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  1. Article ; Online: Cell-Like Synthetic Supramolecular Soft Materials Realized in Multicomponent, Non-/Out-of-Equilibrium Dynamic Systems.

    Kubota, Ryou / Hamachi, Itaru

    Advanced science (Weinheim, Baden-Wurttemberg, Germany)

    2023  Volume 11, Issue 8, Page(s) e2306830

    Abstract: Living cells are complex, nonequilibrium supramolecular systems capable of independently and/or cooperatively integrating multiple bio-supramolecules to execute intricate physiological functions that cannot be accomplished by individual biomolecules. ... ...

    Abstract Living cells are complex, nonequilibrium supramolecular systems capable of independently and/or cooperatively integrating multiple bio-supramolecules to execute intricate physiological functions that cannot be accomplished by individual biomolecules. These biological design strategies offer valuable insights for the development of synthetic supramolecular systems with spatially controlled hierarchical structures, which, importantly, exhibit cell-like responses and functions. The next grand challenge in supramolecular chemistry is to control the organization of multiple types of supramolecules in a single system, thus integrating the functions of these supramolecules in an orthogonal and/or cooperative manner. In this perspective, the recent progress in constructing multicomponent supramolecular soft materials through the hybridization of supramolecules, such as self-assembled nanofibers/gels and coacervates, with other functional molecules, including polymer gels and enzymes is highlighted. Moreover, results show that these materials exhibit bioinspired responses to stimuli, such as bidirectional rheological responses of supramolecular double-network hydrogels, temporal stimulus pattern-dependent responses of synthetic coacervates, and 3D hydrogel patterning in response to reaction-diffusion processes are presented. Autonomous active soft materials with cell-like responses and spatially controlled structures hold promise for diverse applications, including soft robotics with directional motion, point-of-care disease diagnosis, and tissue regeneration.
    MeSH term(s) Polymers/chemistry ; Hydrogels/chemistry
    Chemical Substances Polymers ; Hydrogels
    Language English
    Publishing date 2023-11-28
    Publishing country Germany
    Document type Journal Article
    ZDB-ID 2808093-2
    ISSN 2198-3844 ; 2198-3844
    ISSN (online) 2198-3844
    ISSN 2198-3844
    DOI 10.1002/advs.202306830
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  2. Article ; Online: Emergence of Dynamic Instability by Hybridizing Synthetic Self-Assembled Dipeptide Fibers with Surfactant Micelles.

    Torigoe, Shogo / Nagao, Kazutoshi / Kubota, Ryou / Hamachi, Itaru

    Journal of the American Chemical Society

    2024  Volume 146, Issue 9, Page(s) 5799–5805

    Abstract: Supramolecular chemistry currently faces the challenge of controlling nonequilibrium dynamics such as the dynamic instability of microtubules. In this study, we explored the emergence of dynamic instability through the hybridization of peptide-type ... ...

    Abstract Supramolecular chemistry currently faces the challenge of controlling nonequilibrium dynamics such as the dynamic instability of microtubules. In this study, we explored the emergence of dynamic instability through the hybridization of peptide-type supramolecular nanofibers with surfactant micelles. Using real-time confocal imaging, we discovered that the addition of micelles to nanofibers induced the simultaneous but asynchronous growth and shrinkage of nanofibers during which the total number of fibers decreased monotonically. This dynamic phenomenon unexpectedly persisted for 6 days and was driven not by chemical reactions but by noncovalent supramolecular interactions between peptide-type nanofibers and surfactant micelles. This study demonstrates a strategy for inducing autonomous supramolecular dynamics, which will open up possibilities for developing soft materials applicable to biomedicine and soft robotics.
    Language English
    Publishing date 2024-02-26
    Publishing country United States
    Document type Journal Article
    ZDB-ID 3155-0
    ISSN 1520-5126 ; 0002-7863
    ISSN (online) 1520-5126
    ISSN 0002-7863
    DOI 10.1021/jacs.3c14565
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  3. Article ; Online: Post-click labeling enables highly accurate single cell analyses of glucose uptake ex vivo and in vivo.

    Tsuchiya, Masaki / Tachibana, Nobuhiko / Hamachi, Itaru

    Communications biology

    2024  Volume 7, Issue 1, Page(s) 459

    Abstract: Cellular glucose uptake is a key feature reflecting metabolic demand of cells in physiopathological conditions. Fluorophore-conjugated sugar derivatives are widely used for monitoring glucose transporter (GLUT) activity at the single-cell level, but have ...

    Abstract Cellular glucose uptake is a key feature reflecting metabolic demand of cells in physiopathological conditions. Fluorophore-conjugated sugar derivatives are widely used for monitoring glucose transporter (GLUT) activity at the single-cell level, but have limitations in in vivo applications. Here, we develop a click chemistry-based post-labeling method for flow cytometric measurement of glucose uptake with low background adsorption. This strategy relies on GLUT-mediated uptake of azide-tagged sugars, and subsequent intracellular labeling with a cell-permeable fluorescent reagent via a copper-free click reaction. Screening a library of azide-substituted monosaccharides, we discover 6-azido-6-deoxy-D-galactose (6AzGal) as a suitable substrate of GLUTs. 6AzGal displays glucose-like physicochemical properties and reproduces in vivo dynamics similar to
    MeSH term(s) Azides/chemistry ; Glucose/metabolism ; Single-Cell Analysis
    Chemical Substances Azides ; Glucose (IY9XDZ35W2)
    Language English
    Publishing date 2024-04-16
    Publishing country England
    Document type Journal Article
    ISSN 2399-3642
    ISSN (online) 2399-3642
    DOI 10.1038/s42003-024-06164-y
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  4. Article ; Online: Quantitative Analysis of the Endoplasmic Reticulum-Associated Proteins Using ER-Localizable Reactive Molecules.

    Tamura, Tomonori / Hamachi, Itaru

    Methods in molecular biology (Clifton, N.J.)

    2022  Volume 2603, Page(s) 139–150

    Abstract: The endoplasmic reticulum (ER) is an essential organelle responsible for many cellular functions, including protein synthesis and folding, lipid synthesis, membrane trafficking, and storage of Ca ... 2+ ... Therefore, global profiling of ER-associated ...

    Abstract The endoplasmic reticulum (ER) is an essential organelle responsible for many cellular functions, including protein synthesis and folding, lipid synthesis, membrane trafficking, and storage of Ca<sup>2+</sup>. Therefore, global profiling of ER-associated proteins should be invaluable for understanding these biological processes. However, the difficulty of isolating the intact ER hampered proteome-wide analysis of ER proteins. This chapter describes a chemoproteomic approach for ER proteome analysis using ER-localizable reactive molecules (ERMs), which need neither ER fractionation nor genetic transformation. ERMs spontaneously accumulate in the ER of live cells, and the resultant high concentration of ERMs facilitates spatially limited chemical modification of ER-localized proteins with a detection/purification tag via simple intermolecular reactions. This enables the tag-mediated enrichment and quantitative analysis of the ER-associated proteins using liquid chromatography-tandem mass spectrometry (LC-MS/MS) coupled with SILAC technology.
    MeSH term(s) Proteome/metabolism ; Chromatography, Liquid ; Tandem Mass Spectrometry ; Endoplasmic Reticulum/metabolism ; Protein Biosynthesis ; Transcription Factors/metabolism ; Endoplasmic Reticulum Stress ; Unfolded Protein Response
    Chemical Substances Proteome ; Transcription Factors
    Language English
    Publishing date 2022-11-12
    Publishing country United States
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ISSN 1940-6029
    ISSN (online) 1940-6029
    DOI 10.1007/978-1-0716-2863-8_11
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  5. Article ; Online: Chemical biology tools for imaging-based analysis of organelle membranes and lipids.

    Tamura, Tomonori / Hamachi, Itaru

    Current opinion in chemical biology

    2022  Volume 70, Page(s) 102182

    Abstract: Membrane biology studies have revealed that in addition to providing structural support for compartment formation and membrane protein function, subcellular biomembranes are also critically involved in many biological events. To facilitate our ... ...

    Abstract Membrane biology studies have revealed that in addition to providing structural support for compartment formation and membrane protein function, subcellular biomembranes are also critically involved in many biological events. To facilitate our understanding of the functions, biophysical properties and structural dynamics of organelle membranes, various exciting chemical biology tools have recently emerged. This short review aims to describe the latest molecular probes for organelle membrane studies. In particular, we will feature chemical strategies to visualize and quantitatively analyze the dynamic propeties of organelle membranes and lipids and discuss current limitations and potential future directions of this challenging research area.
    MeSH term(s) Biology ; Lipids/chemistry ; Membrane Proteins/metabolism ; Molecular Probes/metabolism ; Organelles/metabolism
    Chemical Substances Lipids ; Membrane Proteins ; Molecular Probes
    Language English
    Publishing date 2022-06-29
    Publishing country England
    Document type Journal Article ; Review ; Research Support, Non-U.S. Gov't
    ZDB-ID 1439176-4
    ISSN 1879-0402 ; 1367-5931
    ISSN (online) 1879-0402
    ISSN 1367-5931
    DOI 10.1016/j.cbpa.2022.102182
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  6. Article ; Online: Endogenous Cell-Surface Receptor Modification by Metal Chelation-Assisted Pyridinium Oxime Catalyst.

    Thimaradka, Vikram / Utsunomiya, Hayata / Tamura, Tomonori / Hamachi, Itaru

    Organic letters

    2023  Volume 25, Issue 12, Page(s) 2118–2122

    Abstract: Organocatalyst-mediated acyl transfer reactions hold promise for selective protein labeling in biological milieu. However, they often suffer from off-target reactions and high background signals because of the requirement of high concentrations of ... ...

    Abstract Organocatalyst-mediated acyl transfer reactions hold promise for selective protein labeling in biological milieu. However, they often suffer from off-target reactions and high background signals because of the requirement of high concentrations of substrates. Here, we report a new catalytic protein acylation strategy promoted by the His-tag/NiNTA interaction. The recognition-assisted activation mechanism allows efficient protein labeling even with >10-fold lower substrate concentrations than conventional reactions, thereby enabling highly selective and efficient cell-surface receptor modification in live cells.
    MeSH term(s) Oximes ; Proteins
    Chemical Substances Oximes ; Proteins
    Language English
    Publishing date 2023-03-22
    Publishing country United States
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ISSN 1523-7052
    ISSN (online) 1523-7052
    DOI 10.1021/acs.orglett.3c00541
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  7. Article ; Online: Flow cytometric analysis of phosphatidylcholine metabolism using organelle-selective click labeling.

    Tsuchiya, Masaki / Tachibana, Nobuhiko / Hamachi, Itaru

    STAR protocols

    2023  Volume 4, Issue 3, Page(s) 102525

    Abstract: Here, we present a protocol to analyze phosphatidylcholine (PC) metabolism in mammalian cells using organelle-selective click labeling coupled with flow cytometry (O-ClickFC). We describe steps for the metabolic incorporation of azide-choline into PC. We ...

    Abstract Here, we present a protocol to analyze phosphatidylcholine (PC) metabolism in mammalian cells using organelle-selective click labeling coupled with flow cytometry (O-ClickFC). We describe steps for the metabolic incorporation of azide-choline into PC. We then detail fluorescent labeling of the azide-modified PC with organelle-targeting clickable dyes in the ER-Golgi, plasma membrane, and mitochondria, and by flow cytometry. This protocol is optimized for flow cytometric quantification of the labeled PC at the organelle level within single live cells. For complete details on the use and execution of this protocol, please refer to Tsuchiya et al. (2023).
    MeSH term(s) Animals ; Azides ; Flow Cytometry/methods ; Golgi Apparatus ; Mitochondria ; Phosphatidylcholines ; Mammals
    Chemical Substances Azides ; Phosphatidylcholines
    Language English
    Publishing date 2023-08-26
    Publishing country United States
    Document type Journal Article
    ISSN 2666-1667
    ISSN (online) 2666-1667
    DOI 10.1016/j.xpro.2023.102525
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  8. Article ; Online: Four distinct network patterns of supramolecular/polymer composite hydrogels controlled by formation kinetics and interfiber interactions.

    Nakamura, Keisuke / Kubota, Ryou / Aoyama, Takuma / Urayama, Kenji / Hamachi, Itaru

    Nature communications

    2023  Volume 14, Issue 1, Page(s) 1696

    Abstract: Synthetic composite hydrogels comprising supramolecular fibers and covalent polymers have attracted considerable attention because their properties are similar to biological connective tissues. However, an in-depth analysis of the network structures has ... ...

    Abstract Synthetic composite hydrogels comprising supramolecular fibers and covalent polymers have attracted considerable attention because their properties are similar to biological connective tissues. However, an in-depth analysis of the network structures has not been performed. In this study, we discovered the composite network can be categorized into four distinct patterns regarding morphology and colocalization of the components using in situ, real-time confocal imaging. Time-lapse imaging of the network formation process reveals that the patterns are governed by two factors, the order of the network formation and the interactions between the two different fibers. Additionally, the imaging studies revealed a unique composite hydrogel undergoing dynamic network remodeling on the scale of a hundred micrometers to more than one millimeter. Such dynamic properties allow for fracture-induced artificial patterning of a network three dimensionally. This study introduces a valuable guideline to the design of hierarchical composite soft materials.
    Language English
    Publishing date 2023-03-27
    Publishing country England
    Document type Journal Article
    ZDB-ID 2553671-0
    ISSN 2041-1723 ; 2041-1723
    ISSN (online) 2041-1723
    ISSN 2041-1723
    DOI 10.1038/s41467-023-37412-0
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  9. Article ; Online: Visualizing Formation and Dynamics of a Three-Dimensional Sponge-like Network of a Coacervate in Real Time.

    Kubota, Ryou / Hiroi, Taro / Ikuta, Yuriki / Liu, Yuchong / Hamachi, Itaru

    Journal of the American Chemical Society

    2023  Volume 145, Issue 33, Page(s) 18316–18328

    Abstract: Coacervates, which are formed by liquid-liquid phase separation, have been extensively explored as models for synthetic cells and membraneless organelles, so their in-depth structural analysis is crucial. However, both the inner structure dynamics and ... ...

    Abstract Coacervates, which are formed by liquid-liquid phase separation, have been extensively explored as models for synthetic cells and membraneless organelles, so their in-depth structural analysis is crucial. However, both the inner structure dynamics and formation mechanism of coacervates remain elusive. Herein, we demonstrate real-time confocal observation of a three-dimensional sponge-like network in a dipeptide-based coacervate.
    Language English
    Publishing date 2023-08-10
    Publishing country United States
    Document type Journal Article
    ZDB-ID 3155-0
    ISSN 1520-5126 ; 0002-7863
    ISSN (online) 1520-5126
    ISSN 0002-7863
    DOI 10.1021/jacs.3c03793
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  10. Article ; Online: Organelle-selective click labeling coupled with flow cytometry allows pooled CRISPR screening of genes involved in phosphatidylcholine metabolism.

    Tsuchiya, Masaki / Tachibana, Nobuhiko / Nagao, Kohjiro / Tamura, Tomonori / Hamachi, Itaru

    Cell metabolism

    2023  Volume 35, Issue 6, Page(s) 1072–1083.e9

    Abstract: Cellular lipid synthesis and transport are governed by intricate protein networks. Although genetic screening should contribute to deciphering the regulatory networks of lipid metabolism, technical challenges remain-especially for high-throughput ... ...

    Abstract Cellular lipid synthesis and transport are governed by intricate protein networks. Although genetic screening should contribute to deciphering the regulatory networks of lipid metabolism, technical challenges remain-especially for high-throughput readouts of lipid phenotypes. Here, we coupled organelle-selective click labeling of phosphatidylcholine (PC) with flow cytometry-based CRISPR screening technologies to convert organellar PC phenotypes into a simple fluorescence readout for genome-wide screening. This technique, named O-ClickFC, was successfully applied in genome-scale CRISPR-knockout screens to identify previously reported genes associated with PC synthesis (PCYT1A, ACACA), vesicular membrane trafficking (SEC23B, RAB5C), and non-vesicular transport (PITPNB, STARD7). Moreover, we revealed previously uncharacterized roles of FLVCR1 as a choline uptake facilitator, CHEK1 as a post-translational regulator of the PC-synthetic pathway, and CDC50A as responsible for the translocation of PC to the outside of the plasma membrane bilayer. These findings demonstrate the versatility of O-ClickFC as an unprecedented platform for genetic dissection of cellular lipid metabolism.
    MeSH term(s) Clustered Regularly Interspaced Short Palindromic Repeats ; Flow Cytometry ; Lipid Metabolism ; Phosphatidylcholines/metabolism ; Organelles/metabolism ; CRISPR-Cas Systems/genetics
    Chemical Substances Phosphatidylcholines
    Language English
    Publishing date 2023-03-13
    Publishing country United States
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ZDB-ID 2176834-1
    ISSN 1932-7420 ; 1550-4131
    ISSN (online) 1932-7420
    ISSN 1550-4131
    DOI 10.1016/j.cmet.2023.02.014
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