Biology subjects

Matsuoka, S.

Publications and source records attributed to Matsuoka, S..

18 recordsLinked to original sources

bioRxiv2026

A phosphorylation-dependent partner-switching-like module controls heterocyst polysaccharide layer formation in Anabaena sp. strain PCC 7120

Heterocysts are specialized cells formed by filamentous cyanobacteria to enable nitrogen fixation under aerobic conditions. These cells are surrounded by a multilayered envelope, including a heterocyst-specific polysaccharide (Hep) layer that restricts oxygen diffusion and protects nitrogenase activity. Although several genes required for Hep biosynthesis have been identified, the regulatory mechanisms controlling Hep layer formation remain poorly understood. Here, we show that Hep layer formation in Anabaena sp. PCC 7120 is regulated by a phosphorylation-dependent partner-switching-like system. The phosphatase HenR and the STAS domain-containing protein All4160 are both required for Hep formation and nitrogen fixation. A nonphosphorylatable All4160 variant restores Hep formation and nitrogen fixation in a henR disruptant, indicating that phosphorylation negatively regulates All4160 function. Using a bacterial two-hybrid assay, we identified two candidate kinases, Alr3423 and All2284, that interact with All4160, and both proteins phosphorylate All4160 in vitro. Genetic analysis revealed that deletion of alr3423, but not all2284, suppresses the nitrogen fixation defect of the henR disruptant, suggesting that Alr3423 is a major kinase regulating All4160 in vivo. These findings uncover a phosphorylation switch that controls heterocyst polysaccharide layer formation and expand the role of partner-switching systems beyond {sigma} factor-dependent transcription to the direct regulation of a biosynthetic enzyme involved in exopolysaccharide (EPS) synthesis. ImportanceExopolysaccharides (EPS) are involved in diverse aspects of bacterial physiology, including environmental acclimation, motility, host association, and cellular differentiation, yet their regulatory mechanisms remain poorly understood. Cyanobacteria play central roles in global carbon and nitrogen cycles through photosynthesis and nitrogen fixation, the latter requiring a micro-oxic environment provided by specialized cells called heterocysts. This study reveals a previously unrecognized mechanism for controlling polysaccharide biosynthesis, in which a partner-switching-like system directly regulates the activity of a biosynthetic enzyme. By expanding the role of partner-switching systems beyond {sigma} factor-dependent transcription, these findings provide a framework for understanding how bacteria control EPS production in response to environmental and developmental cues.

microbiology↗

bioRxiv2025

Biophysical and Colloidal Properties Govern Anti-Drug Antibody Generation: A Case Study Using the Tumoricidal mAb 4713

Anti-drug antibodies (ADA) are a well-recognized clinical problem, but their sporadic occurrence has limited detailed biophysical characterization of causative factors. We investigated how different stress conditions influence aggregation of a murine monoclonal antibody, mAb 4713, and the resulting potential to induce ADA in mice. Dynamic light scattering (DLS) revealed that stirring, freeze-thaw, and heat stress each generated subvisible aggregates (30-700 nm), whereas non-stressed mAb 4713 remained predominantly monomeric. Stirring- and freeze-thaw-induced aggregates retained native-like secondary and tertiary structures, thermal and biochemical stability, and anti-tumor cytotoxicity, as shown by circular dichroism, tryptophan fluorescence, thermal shift assay, limited proteolysis, and cell death assay. In contrast, heat-induced aggregates displayed complete structural denaturation and loss of cytotoxic activity. Immunization of Jcl: ICR mice demonstrated that heat-induced aggregates were only mildly immunogenic, while freeze-thawed and non-stressed mAb 4713 elicited no detectable immune response, and ADA generated under these conditions did not affect anti-tumor activity. Strikingly, stirred aggregates triggered a strong anti-mAb antibody response, as measured by ELISA, and the resulting antisera completely abolished mAb 4713 cytotoxicity against Raji cells, a Burkitt lymphoma cell line, indicating induction of neutralizing ADA. These results show that aggregate immunogenicity depends not simply on aggregate presence but critically on their structural and colloidal properties and formation pathways, highlighting the need to assess both structural and colloidal properties of aggregates in biopharmaceutical development to mitigate ADA risks.

biophysics↗

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

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

SARS-CoV-2 ORF8 is a viral cytokine regulating immune responses

Many patients with severe COVID-19 suffer from pneumonia, and thus elucidation of the mechanisms underlying the development of such severe pneumonia is important. The ORF8 protein is a secreted protein of SARS-CoV-2, whose in vivo function is not well understood. Here, we analyzed the function of ORF8 protein by generating ORF8-knockout SARS-CoV-2. We found that the lung inflammation observed in wild-type SARS-CoV-2-infected hamsters was decreased in ORF8-knockout SARS-CoV-2-infected hamsters. Administration of recombinant ORF8 protein to hamsters also induced lymphocyte infiltration into the lungs. Similar pro-inflammatory cytokine production was observed in primary human monocytes treated with recombinant ORF8 protein. Furthermore, we demonstrate that the serum ORF8 protein levels are correlated well with clinical markers of inflammation. These results demonstrated that the ORF8 protein is a viral cytokine of SARS-CoV-2 involved in the in the immune dysregulation observed in COVID-19 patients, and that the ORF8 protein could be a novel therapeutic target in severe COVID-19 patients.

microbiology↗

bioRxiv2021

Influence of DNA extraction kits on the fungal DNA metabarcoding for freshwater environmental DNA

BackgroundEnvironmental DNA (eDNA) metabarcoding is a common technique for efficient biodiversity monitoring, especially of microbes. Recently, the usefulness of aquatic eDNA in monitoring the diversity of both terrestrial and aquatic fungi has been suggested. In eDNA studies, different experimental factors, such as DNA extraction kits or methods, can affect the subsequent analyses and the results of DNA metabarcoding. However, few methodological studies have been carried out on eDNA of fungi, and little is known about how experimental procedures can affect the results of biodiversity analysis. In this study, we focused on the effect of DNA extraction method on fungal DNA metabarcoding using freshwater samples obtained from rivers and lakes. MethodsDNA was extracted from freshwater samples using the DNeasy PowerSoil kit, which is mainly used to extract microbial DNA from soil, and the DNeasy Blood & Tissue kit, which is commonly used for eDNA studies on animals. We then compared PCR inhibition and fungal DNA metabarcoding results; i.e., operational taxonomic unit (OTU) number and composition of the extracted samples. ResultsNo PCR inhibition was detected in any of the samples, and no significant differences in the number of OTUs and OTU compositions were detected between the samples processed using different kits. These results indicate that both DNA extraction kits may provide similar diversity results for the river and lake samples evaluated in this study. Therefore, it may be possible to evaluate the diversity of fungi using a unified experimental method, even with samples obtained for diversity studies on other taxa such as those of animals.

microbiology↗

bioRxiv2021

The SARS-CoV-2 Delta variant is poised to acquire complete resistance to wild-type spike vaccines

mRNA-based vaccines provide effective protection against most common SARS-CoV-2 variants. However, identifying likely breakthrough variants is critical for future vaccine development. Here, we found that the Delta variant completely escaped from anti-N-terminal domain (NTD) neutralizing antibodies, while increasing responsiveness to anti-NTD infectivity-enhancing antibodies. Although Pfizer-BioNTech BNT162b2-immune sera neutralized the Delta variant, when four common mutations were introduced into the receptor binding domain (RBD) of the Delta variant (Delta 4+), some BNT162b2-immune sera lost neutralizing activity and enhanced the infectivity. Unique mutations in the Delta NTD were involved in the enhanced infectivity by the BNT162b2-immune sera. Sera of mice immunized by Delta spike, but not wild-type spike, consistently neutralized the Delta 4+ variant without enhancing infectivity. Given the fact that a Delta variant with three similar RBD mutations has already emerged according to the GISAID database, it is necessary to develop vaccines that protect against such complete breakthrough variants.

microbiology↗

bioRxiv2020

An infectivity-enhancing site on the SARS-CoV-2 spike protein is targeted by COVID-19 patient antibodies

SARS-CoV-2 infection causes severe symptoms in a subset of patients, suggesting the presence of certain unknown risk factors. Although antibodies against the receptor-binding domain (RBD) of the SARS-CoV-2 spike have been shown prevent SARS-CoV-2 infection, the effects of antibodies against other spike protein domains are largely unknown. Here, we screened a series of anti-spike monoclonal antibodies from COVID-19 patients, and found that some of antibodies against the N-terminal domain (NTD) dramatically enhanced the binding capacity of the spike protein to ACE2, and thus increased SARS-CoV2 infectivity. Surprisingly, mutational analysis revealed that all the infectivity-enhancing antibodies recognized a specific site on the surface of the NTD. The antibodies against this infectivity-enhancing site were detected in all samples of hospitalized COVID-19 patients in the study. However, the ratio of infectivity-enhancing antibodies to neutralizing antibodies differed among patients. Furthermore, the antibodies against the infectivity-enhancing site were detected in 3 out of 48 uninfected donors, albeit at low levels. These findings suggest that the production of antibodies against SARS-CoV-2 infectivity-enhancing site could be considered as a possible exacerbating factors for COVID-19 and that a spike protein lacking such antibody epitopes may be required for safe vaccine development, especially for individuals with pre-existing enhancing antibodies.

microbiology↗

bioRxiv2020

Evaluation of seasonal dynamics of fungal DNA assemblages in a flow-regulated stream in a restored forest using eDNA metabarcoding

Investigation of seasonal variation in fungal communities is essential for understanding biodiversity and ecosystem functions. However, the conventional sampling method, with substrate removal and high spatial heterogeneity of community composition, makes surveying the seasonality of fungal communities challenging. Recently, water environmental DNA (eDNA) analysis has been explored for its utility in biodiversity surveys. In this study, we assessed whether the seasonality of fungal communities can be detected by monitoring eDNA in a forest stream. We conducted monthly water sampling in a forest stream over two years and used DNA metabarcoding to identify fungal eDNA. The stream water contained DNA from functionally diverse aquatic and terrestrial fungi, such as plant decomposers, parasites, and mutualists. The variation in the fungal assemblage showed a regular annual periodicity, meaning that the assemblages in a given season were similar, irrespective of the year or sampling. Furthermore, the strength of the annual periodicity varied among functional groups. Our results suggest that forest streams may act as a "trap" for terrestrial fungal DNA derived from different habitats, allowing the analysis of fungal DNA in stream water to provide information about the temporal variation in fungal communities in both the aquatic and the surrounding terrestrial ecosystems.

ecology↗

bioRxiv2020

Effects of species traits and ecosystem characteristics on species detection by eDNA metabarcoding in lake fish communities

Although environmental DNA (eDNA) metabarcoding is acknowledged to be an exceptionally useful and powerful tool for monitoring surveys, it has limited applicability, particularly for nationwide surveys. To evaluate the performance of eDNA metabarcoding in broad-scale monitoring, we examined the effects of species ecological/biological traits and ecosystem characteristics on species detection rates and the consequences for community analysis. We conducted eDNA metabarcoding on fish communities in 18 Japanese lakes on a country-wide scale. By comparing species records, we found that certain species traits, including body size, body shape, saltwater tolerance, and habitat preferences, influenced eDNA detection. We also found that the proportion of species detected decreased significantly with an increase in lake surface area, owing to an ecosystem-size effect on species detection. We conclude that species traits, including habitat preferences and body size, and ecosystem size should be taken into consideration when assessing the performance of eDNA metabarcoding in broad-scale monitoring.

ecology↗

bioRxiv2020

Illumina iSeq 100 and MiSeq exhibit similar performance in freshwater fish environmental DNA metabarcoding

Environmental DNA (eDNA) analysis is a method of detecting DNA from environmental samples and is used as a biomonitoring tool. In recent studies, Illumina MiSeq has been the most extensively used tool for eDNA metabarcoding. The Illumina iSeq 100 (hereafter, iSeq), one of the high-throughput sequencers (HTS), has a relatively simple workflow and is potentially more affordable than other HTS. However, its utility in eDNA metabarcoding has still not been investigated. In the present study, we applied fish eDNA metabarcoding to 40 water samples from river and lake ecosystems to assess the difference in species detectability and composition between iSeq and MiSeq. To check differences in sequence quality and errors, we also assessed differences in read changes between the two HTS. There were similar sequence qualities between iSeq and MiSeq. Significant difference was observed in the number of species between two HTS, but no difference was observed in species composition between the two HTS. Additionally, the species compositions in common with the conventional method were the same between the two HTS. According to the results, using the same amplicon library for sequencing, two HTS would exhibit a similar performance of fish species detection using eDNA metabarcoding.

ecology↗

bioRxiv2020

Reassembly of soil fungal communities under reforestation and herbivore exclusion

Soil fungi can help improve ecosystem restoration, yet our understanding of how they reassemble in degraded land is limited. Here, we studied fungal community structure using DNA metabarcoding in reforested sites following agricultural abandonment and overgrazing. Two treatments, namely reforestation using different numbers of tree species and deer exclusion, have been applied for multiple decades in the study sites. We found that local fungal richness (alpha diversity) and total fungal richness (gamma diversity) were 1.9-2.9 and 1.3-1.9 times greater, respectively, in reforested stands than in natural forests. These results were regardless of the number of tree species planted in the reforested stands. Conversely, reforested stands had a spatially homogenized community structure with relatively lower degrees of compositional dissimilarity among sites within each stand (beta diversity). These findings were attributable to lower environmental heterogeneity, stronger dispersal limitation, and a comparatively shorter time since the onset of community assembly in reforested stands. Deer exclosures had no detectable effect on fungal community structure. Overall, the agricultural legacy in fungal community structure appears to have persisted for decades, even under proactive restoration of aboveground vegetation. Direct human intervention belowground may therefore be necessary for the recovery of soil biota once altered.

ecology↗

bioRxiv2019

Evaluation of host effects on ectomycorrhizal fungal community compositions in a forest landscape in northern Japan

Community compositions of ectomycorrhizal (ECM) fungi are similar within the same host taxa. However, careful interpretation is required to determine whether the combination of ECM fungi and plants is explained by the host preference of ECM fungi or by the influence of neighboring conspecific and/or heterospecific hosts. In the present study, we aimed to evaluate the effects of host species on the ECM community compositions in a forest landscape (~ 10 km) where monodominant forest stands of six ECM host species belonging to three families were patchily distributed. The ECM communities were identified with DNA metabarcoding. A total of 180 ECM operational taxonomic units (OTUs) were detected. The ECM community compositions were primarily structured by host species and families, regardless of the soil environments and spatial arrangements of the sampling plots. In addition, 38 ECM OTUs were detected from particular host tree species. Furthermore, the neighboring plots harbored similar fungal compositions, although the host species were different. The relative effect of the spatial factors on the ECM compositions was weaker than that of host species. Our results suggest that the host preference of ECM fungi is a primary determinant of ECM fungal compositions in the forest landscape.

ecology↗

bioRxiv2019

Describing macroecological patterns in microbes: Approaches for comparative analyses of operational taxonomic unit read number distribution with a case study of global oceanic bacteria

Describing the variation in commonness and rarity in a community is a fundamental method of evaluating biodiversity. Such patterns have been studied in the context of species abundance distributions (SADs) among macroscopic organisms in numerous communities. Recently, models for analyzing variation in local SAD shapes along environmental gradients have been constructed. The recent development of high-throughput sequencing enables evaluation of commonness and rarity in local communities of microbes using operational taxonomic unit (OTU) read number distributions (ORDs), which are conceptually similar to SADs. However, few studies have explored the variation in local microbial ORD shapes along environmental gradients. Therefore, the similarities and differences between SADs and ORDs are unclear, clouding any universal rules of global biodiversity patterns. We investigated the similarities and differences in ORD shapes vs. SADs, and how well environmental variables explain the variation in ORDs along latitudinal and depth gradients. Herein, we integrate ORDS into recent comparative analysis methods for SAD shape using datasets generated on the Tara Oceans expedition. About 56% of the variance in skewness of ORDs among global oceanic bacterial communities was explained with this method. Moreover, we confirmed that the parameter combination constraints of Weibull distributions were shared by ORDs of bacterial communities and SADs of tree communities, suggesting common long-term limitation processes such as adaptation and community persistence acting on current abundance variation. On the other hand, skewness was significantly greater for bacterial communities than tree communities, and many ecological predictions did not apply to bacterial communities, suggesting differences in the community assembly rules for microbes and macroscopic organisms. Approaches based on ORDs provide opportunities to quantify macroecological patterns of microbes under the same framework as macroscopic organisms.

ecology↗

bioRxiv2019

Spatial structures of fungal DNA assemblages revealed with eDNA metabarcoding in a forest river network in western Japan

Growing evidence has revealed high diversity and spatial heterogeneity of fungal communities including in local habitats in terrestrial ecosystems. These findings highlight the considerable sampling effort, analysis time, and costs required for the investigation of fungal diversity over large spatial scales. Recently, the analysis of environmental DNA in river water has been undertaken to study the biodiversity of organisms, such as animals and plants, in both aquatic and terrestrial habitats. However, previous studies have not investigated the spatial structure of fungal DNA assemblages in river water. In the present study, we investigate fungal DNA assemblages and their spatial structure using environmental DNA metabarcoding in water across different branches of river over forest landscapes. The river water was found to contain both phylogenetically and functionally diverse fungal DNA, including aquatic and terrestrial fungi, such as plant decomposers and mycorrhizal fungi. These fungal DNA assemblages were more similar within, rather than between, branches. In addition, the assemblages were more similar between spatially closer branches. These results imply that information on the terrestrial and aquatic fungal compositions of watersheds, and therefore their spatial structure can be obtained by investigating the fungal DNA assemblages in river water.

ecology↗

bioRxiv2019

Evaluation of biodiversity metrics through environmental DNA metabarcoding outperforms visual and capturing surveys

O_LIInformation on alpha (local), beta (between habitats), and gamma (regional) diversity is fundamental to understanding biodiversity as well as the function and stability of community dynamics. Methods like environmental DNA (eDNA) metabarcoding are currently considered useful to investigate biodiversity. C_LIO_LIWe compared the performance of eDNA metabarcoding with visual and capture surveys for estimating alpha and gamma diversity of river fish communities, and nestedness and turnover in particular. C_LIO_LIIn five rivers across west Japan, by comparison to visual/capture surveys, eDNA metabarcoding detected more species in the study sites (i.e., alpha diversity). Consequently the overall number of species in the region (i.e., gamma diversity) was higher. In particular, the species found by visual/capture surveys were encompassed by those detected by eDNA metabarcoding. C_LIO_LIEstimates of community diversity within rivers differed between survey methods. Although we found that the methods show similar levels of community nestedness and turnover within the rivers, visual/capture surveys showed more distinct community differences from upstream to downstream. Our results suggest that eDNA metabarcoding may be a suitable method for community assemblage analysis, especially for understanding regional community patterns, for fish monitoring in rivers. C_LI

ecology↗

bioRxiv2019

Biogeographic patterns of ectomycorrhizal fungal communities associated with Castanopsis sieboldii across the Japanese archipelago

Biogeographic patterns in ectomycorrhizal (ECM) fungal communities and their drivers have been elucidated, including effects of host tree species and abiotic (climatic and edaphic) conditions. At these geographic scales, genotypic diversity and composition of single host tree species change with spatial and environmental gradients, reflecting their historical dispersal events. However, whether the host genotypes can be associated with the biogeographic patterns of ECM communities remains unclear. We investigated the biogeographic pattern of ECM fungal community associated with the single host species Castanopsis sieboldii (Fagaceae), whose genotypic diversity and composition across the Japanese archipelago has already been evaluated, and we quantified the effect of host genotypes on the biogeographic pattern. Richness and community composition of ECM fungi changed with latitude and longitude; these biogeographic changes of ECM community were significantly explained by host genotypic variables. Quantitative analyses showed a higher relative explanatory power of climatic and spatial variables than that of host genotypic variables for the biogeographic patterns in the ECM community. Our results suggest the importance of historical events of host dispersal in determining the biogeographic patterns of the ECM fungal community, while their explanation power was lower than that for climatic filtering and/or fungal dispersal.

ecology↗

bioRxiv2018

Excitable dynamics of Ras triggers self-organized PIP3 signaling for spontaneous cell migration

Spontaneous cell movement is underpinned by an asymmetric distribution of signaling molecules including small G proteins and phosphoinositides on the cell membrane. A fundamental question is the molecular mechanism for the spontaneous symmetry breaking. Here we report that GTP bound Ras (Ras-GTP) breaks the symmetry due to excitability even in the absence of extracellular spatial cues and cytoskeletal polarity as well as downstream signaling activities. A stochastic excitation of local and transient Ras activation induced PIP3 accumulation via direct interaction with PI3K, causing tightly coupled traveling waves propagating along the membrane. Comprehensive phase analysis of the waves of Ras-GTP and PIP3 metabolism-related molecules revealed the network structure of the excitable system including positive feedback regulation of Ras-GTP by PIP3. A mathematical model reconstituted a series of the observed symmetry breaking phenomena, illustrating an essential involvement of excitability in the cellular decision-making process.\n\nAuthor contributionsS.F. conducted the experiments; all authors analyzed the data and wrote the manuscript.

cell biology↗