Issue 22, 2023

Visualization of multiple localizations of GLUT4 by fluorescent probes of PYP-tag with designed unnatural warhead

Abstract

Within a cell, multiple copies of the same protein coexist in different pathways and behave differently. Being able to individually analyze the constant actions of proteins in a cell is crucial to know the pathways through which they pass and which physiological functions they are deeply involved in. However, until now, it has been difficult to distinguish protein copies with distinct translocation properties by fluorescence labeling with different colors in living cells. In this study, we have created an unnatural ligand with an unprecedented protein-tag labeling property in living cells and overcome the above-mentioned problem. Of special interest is that some fluorescent probes with the ligand can selectively and efficiently label intracellular proteins without binding to cell-surface proteins, even if the proteins are present on the cell membrane. We also developed a cell-membrane impermeable fluorescent probe that selectively labels cell-surface proteins without labeling of intracellular proteins. These localization-selective properties enabled us to visually discriminate two kinetically distinct glucose transporter 4 (GLUT4) molecules that show different multiple subcellular localization and translocation dynamics in live cells. Taking advantage of the probes, we revealed that N-glycosylation of GLUT4 influences intracellular localization. Furthermore, we were able to visually distinguish active GLUT4 molecules that underwent membrane translocation at least twice within an hour from those that remained intracellularly, discovering previously unrecognized dynamic behaviors of GLUT4. This technology provides not only a valuable tool for study on multiple localization and dynamics of proteins but also important information on diseases caused by protein translocation dysfunction.

Graphical abstract: Visualization of multiple localizations of GLUT4 by fluorescent probes of PYP-tag with designed unnatural warhead

Supplementary files

Article information

Article type
Edge Article
Submitted
09 feb 2023
Accepted
08 maj 2023
First published
15 maj 2023
This article is Open Access

All publication charges for this article have been paid for by the Royal Society of Chemistry
Creative Commons BY-NC license

Chem. Sci., 2023,14, 5925-5935

Visualization of multiple localizations of GLUT4 by fluorescent probes of PYP-tag with designed unnatural warhead

M. Nishiura, Y. Hori, M. Umeno and K. Kikuchi, Chem. Sci., 2023, 14, 5925 DOI: 10.1039/D3SC00724C

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