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Dynamic Changes in Reactive Oxygen Species in the Shoot Apex Contribute to Stem Cell Death in Arabidopsis thaliana
No full textIn monocarpic plants, stem cells are fated to die. However, the potential mechanism of stem cell death has remained elusive. Here, we reveal that the levels of two forms of reactive oxygen species (ROS), superoxide anion free radical (O2・-) and hydrogen peroxide (H2O2), show dynamic changes in the shoot apex during the plant life cycle of Arabidopsis thaliana. We found that the level of O2・- decreased and disappeared at four weeks after bolting (WAB), while H2O2 appeared at 3WAB and showed a burst at 5 WAB. The timing of dynamic changes in O2・- and H2O2 was delayed for approximately three weeks in clv3-2, which has a longer lifespan. Moreover, exogenous application of H2O2 inhibited the expression of the stem cell determinant WUSCHEL (WUS) and promoted the expression of the developmentally programmed cell death (dPCD) marker gene ORESARA 1 (ORE1). These results indicate that H2O2 triggers an important signal inducing dPCD in stem cells. Given that O2・- plays roles in maintaining WUS expression and stem cell activity, we speculate that the dynamic shift from O2・- to H2O2 in the shoot apex results in stem cell death. Our findings provide novel insights for understanding ROS-mediated regulation during plant stem cell death.journal articl
Interaction of Proteins with Biomembranes
No full textMany proteins interact with cell and subcellular membranes. The plasma and intracellular membranes are characterized by their different lipid compositions that enable membrane-binding proteins to localize to distinct subcellular compartments. These lipid?protein interactions also regulate protein conformation and protein?protein interactions, which precisely regulate the activation of molecular complexes at the respective membranes. Furthermore, membrane-bound proteins can control lipid lateral diffusion, membrane tension/fluidity, and lipid phase separation. These membrane properties can induce the intracellular signaling that plays crucial roles in various cellular processes such as cell migration, morphogenesis, membrane trafficking, and signal transduction. However, due to the complexity of the abundant protein?protein interactions within a cell, the exact molecular mechanisms underlying protein interactions with lipids contain a lot of unclarity.This Membranes Special Issue, entitled “Interaction of Proteins with Biomembrane”, discusses the recent progress in lipid?protein interactions from various perspectives, including cell biology, biochemistry, and biophysics. These studies will elucidate the mechanisms by which membranes regulate protein localizations/functions, and thus provide new insights into the fundamental principles of lipid?protein interactions. A summary of the research articles is presented here.Motegi et al. [1] studied the formation of phosphatidylinositol (PI)-induced microdomains on supported lipid bilayers using atomic force microscopy (AFM) and single-particle tracking. The authors found that the PI-induced microdomains had less fluidity than the surrounding regions where lipids freely diffused, and thus functioning as diffusion barriers. These PI-induced microdomains acted as a scaffold to promote the initial clustering of FBP17, one of the membrane-remodeling Bin/Amphiphysin/Rvs (BAR) domain proteins. The surrounding fluid region promoted FBP17 assembly through lipid lateral diffusion. This study suggests the possible role of lipid microdomains in the self-assembly of membrane-binding proteins according to their lipid composition and physical properties.To et al. [2] studied the non-structural (NS) protein, NS4A, a membrane protein critical for virulence, and thus flavivirus membrane morphogenesis. The authors found that a peptide containing an N-terminal cytoplasmic tail and one-third of the first transmembrane domain of Zika virus (ZIKV) NS4A formed homotrimers. The authors propose that the disruption of this oligomerization is essential for in vitro screening assays for the antiviral discovery.By using a single quantum dot tracking approach, Kovtun et al. [3] studied the lateral diffusion, nanodomain formation, and their implications in signal transduction of the D2 subtype dopamine receptor (D2DR), a class A G-protein-coupled receptor (GPCR), which has naturally occurring genetic variants in schizophrenia. The authors found a significant decrease in the diffusion dynamics of the Val96Ala D2L schizophrenia variant. By measuring the relative frequency of D2L?D2L interactions, the authors found a significant fraction of D2L receptors and their variants transiently colocalized. The authors also compared nanoclusters of D2DR to those of phosphatidylinositol 4,5-bisphosphate in the plasma membrane.Aalst et al. [4] studied the cholesterol binding of the CC motif chemokine receptor 3 (CCR3), a class A GPCR, which is mainly responsible for the cellular trafficking in eosinophils. CCR3 plays vital roles in inflammatory conditions such as asthma, arthritis and the cancer metastasis. The authors analyzed lipid?protein contacts to identify the potential cholesterol-binding sites in the transmembrane region of CCR3 by using in silico coarse-grained molecular dynamics (MD) using PyLipID. Several cholesterol-binding sites of CCR3 contain a cholesterol recognition/interaction amino acid consensus (CRAC) motif and its inverted CARC motif. Generally, the CARC motif in the transmembrane region is located in the outer membrane leaflet, and its mirror motif, CRAC, is in the inner membrane leaflet. Based on the sequence alignment, these cholesterol-binding sites are conserved not only in CCR3 but also in other CC and CXC motif chemokine receptors. Furthermore, the functional residues in and near these sites were implicated in receptor dimerization, ligand binding, and signal transduction. The authors propose that their findings provide insights into the mechanisms underlying cholesterol regulation of the class A GPCR subfamily.Taken together, the papers in this Special Issue will update our current knowledge of the interaction between proteins and biomembranes. The technical approaches presented here, such as AFM, single-particle tracking, and supported lipid bilayers, will help us understand the spatiotemporal dynamics of protein/lipid lateral diffusion, compartmentalization of the cell membrane, and microdomain formations and their physical properties. These studies will provide new insights into the fundamental principles underlying physiological functions of membrane proteins such as GPCRs and membrane-remodeling proteins in cells and tissues.journal articl
Investigation of CRISPR-Cas9 as a novel method to generate organ-deficient mouse model for blastocyst complementation
Get PDF奈良先端科学技術大学院大学博士(バイオサイエンス)doctoral thesi
Analysis of H. pylori-induced gastric pathology using mouse ES cells-derived gastric organoids
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Modular head-mounted cortical imaging device for chronic monitoring of intrinsic signals in mice
No full textSignificance: Intrinsic optical signals (IOS) generated in the cortical tissue as a result of various interacting metabolic processes are used extensively to elucidate the underlying mechanisms that govern neurovascular coupling. However, current IOS measurements still often rely on bulky, tabletop imaging systems, and there remains a dearth of studies in freely moving subjects. Lightweight, miniature head-mounted imaging devices provide unique opportunities for investigating cortical dynamics in small animals under a variety of naturalistic behavioral settings.Aim: The aim of this work was to monitor IOS in the somatosensory cortex of wild-type mice by developing a lightweight, biocompatible imaging device that readily lends itself to animal experiments in freely moving conditions.Approach: Herein we describe a method for realizing long-term IOS imaging in mice using a 0.54-g, compact, CMOS-based, head-mounted imager. The two-part module, consisting of a tethered sensor plate and a base plate, allows facile assembly prior to imaging sessions and disassembly when the sensor is not in use. LEDs integrated into the device were chosen to illuminate the cortical mantle at two different wavelengths in the visible regime (λcenter: 535 and 625 nm) for monitoring volume- and oxygenation state-dependent changes in the IOS, respectively. To test whether the system can detect robust cortical responses, we recorded sensory-evoked IOS from mechanical stimulation of the hindlimbs (HL) of anesthetized mice in both acute and long-term implantation conditions.Results: Cortical IOS recordings in the primary somatosensory cortex hindlimb receptive field (S1HL) of anesthetized mice under green and red LED illumination revealed robust, multiphasic profiles that were time-locked to the mechanical stimulation of the contralateral plantar hindpaw. Similar intrinsic signal profiles observed in S1HL at 40 days postimplantation demonstrated the viability of the approach for long-term imaging. Immunohistochemical analysis showed that the brain tissue did not exhibit appreciable immune response due to the device implantation and operation. A proof-of-principle imaging session in a freely behaving mouse showed minimal locomotor impediment for the animal and also enabled estimation of blood flow speed.Conclusions: We demonstrate the utility of a miniature cortical imaging device for monitoring IOS and related hemodynamic processes in both anesthetized and freely moving mice, cueing potential for applications to some neuroscientific studies of sensation and naturalistic behavior.journal articl
Multichannel Bandpass Filters for Reconstructed High-Resolution Spectral Imaging in Near-infrared Fundus Camera
No full textDiagnosis by ocular fundus imaging plays a key role in the monitoring, detection, and diagnosis of several diseases including eye-related, hypertensive, and cardiovascular diseases as well as the detection of brain microvascular and neuronal pathology. The spectral detection of the fundus in the near-infrared (NIR) region can be used to analyze the composition and content of the fundus substance. The aim of this study was to develop a multichannel bandpass filter, which can be assembled on a CMOS image sensor installed in a fundus camera, to realize spectral imaging. We designed the filter in the NIR region according to interference filter theory and fabricated it by sputtering, lithography, and etching. The images acquired by the image sensor with the fabricated bandpass filter confirmed that the pixel values were comparable to the values calculated from spectrometer measurements. In an experimental validation, the number of measurement wavelengths was increased by applying the incident angle dependence of the interference filter. Each fabricated filter had a relatively wide transmittance band for spectral detection, broadening the spectrum curve. Moreover, a high-resolution spectrum was reconstructed using the developed algorithm. The spectrum reconstructed by the proposed filter had a 15% average relative error over the entire wavelength range of interest.research repor
Development of a Miniaturized Light Source with Excitation Filter for in-vivo Fluorescence Imaging
Get PDF奈良先端科学技術大学院大学博士(工学)doctoral thesi
