Biology subjects

Yoshida, S.

Publications and source records attributed to Yoshida, S..

51 records · Page 3Linked to original sources

bioRxiv2021

The Phtheirospermum japonicum isopentenyltransferase PjIPT1a regulates host cytokinin responses in Arabidopsis

The hemiparasitic plant Phtheirospermum japonicum is a nutritional specialist that supplements its nutrient requirements by parasitizing other plants through haustoria. During parasitism, the Phtheirospermum haustorium transfers hypertrophy-inducing cytokinins (CKs) to the infected host root. The CK biosynthesis genes required for haustorium-derived CKs and the induction of hypertrophy are still unknown. We searched for haustorium-expressed isopentenyltransferases (IPTs) that catalyse the first step of CK biosynthesis, confirmed the specific expression by in vivo imaging of a promoter-reporter, and further analysed the subcellular localization, the enzymatic function, and contribution to inducing hypertrophy by studying CRISPR-Cas9 induced Phtheirospermum mutants. PjIPT1a was expressed in intrusive cells of the haustorium close to the host vasculature. PjIPT1a and its closest homolog PjIPT1b located to the cytosol and showed isopentenyltransferases activity in vitro with differences in substrate specificity. Mutating PjIPT1a abolished parasite-induced CK responses in the host. A homolog of PjIPT1a with shared characteristics was also identified in the related weed Striga hermonthica. We propose that PjIPT1a exemplifies how parasitism-related functions evolve through gene duplications and neofunctionalization.

plant biology↗

bioRxiv2020

Computationally-efficient spatiotemporal correlation analysis super-resolves anomalous diffusion

Anomalous diffusion dynamics in confined nanoenvironments govern the macroscale properties and interactions of many biophysical and material systems. Currently, it is difficult to quantitatively link the nanoscale structure of porous media to anomalous diffusion within them. Fluorescence correlation spectroscopy super-resolution optical fluctuation imaging (fcsSOFI) has been shown to extract nanoscale structure and Brownian diffusion dynamics within gels, liquid crystals, and polymers, but has limitations which hinder its wider application to more diverse, biophysically-relevant datasets. Here, we parallelize the least-squares curve fitting step on a GPU improving computation times by up to a factor of 40, implement anomalous diffusion and two-component Brownian diffusion models, and make fcsSOFI more accessible by packaging it in a user-friendly GUI. We apply fcsSOFI to simulations of the protein fibrinogen diffusing in polyacrylamide of varying matrix densities and super-resolve locations where slower, anomalous diffusion occurs within smaller, confined pores. The improvements to fcsSOFI in speed, scope, and usability will allow for the wider adoption of super-resolution correlation analysis to diverse research topics.

biophysics↗

bioRxiv2020

Preclinical study of DNA vaccines targeting SARS-CoV-2

To fight against the worldwide COVID-19 pandemic, the development of an effective and safe vaccine against SARS-CoV-2 is required. As potential pandemic vaccines, DNA/RNA vaccines, viral vector vaccines and protein-based vaccines have been rapidly developed to prevent pandemic spread worldwide. In this study, we designed plasmid DNA vaccine targeting the SARS-CoV-2 Spike glycoprotein (S protein) as pandemic vaccine, and the humoral, cellular, and functional immune responses were characterized to support proceeding to initial human clinical trials. After intramuscular injection of DNA vaccine encoding S protein with alum adjuvant (three times at 2-week intervals), the humoral immunoreaction, as assessed by anti-S protein or anti-receptor-binding domain (RBD) antibody titers, and the cellular immunoreaction, as assessed by antigen-induced IFN{gamma} expression, were up-regulated. In IgG subclass analysis, IgG2b was induced as the main subclass. Based on these analyses, DNA vaccine with alum adjuvant preferentially induced Th1-type T cell polarization. We confirmed the neutralizing action of DNA vaccine-induced antibodies via two different methods, a binding assay of RBD recombinant protein with angiotensin-converting enzyme 2 (ACE2), a receptor of SARS-CoV-2, and pseudovirus assay. Further B cell epitope mapping analysis using a peptide array showed that most vaccine-induced antibodies recognized the S2 and RBD subunits, but not the S1 subunit. In conclusion, DNA vaccine targeting the spike glycoprotein of SARS-CoV-2 might be an effective and safe approach to combat the COVID-19 pandemic.

immunology↗

bioRxiv2020

SARS-CoV-2-induced humoral immunity through B cell epitope analysis and neutralizing activity in COVID-19 infected individuals in Japan

The aim of this study is to understand adaptive immunity to SARS-CoV-2 through the analysis of B cell epitope and neutralizing activity in coronavirus disease 2019 (COVID-19) patients. We obtained serum from thirteen COVID-19 patients. Most individuals revealed neutralizing activity against SARS-CoV-2 assessed by a pseudotype virus-neutralizing assay. The antibody production against the spike glycoprotein (S protein) or receptor-binding domain (RBD) of SARS-CoV-2 was elevated, with large individual differences, as assessed by ELISA. In the analysis of the predicted the linear B cell epitopes, two regions (671-690 aa. and 1146-1164 aa.), which were located in S1 and S2 but not in the RBD, were highly reactive with the sera from patients. In the further analysis of the B cell epitope within the S protein by utilizing a B cell epitope array, a hot spot in the N-terminal domain of the S protein but not the RBD was observed in individuals with neutralizing activity. Overall, the analysis of antibody production and B cell epitopes of the S protein from patient serum may provide a novel target for the vaccine development against SARS-CoV-2.

immunology↗

bioRxiv2020

EXOC1 regulates cell morphology of spermatogonia and spermatocytes in mice

Spermatogenesis requires high regulation of germ cell morphology. The spermatogonia regulates its differentiation state by its own migration. The male germ cells differentiate and mature with the formation of syncytia, failure of forming the appropriate syncytia results in the arrest of spermatogenesis at the spermatocyte stage. However, the detailed molecular mechanisms of male germ cell morphological regulation are unknown. Here, we found that EXOC1 is important for the pseudopod formation of spermatogonia and spermatocyte syncytia in mice. We found that while EXOC1 contributes to the inactivation of Rac1 in the pseudopod formation of spermatogonia, in spermatocyte syncytium formation, EXOC1 and SNAP23 cooperate with STX2. Our results showed that EXOC1 functions in concert with various cell morphology regulators in spermatogenesis. Since EXOC1 is known to bind to several cell morphogenesis factors, this study is expected to be the starting point for the discovery of many morphological regulators of male germ cells.

developmental biology↗

bioRxiv2020

Stable kinetochore-microtubule attachments restrict MTOC position and spindle elongation in acentrosomal oocytes

In mouse oocytes, acentriolar MTOCs functionally replace centrosomes and act as microtubule nucleation sites. Microtubules nucleated from MTOCs initially assemble into an unorganized ball-like structure, which then transforms into a bipolar spindle carrying MTOCs at its poles, a process called spindle bipolarization. In mouse oocytes, spindle bipolarization is promoted by kinetochores but the mechanism by which kinetochore-microtubule attachments contribute to spindle bipolarity remains unclear. This study demonstrates that the stability of kinetochore-microtubule attachment is essential for confining MTOC positions at the spindle poles and for limiting spindle elongation. MTOC sorting is gradual and continues even in the metaphase spindle. When stable kinetochore-microtubule attachments are disrupted, the spindle is unable to restrict MTOCs at its poles and fails to terminate its elongation. Stable kinetochore fibers are directly connected to MTOCs and to the spindle poles, and thus may serve as a measure that defines proper spindle length. These findings reinforce the hypothesis that kinetochores act as scaffolds for acentrosomal spindle bipolarity.

cell biology↗

bioRxiv2020

Three-dimensional reconstructions of the internal structures of haustoria in parasitic Orobanchaceae

Parasitic plants infect other plants by forming haustoria, specialized multicellular organs consisting of several cell types each of which has unique morphological features and physiological roles associated with parasitism. Understanding the spatial organization of cell types is, therefore, of great importance in elucidating the functions of haustoria. Here, we report a three-dimensional (3-D) reconstruction of haustoria from two Orobanchaceae species, the obligate parasite Striga hermonthica infecting rice and the facultative parasite Phtheirospermum japonicum infecting Arabidopsis. Our images reveal the spatial arrangements of multiple cell types inside haustoria and their interaction with host roots. The 3-D internal structures of haustoria highlight differences between the two parasites, particularly at the xylem connection site with the host. Our study provides structural insights into how organs interact between hosts and parasitic plants. One-sentence summaryThree-dimensional image reconstruction was used to visualize the spatial organization of cell types in the haustoria of parasitic plants with special reference to their interaction with host roots.

plant biology↗

bioRxiv2020

Subtilase activity in the intrusive cells mediates haustorium maturation in parasitic plants

Parasitic plants that infect crops are devastating to agriculture throughout the world. They develop a unique inducible organ called the haustorium, which connects the vascular systems of the parasite and host to establish a flow of water and nutrients. Upon contact with the host, the haustorial epidermal cells at the interface with the host differentiate into specific cells called intrusive cells that grow endophytically towards the host vasculature. Then, some of the intrusive cells re-differentiate to form a xylem bridge that connects the vasculatures of the parasite and host. Despite the prominent role of intrusive cells in host infection, the molecular mechanisms mediating parasitism in the intrusive cells are unknown. In this study, we investigated differential gene expression in the intrusive cells of the facultative parasite Phtheirospermum japonicum in the family Orobanchaceae by RNA-Sequencing of laser-microdissected haustoria. We then used promoter analyses to identify genes that are specifically induced in intrusive cells, and used promoter fusions with genes encoding fluorescent proteins to develop intrusive cell-specific markers. Four of the intrusive cell-specific genes encode subtilisin-like serine proteases (SBTs), whose biological functions in parasitic plants are unknown. Expression of an SBT inhibitor in the intrusive cells inhibited their development, inhibited the development of the xylem bridge, and reduced auxin response levels near the site where the xylem bridge normally develops. Therefore, we propose that subtilase activity plays an important role in haustorium development in this parasitic plant. One sentence summaryTissue-specific analysis showed that the subtilases specifically expressed in intrusive cells regulate auxin-mediated host-parasite connections in the parasitic plant Phtheirospermum japonicum.

plant biology↗

bioRxiv2019

Auxin transport network underlies xylem bridge formation between the hemi-parasitic plant Phtheirospermum japonicum and host Arabidopsis

Parasitic plants form vascular connections to host plants for efficient material transport. The haustorium is the responsible organ for host invasion and subsequent vascular connection. After invasion of host tissues, vascular meristem-like cells emerge in the central region of the haustorium, differentiate into tracheary elements, and establish a connection, known as a xylem bridge, between parasite and host xylem systems. Despite the importance of this parasitic connection, the regulatory mechanisms of xylem bridge formation are unknown. Here we show the role of auxin and auxin transporters during the process of xylem bridge formation using an Orobanchaceae hemiparasitic plant, Phtheirospermum japonicum. The auxin response marker DR5 has a similar expression pattern to tracheary element differentiation genes in haustoria. Auxin transport inhibitors alter tracheary element differentiation in haustoria, but biosynthesis inhibitors do not, demonstrating the importance of auxin transport during xylem bridge formation. The expression patterns and subcellular localization of PIN family auxin efflux carriers and AUX/LAX influx carriers correlate with DR5 expression patterns. The cooperative action of auxin transporters is therefore responsible for controlling xylem vessel connections between parasite and host.

plant biology↗

bioRxiv2019

AMP-activated protein kinase and adenylate kinase prevent ATP catastrophe and cytotoxic protein aggregation

ATP at millimolar levels has recently been implicated in the solubilization of cellular proteins. However, the significance of this high ATP level under physiological conditions and the mechanisms that maintain ATP remain unclear. We herein demonstrated that AMP-activated protein kinase (AMPK) and adenylate kinase (ADK) cooperated to maintain cellular ATP levels regardless of glucose levels. Single cell imaging of ATP-reduced yeast mutants revealed that ATP levels in these mutants repeatedly underwent stochastic and transient depletion, which induced the cytotoxic aggregation of endogenous proteins and pathogenic proteins, such as huntingtin and -synuclein. Moreover, pharmacological elevations in ATP levels in an ATP-reduced mutant prevented the accumulation of -synuclein aggregates and its cytotoxicity. The removal of cytotoxic aggregates depended on proteasomes, and proteasomal activity cooperated with AMPK or ADK to resist proteotoxic stresses. The present study is the first to demonstrate that cellular ATP homeostasis ensures proteostasis and revealed that suppressing the high volatility of cellular ATP levels prevented cytotoxic protein aggregation, implying that AMPK and ADK are important factors that prevent proteinopathies, such as neurodegenerative diseases.

cell biology↗

bioRxiv2018

A SOSEKI-based coordinate system interprets global polarity cues in Arabidopsis

Multicellular development requires coordinated cell polarization relative to body axes, and translation to oriented cell division. In plants, it is unknown how cell polarities are connected to organismal axes and translated to division. Here, we identify Arabidopsis SOSEKI (SOK) proteins that integrate apical-basal and radial organismal axes to localize to polar cell edges. Localization does not depend on tissue context, requires cell wall integrity and is defined by a transferrable, protein-specific motif. SOK proteins structurally resemble the DIX oligomerization domain in the animal Dishevelled polarity regulator. The DIX-like domain self-interacts and is required for edge localization and for influencing division orientation. Our work identifies a plant compass, interpreted by SOK proteins. Furthermore, despite fundamental differences, polarity in plants and animals converge upon the same protein domain.

plant biology↗

bioRxiv2018

Characteristic Localization of Neuronatin in Rat Tissues

Neuronatin (Nnat) is expressed in the pituitary, pancreas, and other tissues; however, the function of NNAT is still unclear. Recent studies have demonstrated that NNAT is localized in the sex determining region Y-box 2-positive stem/progenitor cells in the developing rat pituitary primordium and is downregulated during differentiation into mature hormone-producing cells. Moreover, NNAT is widely localized in subcellular organelles, excluding the Golgi. Here, we further evaluated NNAT expression and intracellular localization in embryonic and postnatal rat tissues such as the pancreas, tongue, whisker hair follicle, and testis. Immunohistochemistry showed that NNAT was localized in undifferentiated cells (i.e., epithelial basal cells and basement cells in the papillae of the tongue and round and elongated spermatids of the testis) as well as in differentiated cells (insulin-positive cells and exocrine cells of the pancreas, taste receptor cells of the fungiform papilla, the inner root sheath of whisker hair follicles, and spermatozoa). Additionally, NNAT showed novel intracellular localization in acrosomes in the spermatozoa. Because the endoplasmic reticulum (ER) is excluded from spermatozoa and sarco/ER Ca2+-ATPase isoform 2 (SERCA2) is absent from the inner root sheath, these findings suggested that NNAT localization in the ER and its interaction with SERCA2 were cell-or tissue-specific properties.

developmental biology↗

bioRxiv2018

Dorsal Ruffles Enhance Activation of Akt by Growth Factors

In fibroblasts, platelet-derived growth factor (PDGF) stimulates macropinocytosis and PI 3-kinase (PI3K)-dependent phosphorylation of Akt, leading to activation of mTORC1, a protein complex controlling metabolism and cell growth. PIP3, the phosphoinositide product of PI3K that activates Akt, is frequently concentrated within the macropinocytic cups of growth factor-stimulated cells, which suggests that cup structure enhances phosphorylation of Akt by facilitating PI3K activity. However, inhibitors of the cytoskeleton which block cup formation do not reduce Akt phosphorylation in response to high concentrations of PDGF. Because the dynamics of Akt phosphorylation after stimulation by PDGF can differ from those that follow stimulation with epidermal growth factor (EGF), we analyzed the contributions of the actin and microtubule cytoskeleton to activation of Akt by these two growth factors. Actin-rich, circular dorsal ruffles (CDR), analogous to macropinocytic cups, appeared within several minutes of adding EGF or PDGF and often closed to form macropinosomes. Nocodazole, an inhibitor of microtubule polymerization, blocked both PDGF- and EGF-induced CDR formation, and inhibited phosphorylation of Akt in response to EGF but not PDGF. At concentrations that saturate their cognate receptors, EGF stimulated lower maximal levels of Akt phosphorylation than did PDGF. We hypothesized that weak signals elicited by EGF receptors require cytoskeleton-dependent amplification of PI3K for maximal phosphorylation of Akt. In both PDGF- and EGF-stimulated cells, quantitative immunofluorescence showed increased Akt phosphorylation in cells containing CDR, with PIP3 and Akt concentrated in CDR and ruffles. Stimulation with low concentrations of PDGF elicited lower levels of Akt phosphorylation, which, like responses to EGF, were inhibited by nocodazole. These results indicate that when receptor signaling generates low levels of PI3K activity, CDR facilitate local amplification of PI3K, PIP3 synthesis and phosphorylation of Akt.

cell biology↗

bioRxiv2018

Baculovirus-inducing fast-acting innate immunity kills Plasmodium liver stages

Baculovirus (BV), an enveloped insect virus with a circular double-stranded DNA genome, possesses unique characteristics that induce strong innate immune responses in mammalian cells. Here, we show that BV administration not only sterilely protects BALB/c mice for at least 7 days from subsequent Plasmodium berghei sporozoite infection but also eliminates existing liver-stage parasites completely, effects superior to those of primaquine, and does so in a TLR9-independent manner. Six hours post-BV administration, IFN- and IFN-{gamma} were robustly produced in serum, and RNA transcripts of interferon-stimulated genes were drastically upregulated in the liver. The in vivo passive transfer of post-BV administration serum effectively eliminated liver-stage parasites, and IFN- neutralization abolished this effect, indicating that the BV liver-stage parasite killing mechanism is downstream of the type I IFN signaling pathway. Our results demonstrate that BV is a potent IFN-inducing prophylactic and therapeutic agent with great potential for further development as a new malaria vaccine and/or anti-hypnozoite drug.

immunology↗

bioRxiv2017

Loss Of PTEN Promotes Formation Of Signaling-Specific Clathrin-Coated Pits

Defective endocytosis and vesicular trafficking of signaling receptors has recently emerged as a multifaceted hallmark of malignant cells. Clathrin-coated pits (CCPs), the fundamental unit of clathrin-mediated endocytosis, display highly heterogeneous dynamics on the plasma membrane where they can take from 20 seconds to over a minute to form cytosolic coated-vesicles. Despite the large number of cargo molecules that traffic through CCPs, it is not well understood whether signaling receptors activated in cancer, such as epidermal growth factor receptor (EGFR), are regulated through a specific subset of CCPs. The signaling lipid phosphatidylinositol (3,4,5)-triphosphate (PI(3,4,5)P3), which is dephosphorylated by phosphatase tensin homolog (PTEN), is a potent tumorigenic signaling lipid that is present in excess in many types of cancers. Using total internal reflection fluorescence microscopy and automated tracking and detection of CCPs, we find PTEN and EGF bound EGFR are enriched in a distinct subset of short-lived CCPs that corresponded with clathrin-dependent EGF-induced signaling. By deleting PTEN using CRISPR-Cas9 and reconstituting PTEN, we demonstrate that PTEN plays a role in the regulation of CCP dynamics; this appears to recapitulate CCP dynamics in highly metastatic PTEN-deleted cancer cells where we find a larger proportion of short-lived CCPs and higher initiation density compared to the normal cells. Furthermore, increased PI(3,4,5)P3 results in higher proportion of short-lived CCPs, an effect that recapitulates PTEN deletion. Our findings provide strong evidence for the existence of short-lived signaling-capable CCPs. Altogether, these findings demonstrate the importance of PTEN and PI(3,4,5)P3 in regulating CCP dynamics and assign a new function to PTEN as a modulator of signaling-capable CCPs.

cell biology↗