteins, remains poorly understood. Using multimodal live-cell imaging, here we first show that multiple endogenous and synthetic lipid-anchored proteins, despite maintaining stable tight association with the inner leaflet of the plasma membrane, were unexpectedly depleted from the membrane domains where the signaling network was spontaneously activated such as in the new protrusions as well as within the propagating ventral waves. Although their asymmetric patterns resembled those of standard peripheral "back" proteins such as PTEN, unlike the latter, these lipidated proteins did not dissociate from the membrane upon global receptor activation. Our experiments not only discounted the possibility of recurrent reversible translocation from membrane to cytosol as it occurs for weakly bound peripheral membrane proteins, but also ruled out the necessity of directed vesicular trafficking and cytoskeletal supramolecular structure-based restrictions in driving these dynamic symmetry breaking events. Selective photoconversion-based protein tracking assays suggested that these asymmetric patterns instead originate from the inherent ability of these membrane proteins to "dynamically partition" into distinct domains within the plane of the membrane. Consistently, single-molecule measurements showed that these lipid-anchored molecules have substantially dissimilar diffusion profiles in different regions of the membrane. When these profiles were incorporated into an excitable network-based stochastic reaction-diffusion model of the system, simulations revealed that our proposed "dynamic partitioning" mechanism is sufficient to give rise to familiar asymmetric propagating wave patterns. Moreover, we demonstrated that normally uniform integral and lipid-anchored membrane proteins in Dictyostelium and mammalian neutrophil cells can be induced to partition spatiotemporally to form polarized patterns, by optogenetically recruiting membrane domain-specific peptides to these proteins. Together, our results indicate "dynamic partitioning" as a new mechanism of plasma membrane organization, that can account for large-scale compartmentalization of a wide array of lipid-anchored and integral membrane proteins in different physiological processes.

cell biology↗

bioRxiv2022

Immune Isoform Atlas: Landscape of alternative splicing in human immune cells

Alternative splicing events are a major causal mechanism for complex traits, but they have been understudied due to the limitation of short-read sequencing. Here, we generated a comprehensive full-length isoform annotation of human immune cells, Immune Isoform Atlas, by long-read sequencing for 29 cell subsets. Our atlas contained a number of unannotated transcripts and isoforms such as a read-through transcript of TOMM40-APOE. We profiled functional characteristics of isoforms including encoded domains, inserted repetitive elements, and translational efficiency, and we showed that repetitive elements significantly explained the diversity of unannotated isoforms. Some of the isoforms are expressed in a cell-type specific manner, whose alternative 3-UTRs usage contributed to their specificity. Further, we identified a number of disease-associated isoforms by isoform switch analysis and by integration of several quantitative trait loci analyses with genome-wide association study data. Our findings will promote the elucidation of the pathomechanism of diseases via alternative splicing.

genetics↗

bioRxiv2022

Reconstitution of ribosome self-replication outside a living cell

Ribosome biogenesis, a recursive process of pre-existing ribosomes self-replicating nascent ones, is pivotal in the self-replication of life. In Escherichia coli, three ribosomal RNAs (rRNAs) are transcribed, and 54 ribosomal proteins (r-proteins) are synthesized by pre-existing ribosomes as structural components1, 2. They are cotranscriptionally assembled in a cooperative hierarchy under the support of [~]100 accessory factors1-3. The reconstitution of ribosome biogenesis outside a living cell is an essential goal to understand the self-replication of life. However, this goal could not have been achieved so far due to its complexity. Here, we report the successful in vitro reconstitution of the entire ribosome biogenesis process. We hypothesized that mimicking in vivo ribosome biogenesis1-6 could result in in vitro ribosome biogenesis. Specifically, we found that coactivating the transcription of an rRNA operon, as well as the transcription and translation of 54 r-protein genes encoding r-proteins, and the coordinated ribosomal assembly in a cytoplasm-mimicking reaction solution, resulted in highly efficient in vitro reconstitution of ribosome biogenesis. Our achievement represents a critical step toward revealing fundamental principles underlying the self-replication of life and creating self-replicating artificial cells7. We also succeeded in engineering rRNA and r-proteins by only adding mutant ribosomal genes in the reaction, enabling high-throughput and unconstrained creation of artificial ribosomes with altered or enhanced functionality8-12.

synthetic biology↗

bioRxiv2022

Male-killing-associated bacteriophage WO identified from comparisons of Wolbachia endosymbionts of Homona magnanima

The origin and mechanism of male-killing, an advantageous strategy employed by maternally transmitted symbionts such as Wolbachia, remain unclear. We compared genomes of four Wolbachia strains derived from Homona magnanima, a male-killing strain wHm-t (1.5 Mb), and three non-male-killing strains, wHm-a (1.1 Mb), wHm-b (1.3 Mb), and wHm-c (1.4 Mb). A wHm-t-specific 76-kbp prophage region harboured two tandemly arrayed WO-mediated killing (wmk) gene homologs (wmk-1/wmk-2 and wmk-3/wmk-4). Of these, wmk-1 or wmk-3 killed almost all Drosophila melanogaster individuals when transgenically overexpressed. Dual expression of wmk-3 and wmk-4 killed all males and rescued females. We propose a novel hypothesis wherein horizontally transmitted proto-Wolbachia with a single wmk killed both sexes, and tandem duplication of wmk allowed an evolutionary transition to a vertically transmitted symbiont, causing male-killing. Our study highlights the bacteriophage as a critical driver of the evolution of male-killing and argues for a conserved male-killing mechanism in diverse insects.

evolutionary biology↗

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↗

bioRxiv2021

Membrane cholesterol interferes with tyrosine phosphorylation but facilitates the clustering and signal transduction of EGFR

Epidermal growth factor receptor (EGFR) activates major cell signaling pathways that regulate various cell responses. Its dimerization and clustering coupled with its lateral mobility are critical for EGFR function, but the contribution of the plasma membrane environment to EGFR function is unknown. Here we show, using single-molecule analysis, that EGFR mobility and clustering are altered by the depletion of cholesterol or sphingomyelin, major lipids of membrane subdomains, causing significant changes in EGFR signaling. When cholesterol was depleted, the subdomain boundary in EGFR diffusion disappeared, the fraction of EGFR pre-dimers was increased, and the ligand-induced phosphorylation of EGFR was enhanced. In addition, the depletion of either lipid prevented the formation of immobile clusters after EGF association and decreased the phosphorylation of downstream proteins. Our results revealed that cholesterol plays dichotomous roles in the signaling pathway of EGFR and that clustering in the membrane subdomains is critical for EGFR signal transduction.

biophysics↗