Biology subjects

Hamachi, I.

Publications and source records attributed to Hamachi, I..

16 recordsLinked to original sources

bioRxiv2026

Patterned photocatalytic proximity labelling for spatial interactomics

Mapping protein-protein interactions and proximity within their native spatial context is essential for elucidating molecular mechanisms underlying biological processes; however, methods for spatial interactomics with subanatomical resolution are limited. Here, we introduce digital micromirror device (DMD)-PhoxID, a method that combines photocatalytic proximity labelling with patterned irradiation using a DMD to map the proximal molecular environment of proteins of interest within defined subregions of fixed brain tissue.

neuroscience↗

bioRxiv2026

Native receptor-targeted chemogenetics enables cell-type-specific inhibition of endogenous receptors in freely moving mice

Understanding brain function requires tools that allow precise manipulation of receptor signaling in defined cell types within intact neural circuits. Optogenetics and conventional chemogenetic approaches primarily enable cell-type-specific control of neuronal excitability using engineered receptors or ion channels. However, direct and reversible inhibition of endogenous neurotransmitter receptors in defined cell types has remained technically inaccessible. Here, we introduce native receptor-targeted chemogenetics (NARCH), a chemogenetic strategy that integrates structure-guided receptor engineering with allosteric ligand design to achieve reversible and temporally precise inhibition of endogenous receptor signaling with cell-type specificity in vivo. By applying NARCH to metabotropic glutamate receptor 1 (mGlu1), we demonstrate that mGlu1 signaling in cerebellar Purkinje cells is required for stabilization of motor learning across training sessions in freely moving mice. NARCH thus establishes a receptor-level chemogenetic framework for causal analysis of neural circuits and behavior.

neuroscience↗

bioRxiv2026

Transmembrane PhoxID: Photo-proximity labelling across the plasma membrane

Transmembrane proteins perform essential roles in cellular transport, signalling, and communication. The function and dynamics of these proteins are precisely regulated by interactions on both sides of the plasma membrane; thus, mapping the composition of these interactomes is a fundamental challenge in molecular biology. Proximity labelling methods are powerful tools for this purpose; however, existing approaches that rely on membrane-impermeable reactive species are limited by the ability to detect only one side, either the extracellular or the intracellular region, of transmembrane proteins. Here, we capitalized on the cell permeability of singlet oxygen to carry out proximity labelling of the cytoplasmic side of transmembrane proteins using an extracellularly anchored photosensitizer. We applied this method, termed transmembrane PhoxID (tmPhoxID), to several receptors (GRID2, GABAAR, and GRM1) in the living mouse brain and successfully determined their specific intracellular interactomes. Notably, network analysis of the identified proteins revealed that this method can characterize the native components of transsynaptic nanocolumns formed at parallel fibre-Purkinje cell synapses. Furthermore, our study revealed a previously uncharacterized GABAAR-CAMKV interaction in mice and human brains. Our results provide a proof of concept for transmembrane and transcellular proximity labelling, providing a powerful platform for analysing the interactome of transmembrane proteins.

neuroscience↗

bioRxiv2025

Quantitative analysis of synaptic zinc in the brain by covalent chemistry-based semisynthetic biosensors

Labile zinc ions (Zn{superscript 2}) are stored in glutamatergic vesicles at specific excitatory synapses in the central nervous system and released into synaptic clefts in an activity-dependent manner. Although Zn{superscript 2} is suggested to modulate various neuroreceptor functions, its precise roles remain unclear due to a lack of tools capable of quantitatively analyzing Zn{superscript 2} with synapse-level spatial resolution. Here, we developed neuroreceptor-based semisynthetic sensors that record synaptic Zn{superscript 2} dynamics by covalent chemistry in the living mouse brain. Using a chemical knock-in strategy, we introduced activity-based Zn2+-probes with distinct affinities into endogenous -amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid or {gamma}-aminobutyric acid type A receptors. These in-brain-constructed Zn{superscript 2} sensors enabled, to our knowledge, for the first time, quantitative, region-specific mapping of Zn{superscript 2} released into synaptic clefts. Imaging-based analyses revealed differences in Zn{superscript 2} concentrations at excitatory and inhibitory synapses across hippocampal regions during kainate-induced seizures, providing new insights into the physiological and pathological functions of synaptic Zn{superscript 2}.

neuroscience↗

bioRxiv2025

Discovery of a Selective Inhibitor of ZIP14 with Therapeutic Potential for Cancer-associated Cachexia

ZIP14/SLC39A14, a membrane-bound metal transporter, is essential for systemic metal homeostasis and has been implicated in inflammatory and metabolic disorders, including cancer-associated cachexia. Despite its biological and therapeutic significance, no selective inhibitors have been identified. Here, we identify 1-phenyl-8-(2-phenylethyl)-1,3,8-triazaspiro[4.5]decan-4-one (PPTD) as the first selective small-molecule inhibitor of ZIP14. PPTD efficiently blocks ZIP14-mediated uptake of zinc, iron, manganese, and cadmium, while sparing the closely related transporter ZIP8/SLC39A8. Mechanistically, PPTD binds specifically to a pocket formed at the dimer interface of ZIP14, as revealed by AlphaFold3 structural prediction, ligand-interaction profiling, structure-activity analyses, and site-directed mutagenesis, providing direct evidence for a targeted inhibition mechanism. ZIP14-driven metal influx promotes reactive oxygen species and lipid peroxidation, leading to cytotoxicity, which PPTD effectively reverses. In vivo, PPTD ameliorates major features of cancer cachexia in mice, including weight loss, reduced survival, muscle wasting, impaired locomotor activity, and disease progression. PPTD thus provides both a chemical probe to dissect ZIP14 function and a potential therapeutic candidate for cancer cachexia, establishing a foundation for the development of therapies targeting ZIP14-mediated metal dysregulation.

biochemistry↗

bioRxiv2024

Conversion of silent synapses to AMPA receptor-mediated functional synapses in human cortical organoids

Despite the crucial role of synaptic connections and neural activity in the development and organization of cortical circuits, the mechanisms underlying the formation of functional synaptic connections in the developing human cerebral cortex remain unclear. We investigated the development of -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR)-mediated synaptic transmission using human cortical organoids (hCOs) derived from induced pluripotent stem cells. Two-photon Ca2 imaging revealed an increase in the frequency and amplitude of spontaneous activity in hCOs on day 80 compared to day 50. Additionally, spontaneous neural activity in late-stage hCOs, but not in early-stage hCOs, was blocked by N-methyl-D-aspartate receptor (NMDAR) and AMPAR antagonists. However, transsynaptic circuit tracing with G-deleted rabies viral vectors indicated a similar number of synaptic connections in early- and late-stage hCOs. Notably, chemical labeling demonstrated a significant increase in AMPAR expression on the postsynaptic membrane and colocalization with NMDAR in late-stage hCOs. These results suggest that hCOs progressively organize excitatory synaptic transmission, concurrent with the transition from silent synapses lacking AMPARs to functional synapses containing NMDARs and AMPARs. This in vitro model of human cortical circuits derived from induced pluripotent stem cells reflects the developmental programs underlying physiological transitions, providing valuable insights into human corticogenesis and neurodevelopmental disorders. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=181 SRC="FIGDIR/small/629885v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@1432b6dorg.highwire.dtl.DTLVardef@d8350borg.highwire.dtl.DTLVardef@8273f8org.highwire.dtl.DTLVardef@8bbabd_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2024

Profiling proteins involved in peroxynitrite homeostasis using ROS/RNS conditional proteomics

Peroxynitrite (ONOO-), the product of the diffusion-controlled reaction of superoxide (O2*-) with nitric oxide (NO*), plays a crucial role in oxidative and nitrative stress and modulates key physiological processes such as redox signaling. While biological ONOO- is conventionally analyzed using 3-nitrotyrosine antibodies and fluorescent sensors, such probes lack specificity and sensitivity, making high-throughput and comprehensive profiling of ONOO--associated proteins challenging. In this study, we used a conditional proteomics approach to investigate ONOO- homeostasis by identifying its protein neighbors in cells. We developed Peroxynitrite-responsive protein Labeling reagents (Porp-L) and, for the first time, discovered 2,6-dichlorophenol as an ideal moiety that can be selectively and rapidly activated by ONOO- for labeling of proximal proteins. The reaction of Porp-L with ONOO- generated several short-lived reactive intermediates that can modify Tyr, His, and Lys residues on the protein surface. We have demonstrated the Porp-L-based conditional proteomics in immune-stimulated macrophages, which indeed identified proteins known to be involved in the generation and modification of ONOO- and revealed the endoplasmic reticulum (ER) as a ONOO- hot spot. Moreover, we discovered a previously unknown role for Ero1a, an ER-resident protein, in the formation of ONOO-. Overall, Porp-L represent a promising research tool for advancing our understanding of the biological roles of ONOO-.

biochemistry↗

bioRxiv2024

A fluorophore-ligand conjugate for the rapid and reversible staining of native AMPA receptors in living neurons

The subcellular localizations of neurotransmitter receptors are strictly regulated in neurons. Changes in the trafficking of -amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA)-type glutamate receptors (AMPARs) play an essential role in synaptic plasticity, which is the cellular basis of learning and memory. To explore receptor trafficking, genetically encoded approaches (e.g., the fusion of fluorescent proteins to receptors) are often used. However, concerns remain that genetic approaches cannot fully reproduce the receptor function that is inherent to neurons. Herein, we report a chemical probe, PFQX1(AF488), for the visualization of cell-surface AMPARs without any genetic manipulation to neurons. The rapid and reversible staining features of this probe allowed the visualization of AMPAR accumulation in dendritic spines during synaptic plasticity in living hippocampal neurons. Moreover, the mechanisms of this synaptic accumulation, for which genetically encoded approaches have given controversial results, were revealed by integrating two chemical methods: PFQX1(AF488) and covalent chemical labeling.

neuroscience↗

bioRxiv2024

Nanoscale organization is changed in native, surface AMPARs by mouse brain region and tauopathy

The distribution of synaptic and extra-synaptic AMPA receptors (AMPARs) on neuronal plasma membranes is correlated with learning and memory. Although AMPAR organization has been extensively studied in neuronal cultures, its native cell-surface distribution in intact adult brain tissue across distinct brain regions and in neurodegenerative pathology remains poorly understood. Here, we combine a selective small-molecule labeling strategy with two-color 3D super-resolution dSTORM imaging to map native surface AMPAR organization at the nanoscale in 30 micron thick mouse brain slices. We find that wild-type mice exhibit marked regional differences in AMPAR organization, with the CA1 hippocampus containing a substantially larger extrasynaptic AMPAR pool than the nearby motor and somatosensory cortex. In the PS19 tauopathy mouse model, at an age preceding overt neurodegeneration, AMPAR organization is selectively disrupted in the hippocampus but largely preserved in the cortex. Specifically, we observe depletion of the extrasynaptic receptor pool together with reduced synaptic nanodomain organization, revealing early molecular-scale synaptic remodeling associated with tau pathology. These findings provide direct structural insight into region- and disease-dependent AMPAR organization in intact adult brain tissue and establish a broadly applicable framework for nanoscale investigation of synaptic receptor architecture in health and neurological disease.

neuroscience↗

bioRxiv2024

In-brain construction of receptor-based protease sensors by coupling ligand-directed chemistry and click chemistry

The chemical modification of natural proteins in living systems is highly desirable toward the cutting-edge research in chemistry-biology interface. Recent advances in bioorthogonal protein modification have enabled the production of chemically functional proteins in cultured cell systems. However, few methods are applicable in vivo because of the complexity of the three-dimensional constructs of living systems with diverse, heterogeneous cell populations and flow systems filled with tissue fluids. Here, we report a genetic engineering-free method to modify receptor proteins with various probes in the living mouse brain by combining in-brain ligand-directed chemistry with bioorthogonal click chemistry, and propose a chemical guideline for the reaction design. The rapid and selective tethering of a set of fluorescent peptides to AMPA-type glutamate receptors (AMPARs) allowed the construction of receptor-based fluorescent sensors. These probes enabled mapping of the activity of matrix metalloproteinase-9 proximal to AMPARs in the living brain to be realized with high spatial resolution. Our strategy provides new opportunities for the precise analysis of particular in vivo microenvironments that has not been able to be addressed by conventional methods. Such analysis should contribute to the understanding of the molecular basis for complicated in vivo events, such as the regulation of neuroplasticity, the most important challenge in neuroscience.

biochemistry↗

bioRxiv2023

Phototriggered profiling of receptor-proximal proteins in vivo in minutes

Neurotransmitter receptors are regulated by an extensive and dynamic network of protein-protein interactions. Understanding how these networks control synaptic transmission and give rise to higher-order brain functions necessitates their investigation in the live mammalian brain. However, tools available for this purpose lack the temporal resolution necessary to capture rapid changes in the interactome in live animals and require potentially disruptive genetic modifications to the animal under study. Here, we describe a method for light-activated proximity labelling in the live mouse brain that relies solely on small-molecule reagents and achieves a minute-order temporal resolution. Named PhoxID (photooxidation-driven proximity labelling for protein identification), this method involves selectively tethering a chemical photosensitiser to neurotransmitter receptors of interest and enabled us to identify characteristic as well as less studied interactors of the endogenous -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) and the {psi}-aminobutyric acid type A receptor (GABAAR) with just minutes of in-brain green light irradiation. Furthermore, PhoxIDs temporal precision allowed us to capture molecular snapshots of the AMPAR-proximal proteome in the postnatal developing cerebellum, leading to the discovery of age-dependent shifts. Overall, this work establishes a highly flexible and generalisable platform to study receptor interactomes and proximal microenvironments in genetically intact specimens with an unprecedented temporal resolution.

neuroscience↗

bioRxiv2023

Bioorthogonal chemical labelling of endogenous neurotransmitter receptors in living mouse brains

Neurotransmitter receptors are essential components of synapses for communication between neurons in the brain. Because the spatiotemporal expression profiles and dynamics of neurotransmitter receptors involved in many functions are delicately governed in the brain, in vivo research tools with high spatiotemporal resolution for receptors in intact brains are highly desirable. Covalent chemical labelling of proteins without genetic manipulation is now a powerful method for analyzing receptors in vitro. However, selective target receptor labelling in the brain has not yet been achieved. This study shows that ligand-directed alkoxyacylimidazole (LDAI) chemistry can be used to selectively tether synthetic probes to target endogenous receptors in living mouse brains. The reactive LDAI reagents with negative charges were found to diffuse well over the whole brain and could selectively label target endogenous receptors, including AMPAR, NMDAR, mGlu1, and GABAAR. This simple and robust labelling protocol was then used for various applications: three-dimensional spatial mapping of endogenous receptors in the brains of healthy and disease-model mice; multi-colour receptor imaging; and pulse-chase analysis of the receptor dynamics in postnatal mouse brains. Here, results demonstrated that bioorthogonal receptor modification in living animal brains may provide innovative molecular tools that contribute to the in-depth understanding of complicated brain functions.

biochemistry↗

bioRxiv2022

Organelle-selective click labeling coupled with flow cytometry allows high-throughput CRISPR screening of genes involved in phosphatidylcholine metabolism

Lipids comprise biomembranes and are involved in many crucial cell functions. While cellular lipid synthesis and transport appear to be governed by intricate protein networks, the whole scheme is insufficiently understood. Although functional genome-wide 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, this work revealed previously uncharacterized roles of FLVCR1 as a new choline transporter; CHEK1 as a post-translational regulator of the PC-synthetic pathway, and TMEM30A as responsible for 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.

cell biology↗

bioRxiv2021

Revisiting PFA-mediated tissue fixation chemistry: FixEL enables trapping of small molecules in the brain to visualize their distribution dynamics

Various small molecules have been used as functional probes for tissue imaging in medical diagnosis and pharmaceutical drugs for disease treatment. The spatial distribution, target selectivity, and diffusion/extrusion kinetics of small molecules in structurally complicated specimens are critical for function. However, robust methods for precisely evaluating these parameters in the brain have been limited. Herein we report a new method termed "Fixation-driven chemical crosslinking of exogenous ligands (FixEL)" which traps and images exogenously administered molecules-of-interest (MOI) in complex tissues. This method relies on proteins-MOI interactions, and chemical crosslinking of amine-tethered MOI with paraformaldehyde used for perfusion fixation. FixEL is used to obtain images of the distribution of the small molecules and their dynamics, which addresses selective/nonselective binding to proteins, time-dependent localization changes, and diffusion/retention kinetics of MOI such as PET tracer derivatives or drug-like small molecules. Clear imaging of a nanobody distributed in the whole brain was also achieved with high spatial resolution using 2D/3D mode.

neuroscience↗

bioRxiv2021

Coordination chemogenetics for activation of GPCR-type glutamate receptors in brain tissue

Direct activation of cell-surface receptors is highly desirable for elucidating the physiological roles of receptors. However, subtype-selective ligands are very limited because of the high homology among receptor subtypes. A potential approach for selective activation of a receptor subtype is chemogenetics, in which both point mutagenesis of the receptors and designed ligands are used. However, ligand-binding properties are affected in most current methods. Here, we developed a chemogenetic method for direct activation of metabotropic glutamate receptor 1 (mGlu1), which plays essential roles in cerebellar functions in the brain. Our screening identified a mGlu1 mutant, mGlu1(N264H), that was directly activated by palladium complexes. Notably, a palladium complex showing low cytotoxicity successfully activated mGlu1 in mGlu1(N264H) knock-in mice, revealing that activation of endogenous mGlu1 is sufficient to evoke the critical cellular mechanism of synaptic plasticity, a basis of motor learning in the cerebellum.

neuroscience↗

bioRxiv2020

Ligand-directed two-step labeling to quantify neuronal glutamate receptor trafficking

The regulation of glutamate receptor localization is critical for development and synaptic plasticity in the central nervous system. Conventional biochemical and molecular biological approaches have been widely used to analyze glutamate receptor trafficking, especially for AMPA-type glutamate receptors (AMPARs). However, conflicting findings have been reported because of a lack of useful tools for analyzing endogenous AMPARs. Here, we develop a new method for the rapid and selective labeling of chemical probes to AMPARs by combining affinity-based protein labeling and bioorthogonal click chemistry under physiological conditions. This method allowed us to quantify AMPAR distribution and trafficking, which revealed some unique features of AMPARs, such as a long lifetime and a rapid recycling in neurons. This method was also successfully expanded to selectively label NMDA-type glutamate receptors. Thus, bioorthogonal two-step labeling may be a versatile tool for investigating the physiological and pathophysiological roles of glutamate receptors in neurons.

biochemistry↗