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Masahiro Ueda - One of the best experts on this subject based on the ideXlab platform.

  • Single-Molecule Imaging of PI(4,5)P 2 and PTEN in vitro reveals a positive feedback mechanism for PTEN membrane binding
    Communications biology, 2020
    Co-Authors: Daisuke Yoshioka, Satomi Matsuoka, Seiya Fukushima, Hiroyasu Koteishi, Daichi Okuno, Toru Ide, Masahiro Ueda
    Abstract:

    PTEN, a 3-phosphatase of phosphoinositide, regulates asymmetric PI(3,4,5)P3 signaling for the anterior-posterior polarization and migration of motile cells. PTEN acts through posterior localization on the plasma membrane, but the mechanism for this accumulation is poorly understood. Here we developed an in vitro Single-Molecule Imaging assay with various lipid compositions and use it to demonstrate that the enzymatic product, PI(4,5)P2, stabilizes PTEN's membrane-binding. The dissociation kinetics and lateral mobility of PTEN depended on the PI(4,5)P2 density on artificial lipid bilayers. The basic residues of PTEN were responsible for electrostatic interactions with anionic PI(4,5)P2 and thus the PI(4,5)P2-dependent stabilization. Single-Molecule Imaging in living Dictyostelium cells revealed that these interactions were indispensable for the stabilization in vivo, which enabled efficient cell migration by accumulating PTEN posteriorly to restrict PI(3,4,5)P3 distribution to the anterior. These results suggest that PI(4,5)P2-mediated positive feedback and PTEN-induced PI(4,5)P2 clustering may be important for anterior-posterior polarization.

  • Large-scale Single-Molecule Imaging aided by artificial intelligence
    Microscopy (Oxford England), 2020
    Co-Authors: Michio Hiroshima, Masato Yasui, Masahiro Ueda
    Abstract:

    Single-Molecule Imaging analysis has been applied to study the dynamics and kinetics of molecular behaviors and interactions in living cells. In spite of its high potential as a technique to investigate the molecular mechanisms of cellular phenomena, Single-Molecule Imaging analysis has not been extended to a large scale of Molecules in cells due to the low measurement throughput as well as required expertise. To overcome these problems, we have automated the Imaging processes by using computer operations, robotics and artificial intelligence (AI). AI is an ideal substitute for expertise to obtain high-quality images for quantitative analysis. Our automated in-cell Single-Molecule Imaging system, AiSIS, could analyze 1600 cells in 1 day, which corresponds to ∼ 100-fold higher efficiency than manual analysis. The large-scale analysis revealed cell-to-cell heterogeneity in the molecular behavior, which had not been recognized in previous studies. An analysis of the receptor behavior and downstream signaling was accomplished within a significantly reduced time frame and revealed the detailed activation scheme of signal transduction, advancing cell biology research. Furthermore, by combining the high-throughput analysis with our previous finding that a receptor changes its behavioral dynamics depending on the presence of a ligand/agonist or inhibitor/antagonist, we show that AiSIS is applicable to comprehensive pharmacological analysis such as drug screening. This AI-aided automation has wide applications for Single-Molecule analysis.

  • Automated Single-Molecule Imaging in living cells
    Nature communications, 2018
    Co-Authors: Masato Yasui, Jun Kozuka, Michio Hiroshima, Yasushi Sako, Masahiro Ueda
    Abstract:

    An automated Single-Molecule Imaging system developed for live-cell analyses based on artificial intelligence-assisted microscopy is presented. All significant procedures, i.e., searching for cells suitable for observation, detecting in-focus positions, and performing image acquisition and Single-Molecule tracking, are fully automated, and numerous highly accurate, efficient, and reproducible Single-Molecule Imaging experiments in living cells can be performed. Here, the apparatus is applied for Single-Molecule Imaging and analysis of epidermal growth factor receptors (EGFRs) in 1600 cells in a 96-well plate within 1 day. Changes in the lateral mobility of EGFRs on the plasma membrane in response to various ligands and drug concentrations are clearly detected in individual cells, and several dynamic and pharmacological parameters are determined, including the diffusion coefficient, oligomer size, and half-maximal effective concentration (EC50). Automated Single-Molecule Imaging for systematic cell signaling analyses is feasible and can be applied to Single-Molecule screening, thus extensively contributing to biological and pharmacological research.

  • Intracellular protein-labeling probes for multicolor Single-Molecule Imaging of immune receptor-adaptor molecular dynamics
    Journal of the American Chemical Society, 2017
    Co-Authors: Ryota Sato, Jun Kozuka, Masahiro Ueda, Reiko Mishima, Yutaro Kumagai, Akimasa Yoshimura, Masafumi Minoshima, Shin Mizukami, Kazuya Kikuchi
    Abstract:

    Single-Molecule Imaging (SMI) has been widely utilized to investigate biomolecular dynamics and protein–protein interactions in living cells. However, multicolor SMI of intracellular proteins is challenging because of high background signals and other limitations of current fluorescence labeling approaches. To achieve reproducible intracellular SMI, a labeling probe ensuring both efficient membrane permeability and minimal non-specific binding to cell components is essential. We developed near-infrared fluorescent probes for protein labeling that specifically bind to a mutant β-lactamase tag. By structural fine-tuning of cell permeability and minimized non-specific binding, SiRcB4 enabled multicolor SMI in combination with a HaloTag-based red-fluorescent probe. Upon addition of both chemical probes at sub-nanomolar concentrations, Single-Molecule Imaging revealed the dynamics of TLR4 and its adaptor protein, TIRAP, which are involved in the innate immune system. Statistical analysis of the quantitative pr...

  • Intracellular Protein-Labeling Probes for Multicolor Single-Molecule Imaging of Immune Receptor–Adaptor Molecular Dynamics
    2017
    Co-Authors: Ryota Sato, Jun Kozuka, Masahiro Ueda, Reiko Mishima, Yutaro Kumagai, Akimasa Yoshimura, Masafumi Minoshima, Shin Mizukami, Kazuya Kikuchi
    Abstract:

    Single-Molecule Imaging (SMI) has been widely utilized to investigate biomolecular dynamics and protein–protein interactions in living cells. However, multicolor SMI of intracellular proteins is challenging because of high background signals and other limitations of current fluorescence labeling approaches. To achieve reproducible intracellular SMI, a labeling probe ensuring both efficient membrane permeability and minimal non-specific binding to cell components is essential. We developed near-infrared fluorescent probes for protein labeling that specifically bind to a mutant β-lactamase tag. By structural fine-tuning of cell permeability and minimized non-specific binding, SiRcB4 enabled multicolor SMI in combination with a HaloTag-based red-fluorescent probe. Upon addition of both chemical probes at sub-nanomolar concentrations, Single-Molecule Imaging revealed the dynamics of TLR4 and its adaptor protein, TIRAP, which are involved in the innate immune system. Statistical analysis of the quantitative properties and time-lapse changes in dynamics revealed a protein–protein interaction in response to ligand stimulation

Yukihiro Miyanaga - One of the best experts on this subject based on the ideXlab platform.

  • video rate confocal microscopy for Single Molecule Imaging in live cells and superresolution fluorescence Imaging
    Biophysical Journal, 2012
    Co-Authors: Jinwoo Lee, Masahiro Ueda, Yukihiro Miyanaga, Sungchul Hohng
    Abstract:

    There is no confocal microscope optimized for Single-Molecule Imaging in live cells and superresolution fluorescence Imaging. By combining the swiftness of the line-scanning method and the high sensitivity of wide-field detection, we have developed a, to our knowledge, novel confocal fluorescence microscope with a good optical-sectioning capability (1.0 μm), fast frame rates (<33 fps), and superior fluorescence detection efficiency. Full compatibility of the microscope with conventional cell-Imaging techniques allowed us to do Single-Molecule Imaging with a great ease at arbitrary depths of live cells. With the new microscope, we monitored diffusion motion of fluorescently labeled cAMP receptors of Dictyostelium discoideum at both the basal and apical surfaces and obtained superresolution fluorescence images of microtubules of COS-7 cells at depths in the range 0–85 μm from the surface of a coverglass.

  • Video-Rate Confocal Microscopy for Single-Molecule Imaging in Live Cells and Superresolution Fluorescence Imaging
    Biophysical journal, 2012
    Co-Authors: Jinwoo Lee, Masahiro Ueda, Yukihiro Miyanaga, Sungchul Hohng
    Abstract:

    There is no confocal microscope optimized for Single-Molecule Imaging in live cells and superresolution fluorescence Imaging. By combining the swiftness of the line-scanning method and the high sensitivity of wide-field detection, we have developed a, to our knowledge, novel confocal fluorescence microscope with a good optical-sectioning capability (1.0 μm), fast frame rates (

  • Single-Molecule Imaging Techniques to Visualize Chemotactic Signaling Events on the Membrane of Living Dictyostelium Cells
    Methods in molecular biology (Clifton N.J.), 2009
    Co-Authors: Yukihiro Miyanaga, Satomi Matsuoka, Masahiro Ueda
    Abstract:

    In this chapter, we describe methods to monitor signaling events at the Single-Molecule level on the membrane of living cells by using total internal reflection fluorescence microscopy (TIRFM). The techniques provide a powerful tool for elucidating the stochastic properties of signaling Molecules involved in chemotaxis of the cellular slime mold Dictyostelium discoideum. Taking cAMP receptor 1 (cAR1) as an example of a target protein for Single-Molecule Imaging, we describe the experimental setup of TIRFM, a method for labeling cAR1 with a fluorescent dye, and a method for investigating the receptor's lateral mobility. We discuss how the developmental progression of cells modulates both cAR1 behavior and the phenotypic variability in cAR1 mobility for different cell populations.

  • Stochastic signal inputs for chemotactic response in Dictyostelium cells revealed by Single Molecule Imaging techniques
    Bio Systems, 2006
    Co-Authors: Yukihiro Miyanaga, Toshio Yanagida, Satomi Matsuoka, Masahiro Ueda
    Abstract:

    Chemotactic cells can exhibit extreme sensitivity to chemical gradients. Theoretical estimations of the signal inputs required for chemotaxis suggest that the response can be achieved under the strong influence of stochastic input noise generated by the receptors during the transmembrane signaling. This arises a fundamental question regarding the mechanisms for directional sensing: how do cells obtain reliable information regarding gradient direction by using stochastically operating receptors and the downstream Molecules? To address this question, we have developed Single Molecule Imaging techniques to visualize signaling Molecules responsible for chemotaxis in living Dictyostelium cells, allowing us to monitor the stochastic signaling processes directly. Single Molecule Imaging of a chemoattractant bound to a receptor demonstrates that signal inputs fluctuate with time and space. Downstream signaling Molecules, such as PTEN and a PH domain-containing protein that are constituent parts of chemotactic signaling system, can also be followed at Single Molecule level in living cells, illuminating the stochastic nature of chemotactic signaling processes. In this report, we start with a brief introduction of chemotactic response of the eukaryotic cells, followed by an explanation for Single Molecule Imaging techniques, and finally discuss these applications to chemotactic signaling system of Dictyostelium cells.

Sungchul Hohng - One of the best experts on this subject based on the ideXlab platform.

Jinwoo Lee - One of the best experts on this subject based on the ideXlab platform.

Olivier Thoumine - One of the best experts on this subject based on the ideXlab platform.

  • A super-resolution platform for correlative live Single-Molecule Imaging and STED microscopy
    Nature Methods, 2019
    Co-Authors: V. V. G. Krishna Inavalli, Martin O. Lenz, Corey Butler, Julie Angibaud, Benjamin Compans, Florian Levet, Jan Tønnesen, Olivier Rossier, Grégory Giannone, Olivier Thoumine
    Abstract:

    Super-resolution microscopy offers tremendous opportunities to unravel the complex and dynamic architecture of living cells. However, current super-resolution microscopes are well suited for revealing protein distributions or cell morphology, but not both. We present a super-resolution platform that permits correlative Single-Molecule Imaging and stimulated emission depletion microscopy in live cells. It gives nanoscale access to the positions and movements of synaptic proteins within the morphological context of growth cones and dendritic spines. Seamless integration of Single-Molecule localization microscopy and STED allows for correlative live Imaging of protein position and movement at the nanoscale in the context of fine morphological features.

  • A super-resolution platform for correlative live Single-Molecule Imaging and STED microscopy.
    Nature methods, 2019
    Co-Authors: V. V. G. Krishna Inavalli, Martin O. Lenz, Corey Butler, Julie Angibaud, Benjamin Compans, Florian Levet, Jan Tønnesen, Olivier Rossier, Grégory Giannone, Olivier Thoumine
    Abstract:

    Super-resolution microscopy offers tremendous opportunities to unravel the complex and dynamic architecture of living cells. However, current super-resolution microscopes are well suited for revealing protein distributions or cell morphology, but not both. We present a super-resolution platform that permits correlative Single-Molecule Imaging and stimulated emission depletion microscopy in live cells. It gives nanoscale access to the positions and movements of synaptic proteins within the morphological context of growth cones and dendritic spines.