Biology subjects

Makino, A.

Publications and source records attributed to Makino, A..

8 recordsLinked to original sources

bioRxiv2024

Store-operated Ca2+ entry is involved in endothelium-to-mesenchymal transition in lung vascular endothelial cells

Endothelial-to-mesenchymal transition (EndMT) is a biological process that converts endothelial cells to mesenchymal cells with increased proliferative and migrative abilities. EndMT has been implicated in the development of pulmonary vascular remodeling in pulmonary arterial hypertension (PAH), a fatal and progressive lung vascular disease. Transforming growth factor {beta}1 (TGF-{beta}1), an inflammatory cytokine, is known to induce EndMT in many types of endothelial cells including lung vascular endothelial cells (LVEC). An increase in cytosolic free Ca2+ concentration ([Ca2+]cyt) is a major stimulus for cellular proliferation and phenotypic transition, but it is unknown whether Ca2+ signaling is involved in EndMT. In this study we tested the hypothesis that TGF-{beta}1-induced EndMT in human LVEC is Ca2+-dependent. Treatment of LVEC with TGF-{beta}1 for 5-7 days resulted in increase in SNAI1/2 expression, induction of EndMT, upregulation of STIM/Orai1 and enhancement of store-operated Ca2+ entry (SOCE). Removal (or chelation) of extracellular or intracellular Ca2+ with EGTA or BAPTA-AM respectively abolished EndMT in response to TGF-{beta}1. Moreover, EGTA diminished TGF-{beta}1-induced increase in SNAI in a dose-dependent manner. Knockdown of either STIM1 or Orai1 was sufficient to prevent TGF-{beta}-mediated increase in SNAI1/2 and EndMT, but did not rescue the continuous adherent junctions. Blockade of Orai1 channels by AnCoA4 inhibited TGF-{beta}-mediated EndMT and restored PECAM1-positive continuous adherent junctions. In conclusion, intracellular Ca2+ signaling plays a critical role in TGF-{beta}-associated EndMT through enhanced SOCE and STIM1-Orai1 interaction. Thus, targeting Ca2+ signaling pathways regulating EndMT may be a novel therapeutic approach to treat PAH and other forms of pre-capillary pulmonary hypertension. New & NoteworthyEndMT has been reported to contribute to the pathogenesis of PH. In this study we aimed to determine the role of Ca2+ signaling in the development of EndMT in human lung vascular endothelial cells. Our data suggest that TGF-{beta}1 requires store-operated Ca2+ entry through STIM1/Orai channels to induce SNAI-mediated EndMT. For the first time we demonstrated that TGF-{beta}1-induced EndMT is Ca2+-dependent event while inhibition of STIM1/Orai interaction attenuated EndMT in response to TGF-{beta}1.

cell biology↗

bioRxiv2024

A single amino acid substitution in the Borna disease virus glycoprotein enhances the infectivity titer of vesicular stomatitis virus pseudotyped virus by altering membrane fusion activity

Borna disease virus 1 (BoDV-1) causes acute fatal encephalitis in mammals, including humans. Despite its importance, research on BoDV-1 cell entry has been hindered by low viral particle production in cells and the lack of cytopathic effects. To address these issues, we developed a method to efficiently produce vesicular stomatitis virus (VSV) pseudotyped with glycoprotein (G) of the genus Orthobornavirus, including BoDV-1. We discovered that optimal G expression is required to obtain a high infectivity titer of the VSV pseudotyped virus. Remarkably, the infectivity of the VSV pseudotyped virus with G from the BoDV-1 strain huP2br was significantly higher than that of the VSV pseudotyped virus with G from the He/80 strain. Mutational analysis demonstrated that the BoDV-1-G residue 307 determines the infectivity titer of VSV pseudotyped with BoDV-1-G (VSV-BoDV-1-G). A cell-cell fusion assay indicated that this residue plays a pivotal role in membrane fusion, thus suggesting that high membrane fusion activity and a broad pH range for membrane fusion are crucial for achieving a high infectivity titer of VSV-BoDV-1-G. This finding may be extended to increase the infectivity titer of VSV pseudotyped virus with other orthobornavirus G. Our study also contributes to identifying functional domains of BoDV-1-G and provides insight into G-mediated cell entry.

microbiology↗

bioRxiv2023

A non-toxic equinatoxin-II reveals the dynamics of sphingomyelin in the cytosolic leaflet of the plasma membrane

Super-resolution microscopic observation of a novel non-toxic sphingomyelin probe revealed the formation of dynamic small domains including sphingomyelin and cholesterol in the cytosolic leaflet of living cell plasma membranes. AbstractSphingomyelin (SM) is a major sphingolipid in mammalian cells. SM is enriched in the extracellular leaflet of the plasma membrane (PM). Besides this localization, recent electron microscopic and biochemical studies suggest the presence of SM in the cytosolic leaflet of the PM. In the present study, we generated a non-toxic SM-binding variant (NT-EqtII) based on equinatoxin-II (EqtII) from the sea anemone Actinia equina, and examined the dynamics of SM in the cytosolic leaflet of living cell PMs. NT-EqtII with two point mutations (Leu26Ala and Pro81Ala) had essentially the same specificity and affinity to SM as wild-type EqtII. NT-EqtII expressed in the cytosol was recruited to the PM in various cell lines. Super-resolution microscopic observation revealed that NT-EqtII formed tiny domains that were significantly colocalized with cholesterol and N-terminal Lyn. Meanwhile, all the examined lipid probes including NT-EqtII underwent apparent fast simple Brownian diffusion, exhibiting that SM and other lipids in the cytosolic leaflet rapidly moved in and out of domains. Thus, the novel SM-binding probe demonstrated the presence of the raft-like domain in the cytosolic leaflet of living cell PMs.

cell biology↗

bioRxiv2023

Genome-scale CRISPR-Cas9 screen identifies novel host factors as potential therapeutic targets for SARS-CoV-2 infection.

Although many host factors important for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection have been reported, the mechanisms by which the virus interacts with host cells remain elusive. Here, we identified tripartite motif containing (TRIM) 28, TRIM33, euchromatic histone lysine methyltransferase (EHMT) 1, and EHMT2 as novel proviral factors involved in SARS-CoV-2 infection by CRISPR-Cas9 screening. We demonstrated that TRIM28 plays a role(s) in viral particle formation and that TRIM33, EHMT1, and EHMT2 are involved in viral transcription and replication using cells with suppressed gene expression. UNC0642, a compound that specifically inhibits the methyltransferase activity of EHMT1/2, strikingly suppressed SARS-CoV-2 growth in cultured cells and reduced disease severity in a hamster infection model. This study suggests that EHMT1/2 may be a novel therapeutic target for SARS-CoV-2 infection.

microbiology↗

bioRxiv2022

Contact-number-driven virus evolution: a multi-level modeling framework for the evolution of acute or persistent RNA virus infection

Viruses evolve in infected host populations, and host population dynamics affect viral evolution. RNA viruses with a short duration of infection and a high peak viral load, such as and SARS-CoV-2, are maintained in human populations. By contrast, RNA viruses characterized by a long infection duration and a low peak viral load (e.g., borna disease virus) can be maintained in nonhuman populations, and why the persistent viruses evolved has been rarely explored. Here, using a multi-level modeling approach including both individual-level virus infection dynamics and population-scale transmission, we consider virus evolution based on the host environment, specifically, the effect of the contact history of infected hosts. We found that, with a highly dense contact history, viruses with a high virus production rate but low accuracy are likely to be optimal, resulting in a short infectious period with a high peak viral load. In contrast, with a low-density contact history, viral evolution is toward low virus production but high accuracy, resulting in long infection durations with low peak viral load. Our study sheds light on the origin of persistent viruses and why acute viral infections but not persistent virus infection tends to prevail in human society.

evolutionary biology↗

bioRxiv2022

Ban on wild bird importation accelerated the spread of global viral outbreaks in parrots.

Since the isolation of the first parrot bornavirus (PaBV), which causes slow-onset, fatal neurological disease in various parrot species, in the United States in 2008, PaBVs have spread rapidly worldwide; however, the reason remains unknown. In this study, we show that the most dominant lethal genotype, PaBV-4, spread via the global trade of captive birds. Analyses of traded parrot numbers in the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) database and PaBV-4 phylodynamics suggested that the ban of wild imported birds in the European Union (EU) in 2007 facilitated the international trade of captive parrots, which resulted in an increase in the effective population size of PaBV-4. This change coincided with a historical PaBV-4 epidemic. These data suggest that due to the low transmission efficiency and long incubation period of PaBV-4, the majority of PaBV-4 transmission occurred in breeding facilities and the increased trade of captive parrots accelerated the global spread of PaBV-4 infection. Our results indicate that interventions for the protection of wild animals and prevention of infectious diseases may conversely cause epidemics of infectious diseases in the global system. Conservation of ecosystems requires not only the establishment of importation restrictions and maintenance of the diversity of wild animals but also the implementation of multifaceted management measures, such as quarantine policies and breeding control in captive animals.

microbiology↗

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↗

bioRxiv2021

Manganese toxicity disrupts indole acetic acid homeostasis and suppresses CO2 assimilation reaction in rice plants

Despite the essentiality of Mn in terrestrial plants, its excessive accumulation in plant tissues causes growth defects, known as Mn toxicity. Mn toxicity can be divided into apoplastic and symplastic types depending on its onset. For growth defects, symplastic rather than apoplastic Mn toxicity is hypothesised to be more critical. However, details of the relationship between growth defects and symplastic Mn toxicity remains elusive. In this study, we aimed to elucidate the molecular mechanisms of symplastic Mn toxicity in rice plants. We found that under excess Mn conditions, CO2 assimilation was inhibited by stomatal closure, and both carbon anabolic and catabolic activities were decreased. In addition to stomatal dysfunction, stomatal and leaf anatomical development were also altered by excess Mn accumulation. Furthermore, the indole acetic acid (IAA) concentration was decreased, and auxin-responsive gene expression analyses showed IAA-deficient symptoms in leaves due to excess Mn accumulation. These results suggest that excessive Mn accumulation causes IAA deficiency, and low IAA concentrations suppress plant growth by suppressing stomatal opening and leaf anatomical development for efficient CO2 assimilation in leaves. HIGHLIGHTIncreased Mn concentration lowers auxin concentrations in rice leaves, which suppresses photosynthesis by changing stomatal function and development.

plant biology↗