Biology subjects

Watanabe, S.

Publications and source records attributed to Watanabe, S..

At least 91 records · Page 5Linked to original sources

bioRxiv2021

Metastatic profiling of HER2-positive breast cancer cell lines in xenograft models

Most studies on breast cancer metastasis have been performed using triple-negative breast cancer (TNBC) cells; thus, subtype-dependent metastatic ability of breast cancer is poorly understood. In this research, we performed intravenous injection (IVI) and intra-caudal arterial injections (CAI) using nine human epidermal growth factor receptor-2 (HER2)-positive breast cancer cell lines for evaluating their metastatic abilities. Our results showed that MDA-MB-453, UACC-893, and HCC-202 had strong bone metastatic abilities, whereas HCC-2218 and HCC-1419 did not show bone metastasis. HER2-positive cell lines could hardly metastasize to the lung through IVI. From the genomic analysis, gene signatures were extracted according to the breast cancer subtypes and their metastatic preferences. The UACC-893 cell line was identified as a useful model for the metastasis study of HER2-positive breast cancer. Combined with our previous result on brain proliferation ability, we provide a characteristic metastasis profile of HER2-positive breast cancer cell lines in this study. Statements and DeclarationsO_ST_ABSFundingC_ST_ABSThis study was supported by JSPS KAKENHI (grant no. 18K16269: Grant-in-Aid for Early-Career Scientist to J.N.; grant no. 20J01794, Grant-in-Aid for JSPS fellows to J.N.; grant no. 20J23297, Grant-in-Aid for JSPS fellows to Y.H.) and partially supported by the grants for translational research programs from Fukushima Prefecture (S.W. and K.S.). AuthorshipYH and KA performed the in vivo experiments and bioinformatical analyses. SW, and KS interpreted the data. YH, KA, and JN wrote the manuscript. JN conceived and designed the study. All the authors reviewed and edited the manuscript. Competing InterestsThe authors declare that they have no competing interests. Ethical approvalThe animal experiments were conducted under the approval of the ethics committee of Waseda University (2020-A067, 2021-A074).

cancer biology↗

bioRxiv2021

Guanosine-specific single-stranded ribonuclease effectors of a phytopathogenic fungus potentiate host immune responses

Plants activate immunity upon recognition of pathogen-associated molecular patterns. Although phytopathogens have evolved a set of effector proteins to counteract plant immunity, some effectors are perceived by hosts and induce immune responses. Here, we show that two secreted ribonuclease effectors, SRN1 and SRN2, encoded in a phytopathogenic fungus, Colletotrichum orbiculare, induce cell death in a signal peptide- and catalytic residue-dependent manner, when transiently expressed in Nicotiana benthamiana. The pervasive presence of SRN genes across Colletotrichum species suggested the conserved roles. Using a transient gene expression system in cucumber (Cucumis sativus), an original host of C. orbiculare, we show that SRN1 and SRN2 potentiate host pattern-triggered immunity. Consistent with this, C. orbiculare SRN1 and SRN2 deletion mutants exhibited increased virulence on the host. In vitro analysis revealed that SRN1 specifically cleaves single-stranded RNAs at guanosine, leaving a 3'-end phosphate. This activity has not been reported in plants. Importantly, the potentiation of C. sativus responses by SRN1 and SRN2 depends on the signal peptide and ribonuclease catalytic residues, suggesting that secreted SRNs cleave RNAs in apoplast and are detected by the host. We propose that the pathogen-derived apoplastic guanosine-specific single-stranded endoribonucleases lead to immunity potentiation in plants.

microbiology↗

bioRxiv2021

Defunctionalizing Intracellular Organelles with Genetically-Encoded Molecular Tools Based on Engineered Phospholipase A/Acyltransferases (PLAATs)

Organelles vitally achieve multifaceted functions to maintain cellular homeostasis. Genetic and pharmacological approaches to manipulate individual organelles are powerful in probing their physiological roles. However, many of them are either slow in action, limited to certain organelles, or rely on toxic agents. Here, we designed a generalizable molecular tool utilizing phospholipase A/acyltransferases (PLAATs) for rapid induction of organelle defunctionalization via remodeling of the membrane phospholipid composition. In particular, we identified a minimal, fully catalytic PLAAT with no unfavorable side effects. Chemically-induced translocation of the engineered PLAAT to the mitochondria surface resulted in their rapid deformation in a phospholipase activity dependent manner, followed by loss of luminal proteins as well as dissipated membrane potential, thus invalidating the functionality. To demonstrate wide applicability, we then adapted the molecular tool in peroxisomes, and observed leakage of matrix-resident functional proteins. The technique was compatible with optogenetic control, viral delivery and operation in primary neuronal cultures. Due to such versatility, the PLAAT strategy should present a novel utility in organelle biology of diverse contexts.

cell biology↗

bioRxiv2021

Lysophosphatidylcholine acyltransferase 1 controls the mitochondrial reactive oxygen species generation and survival of the retinal photoreceptor cells

Due to the high energy demands and characteristic morphology, retinal photoreceptor cells require a specialized lipid metabolism for survival and function. Accordingly, dysregulation of lipid metabolism leads to the photoreceptor cell death and retinal degeneration. Mice with a frameshift mutation of lysophosphatidylcholine acyltransferase 1 (Lpcat1), which produces saturated phosphatidylcholine (PC) composed of two saturated fatty acids, has been reported to cause spontaneous retinal degeneration (rd11 mice). In this study, we performed a detailed characterization of LPCAT1 in the retina and found that genetic deletion of Lpcat1 induces light-independent and photoreceptor-specific apoptosis in mice. Lipidomic analyses of the retina and isolated photoreceptor outer segment (OS) suggested that loss of Lpcat1 decreases saturated PC production and affects the proper cellular fatty acid flux, presumably by altering saturated fatty acyl-CoA availabilities. Furthermore, we demonstrated that Lpcat1 deletion increased mitochondrial reactive oxygen species (ROS) levels in photoreceptor cells, but not in other retinal cells without affecting the OS structure and trafficking of OS-localized proteins. These results suggest that the LPCAT1-dependent production of saturated PC is critical for metabolic adaptation during photoreceptor maturation. Our findings highlight the therapeutic potential of saturated fatty acid metabolism in photoreceptor cell degeneration-related retinal diseases.

biochemistry↗

bioRxiv2021

Cryo-sensitive aggregation triggers NLRP3 inflammasome assemblyin cryopyrin-associated periodic syndrome

Cryopyrin-associated periodic syndrome (CAPS) is an autoinflammatory syndrome caused by mutations of NLRP3, which was originally identified as cryopyrin. Familial cold autoinflammatory syndrome (FCAS), the mildest form of CAPS, is characterized by cold-induced inflammation induced by the overproduction of IL-1{beta}. However, the molecular mechanism of how mutated NLRP3 causes inflammasome activation in CAPS remains unclear. Here, we found that CAPS-associated NLRP3 mutants form cryo-sensitive aggregates that function as a scaffold for inflammasome activation. Cold exposure promoted inflammasome assembly and subsequent IL-1{beta} release triggered by mutated NLRP3. While K+ efflux was dispensable, Ca2+ was indispensable for mutated NLRP3-mediated inflammasome assembly. Notably, Ca2+ influx was induced during mutated NLRP3-mediated inflammasome assembly. Furthermore, caspase-1 inhibition prevented Ca2+ influx and inflammasome assembly induced by the mutated NLRP3, suggesting a feed-forward Ca2+ influx loop triggered by mutated NLRP3. Thus, the mutated NLRP3 forms cryo-sensitive aggregates to promote inflammasome assembly distinct from canonical NLRP3 inflammasome activation.

immunology↗

bioRxiv2021

Aberrant accumulation of NIK promotes tumorigenicity by dysregulating post-translational modifications in breast cancer

Post-translational modifications and mRNA translation are frequently altered in human cancers. However, investigations to understand their roles in the cancer progression mechanism remain insufficient. In this research, we explored protein levels altered by translational or post-translational regulation by analyzing transcriptome and western blotting data of the highly malignant breast cancer cell lines. From these analyses, NIK was found to be upregulated at the protein level to predominantly activate the non-canonical NF-{kappa}B pathway in a breast cancer cell line. Furthermore, the increase in NIK protein production was attributed to the dysregulation of ubiquitin-proteasome system caused by a decrease in the translation of cIAP1. NIK upregulation contributed to tumorigenicity by regulating the expression of inflammatory response-related genes. Collectively, our study suggests that NIK is post-translationally modified and has the potential to be a therapeutic target and diagnostic marker for breast cancer.

cancer biology↗

bioRxiv2021

Predicting behavior through dynamic modes in resting-state fMRI data

Dynamic properties of resting-state functional connectivity (FC) provide rich information on brainbehavior relationships. Dynamic mode decomposition (DMD) has been used as a method to characterize FC dynamics. However, it remains unclear whether dynamic modes (DMs), spatial-temporal coherent patterns computed by DMD, provide information about individual behavioral differences. This study established a methodological approach to predict individual differences in behavior using DMs. Furthermore, we investigated the contribution of DMs within each of seven specific frequency bands (0-0.1,...,0.6-0.7 Hz) for prediction. To validate our approach, we confirmed whether each of 59 behavioral measures could be predicted by performing multivariate pattern analysis on a gram matrix, which was created using subject-specific DMs computed from resting-state functional magnetic resonance imaging (rs-fMRI) data of individuals. The prediction was successful, and DMD outperformed temporal independent component analysis, a conventional data decomposition method for extracting spatial activity patterns. Most of the behavioral measures that showed significant prediction accuracies in a permutation test were cognitive-behavioral measures. Our results suggested that DMs within frequency bands <0.2 Hz primarily contributed to prediction. In addition, we found that DMs <0.2 Hz had spatial structures similar to several common resting-state networks. We demonstrated the effectiveness of DMs, indicating that DMD is a key approach for extracting spatiotemporal features from rs-fMRI data.

neuroscience↗

bioRxiv2021

HOXB7 induces oncogenic transformation in NMuMG cells via JAK2-STAT3 signaling

The homeobox family genes are often dysregulated in a various cancer type. Particularly HOXB7 amplification and overexpression correlate with poor prognosis in various cancer such as gastric, pancreatic, and lung cancers. Moreover, HOXB7 is known to contribute to cancer progression by promoting epithelial to mesenchymal transition, anti-cancer drug resistance, and angiogenesis. In this study, we show that HOXB7 is coamplified with ERBB2 in a subset of breast cancer patients and HOXB7 expression correlates with poor prognosis in HER2-positive breast cancer patients. This clinical observation is supported by the following results: HOXB7 overexpression in an immortalized murine mammary gland epithelial cell line NMuMG induces cellular transformation in vitro, tumorigenesis and lung metastasis through the activation of JAK-STAT signaling.

cancer biology↗

bioRxiv2021

Dynamin is primed at endocytic sites for ultrafast endocytosis

Dynamin mediates fission of vesicles from the plasma membrane during endocytosis. Typically, dynamin is recruited from the cytosol to endocytic sites, requiring seconds to tens of seconds. However, ultrafast endocytosis in neurons internalizes vesicles as quickly as 50 ms during synaptic vesicle recycling. Here we demonstrate that Dynamin 1 is pre-recruited to endocytic sites for ultrafast endocytosis. Specifically, Dynamin 1xA, a splice variant of Dynamin 1, interacts with Syndapin 1 to form molecular condensates on the plasma membrane when the proline-rich domain of this variant is dephosphorylated. When this domain is mutated to include phosphomimetic residues or Syndapin 1s dynamin-interacting domain is mutated, Dynamin 1xA becomes diffuse, and consequently, ultrafast endocytosis slows down by [~]100-fold. Mechanistically, Syndapin 1 acts as an adaptor by binding the plasma membrane and stores Dynamin 1xA at endocytic sites. This cache bypasses the recruitment step and accelerates endocytosis at synapses.

neuroscience↗

bioRxiv2021

Synaptotagmin 7 is enriched at the plasma membrane to promote vesicle docking and control synaptic plasticity

Synaptotagmin (SYT) 7 has emerged as key regulator of presynaptic function, but its localization and precise function in the synaptic vesicle cycle remain unclear. Here, we used iGluSnFR to optically and directly interrogate glutamate release, at the single bouton level, in SYT7 KO dissociated mouse hippocampal neurons. We analyzed asynchronous release, paired pulse facilitation, and synaptic vesicle replenishment, and found that SYT7 contributes to each of these processes to different degrees. Zap-and-freeze electron microscopy revealed that loss of SYT7 impairs the docking of synaptic vesicles after a stimulus and the recovery of depleted synaptic vesicles after a stimulus train. To execute these functions, SYT7 must be targeted to the plasma membrane via {gamma}-secretase-mediated cleavage of the amino terminus, followed by palmitoylation. The complex sorting itinerary of SYT7 endows this Ca2+-sensor with the ability to control crucial forms of synaptic function and plasticity. O_LISYT7 mediated asynchronous release, paired pulse facilitation, and synaptic vesicle replenishment was observed optically at individual hippocampal synapses C_LIO_LILocalization, trafficking, and stability of SYT7 is dependent on processing by {gamma}-secretase C_LIO_LIShort term plasticity defects arise in SYT7KOs due to decreased docking of synaptic vesicles after stimulation C_LIO_LISYT7 promotes paired-pulse facilitation and asynchronous release via distinct mechanisms C_LI

neuroscience↗

bioRxiv2021

Frequent coexistence of closely related tree species on a small scale in temperate evergreen forests in Japan

Understanding how biotic interaction affects species composition and distribution is a major challenge in community ecology. In plants, negative reproductive interaction among closely related species (i.e., reproductive interference) is known to hamper the coexistence of congenic species. Since the magnitude of reproductive interference in plants depends on pollen flow distance, we hypothesized that the coexistence of congeners on a small spatial scale would be less likely to occur by chance but that such coexistence would be likely to occur on a scale larger than pollen flow distance. In the present study, we tested this hypothesis using spatially explicit woody plant survey data. Contrary to our prediction, congenic tree species often coexisted at the finest spatial scale and significant exclusive distribution was not detected. Our results suggest that cooccurrence of congenic tree species is not structured by reproductive interference, and they indicate the need for further research to explore the factors that mitigate the effects of reproductive interference.

ecology↗

bioRxiv2021

Biosynthesis system of Synechan, a sulfated exopolysaccharide, in the model cyanobacterium Synechocystis sp. PCC 6803

Extracellular polysaccharides of bacteria contribute to biofilm formation, stress tolerance, and infectivity. Cyanobacteria, the oxygenic photoautotrophic bacteria, uniquely and widely have sulfated extracellular polysaccharides and they may utilize the polysaccharides for survival in nature. In addition, sulfated polysaccharides of cyanobacteria and other organisms have been focused as beneficial biomaterial. However, very little is known about their biosynthesis machinery and function in cyanobacteria. Here we found that the model cyanobacterium, Synechocystis sp. PCC 6803, formed bloom-like cell aggregates using sulfated extracellular polysaccharides (designated as synechan) and identified whole set of genes responsible for synechan biosynthesis and its transcriptional regulation, thereby suggesting a model for the synechan biosynthesis apparatus. Because similar genes are found in many cyanobacterial genomes with wide variation, our findings may lead elucidation of various sulfated polysaccharides, their functions, and their potential application in biotechnology.

microbiology↗

bioRxiv2020

Light-dependent induction of Edn2 expression and attenuation of retinal pathology by endothelin receptor antagonists in Prominin-1- deficient mice

Retinitis pigmentosa (RP) and macular dystrophy (MD) are prevalent retinal degenerative diseases associated with gradual photoreceptor death. These diseases are often caused by genetic mutations that result in degeneration of the retina postnatally after it has fully developed. The Prominin-1 gene (Prom1) is a causative gene for RP and MD, and Prom1- knockout (KO) mice recapitulate key features of these diseases including light-dependent retinal degeneration and stenosis of retinal blood vessels. The mechanisms underlying progression of such degeneration have remained unknown, however. We here analysed early events associated with retinal degeneration in Prom1-KO mice. We found that photoreceptor cell death and glial cell activation occur between 2 and 3 weeks after birth. High-throughput analysis revealed that expression of the endothelin-2 gene (Edn2) was markedly up-regulated in the Prom1-deficient retina during this period. Expression of Edn2 was also induced by light stimulation in Prom1-KO mice that had been reared in the dark. Finally, treatment with endothelin receptor antagonists attenuated photoreceptor cell death, gliosis, and retinal vessel stenosis in Prom1-KO mice. Our findings suggest that inhibitors of endothelin signalling may delay the progression of RP and MD and therefore warrant further study as potential therapeutic agents for these diseases.

neuroscience↗

bioRxiv2020

Disruption of augmin-mediated microtubule nucleation in neural stem cells causes p53-dependent apoptosis and aborts brain development.

Microtubules that assemble the mitotic spindle are generated by three different mechanisms: centrosomal nucleation, chromatin-mediated nucleation, and nucleation from the surface of other microtubules mediated by the augmin complex. Impairment of centrosomal nucleation in apical progenitors of the developing mouse brain induces p53-dependent apoptosis and causes non-lethal microcephaly. Whether disruption of non-centrosomal nucleation has similar effects is unclear. Here we show, using mouse embryos, that conditional knockout of the augmin subunit Haus6 in apical progenitors led to spindle defects and mitotic delay. This triggered massive apoptosis and complete abortion of brain development. Co-deletion of p53 rescued cell death, but brain development was still aborted. This could be explained by exacerbated mitotic errors and resulting chromosomal defects including increased DNA damage. Surviving progenitors had lost apico-basal polarity and failed to organize a pseudostratified epithelium. Thus, in contrast to the centrosomal nucleation pathway, augmin is crucial for apical progenitor mitosis, and, even in the absence of p53, for progression of brain development.

cell biology↗

bioRxiv2020

Plasma irradiation efficiently inactivates the coronaviruses mouse hepatitis virus and SARS-CoV-2

Many inactivation methods have been shown to inactivate SARS-CoV-2 for safe and efficient diagnostic methods. COVID-19 is caused by airborne infection of SARS-CoV-2, and therefore, methods of inactivating the virus efficiently and safely are crucial for reducing the risk of airborne infection. In this regard, the effect of plasma discharge on the infectivity of the coronaviruses mouse hepatitis virus (MHV) and SARS-CoV-2 was tested. Plasma discharge efficiently reduced the infectivity of both coronaviruses. Treatment of SARS-CoV-2 in culture medium with a plasma discharge resulted in 95.17% viral inactivation after plasma irradiation after 1 hour (hr), 99.54% inactivation after 2 hrs and 99.93% inactivation after 3 hrs. Similar results were obtained for MHV. The results indicated that plasma discharge effectively and safely inactivated the airborne coronaviruses and may be useful in minimizing the risk of airborne infection of SARS-CoV-2.

microbiology↗

bioRxiv2020

Postharvest properties of ultra-late maturing peach cultivars and their attributions to Melting Flesh (M) locus: Re-evaluation of M locus in association with flesh texture

The postharvest properties of two ultra-late maturing peach cultivars, Tobihaku (TH) and Daijumitsuto (DJ), were investigated. Fruit were harvested at commercial maturity and held at 25{degrees}C. TH exhibited the characteristics of normal melting flesh (MF) peach, including rapid fruit softening associated with an increase in endogenous ethylene production In contrast, DJ did not soften at all during three-week experimental period even though substantial ethylene production was observed. Fruit of TH and DJ were treated with 5000 ppm of propylene, an ethylene analog, continuously for seven days. TH softened rapidly whereas DJ maintained high flesh firmness in spite of an increase in endogenous ethylene production, suggesting that DJ but not TH lacked the ability to be softened in response to endogenous and exogenous ethylene/propylene. DNA-seq analysis showed that tandem endo-polygalacturonase (endoPG) genes located at melting flesh (M) locus, Pp-endoPGM (PGM) and Pp-endoPGF (PGF), were deleted in DJ. The endoPG genes at M locus are known to control flesh texture of peach fruit, and it was suggested that the non-softening property of DJ is due to the lack of endoPG genes. On the other hand, TH possessed an unidentified M haplotype that is involved in determination of MF phenotype. Structural identification of the unknown M haplotype, designated as M0, through comparison with previously reported M haplotypes revealed distinct differences between PGM on M0 haplotype (PGM-M0) and PGM on other haplotypes (PGM-M1). Peach M haplotypes were classified into four main haplotypes: M0 with PGM-M0; M1 with both PGM-M1 and PGF; M2 with PGM-M1; and M3 lacking both PGM and PGF. Re-evaluation of M locus in association with MF/non-melting flesh (NMF) phenotypes in more than 400 accessions by using whole genome shotgun sequencing data on database and/or by PCR genotyping demonstrated that M0 haplotype was the common haplotype in MF accessions, and M0 and M1 haplotypes were dominant over M2 and M3 haplotypes and co-dominantly determined the MF trait. It was also assumed on the basis of structural comparison of M haplotypes among Prunus species that the ancestral haplotype of M0 diverged from those of the other haplotypes before the speciation of Prunus persica.

plant biology↗

bioRxiv2020

Ccr2 suppression by minocycline in Cx3cr1/Ccr2-visualized inherited retinal degeneration

Retinal inflammation accelerates photoreceptor cell death (PCD) caused by retinal degeneration. Minocycline, a semisynthetic broad-spectrum tetracycline antibiotic, has previously been reported to show PCD rescue effect in retinal degeneration. The purpose of this study was to assess the effect of minocycline on Cx3cr1 and Ccr2 expression in retinal degeneration. Mertk-/-Cx3cr1GFP/+Ccr2RFP/+ mice, which enabled observation of Cx3cr1- and Ccr2-expression pattern in inherited retinal degeneration, were used to test the effect of minocycline. Minocycline was systemically administered to Mertk-/-Cx3cr1GFP/+Ccr2RFP/+ mice. For observing the effect of minocycline on Cx3cr1 and Ccr2 expression, administration was started on 4-week-old mice and continued for 2 weeks. To assess the PCD rescue effect, minocycline was administered to 6-week-old mice for 2 weeks. The expression pattern of Cx3cr1-GFP and Ccr2-RFP were observed on retinal and retinal pigment epithelium (RPE) flat-mounts. The severity of retinal degeneration was assessed on retinal sections. Minocycline administration suppressed Ccr2 expression in Mertk-/-Cx3cr1GFP/+Ccr2RFP/+ mice as observed in retinal and RPE flat-mounts. On the contrary, Cx3cr1 expression was not affected by minocycline administration. Retinal degeneration is ameliorated in minocycline administered Mertk-/-Cx3cr1GFP/+Ccr2RFP/+ mice. In conclusions, Minocycline suppression of Ccr2 expression correlates to amelioration of retinal degeneration.

neuroscience↗

bioRxiv2020

SynapsEM: computer-assisted synapse morphometry

The structural features of a synapse, in part, determine its output. Synapses are extremely small and tightly packed with vesicles and other organelles. Visualizing synaptic structure requires imaging by electron microscopy, and the features in micrographs must be quantified using morphometry. Three parameters are typically assessed from each specimen: 1) the sizes of individual vesicles and organelles, 2) the absolute number and densities of organelles, and 3) distances between organelles and key features at synapses such as active zone membranes and dense projections. For data to be valid, the analysis must be repeated from hundreds or thousands of images from several biological replicates, a daunting task. Here we report a custom computer program to analyze these features: SynapsEM. In short, we developed macros for ImageJ/Fiji to record x,y-coordinates of segmented structures; the coordinates are then exported as text files. Independent investigators can reload the images and text files to confirm or re-evaluate the segmentation using ImageJ. The Matlab program calculates and reports key synaptic parameters from the coordinates. Since the values are calculated, rather than measured from each micrograph, other parameters can be extracted in Matlab by additional scripting. Thus, this program can accelerate morphometry of synapses and promote a more comprehensive analysis of synaptic ultrastructure.

neuroscience↗