Publications by authors named "Mikiko Takato"

2 Publications

  • Page 1 of 1

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

Nat Commun 2021 02 5;12(1):831. Epub 2021 Feb 5.

Department of Biomolecular Engineering, Graduate School of Engineering, Nagoya University, Nagoya, 464-8603, Japan.

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 α-amino-3-hydroxy-5-methyl-4-isoxazole-propionate-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 method for the rapid and selective labeling of AMPARs with chemical probes, by combining affinity-based protein labeling and bioorthogonal click chemistry under physiological temperature in culture medium. This method allows us to quantify AMPAR distribution and trafficking, which reveals some unique features of AMPARs, such as a long lifetime and a rapid recycling in neurons. This method is also successfully expanded to selectively label N-methyl-D-aspartate-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.
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http://dx.doi.org/10.1038/s41467-021-21082-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7864911PMC
February 2021

Imaging and Profiling of Proteins under Oxidative Conditions in Cells and Tissues by Hydrogen-Peroxide-Responsive Labeling.

J Am Chem Soc 2020 09 3;142(37):15711-15721. Epub 2020 Sep 3.

Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan.

Reactive oxygen species (ROS) such as hydrogen peroxide (HO) can inflict damage to biomolecules under oxidative stress and also act as signaling molecules at physiological levels. Here we developed a unique chemical tool to elucidate the biological roles of ROS using both fluorescence imaging and conditional proteomics. HO-responsive protein labeling reagents (Hyp-L) were designed to selectively tag proteins under the oxidative conditions in living cells and tissues. The Hyp-L signal remained even after sample fixation, which was compatible with conventional immunostaining. Moreover, Hyp-L allowed proteomic profiling of the labeled proteins using a conditional proteomics workflow. The integrative analysis enabled the identification of ROS generation and/or accumulation sites with a subcellular resolution. For the first time, we characterized that autophagosomes were enriched with HO in activated macrophages. Hyp-L was further applied to mouse brain tissues and clearly revealed oxidative stress within mitochondria by the conditional proteomics.
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http://dx.doi.org/10.1021/jacs.0c02547DOI Listing
September 2020