Development of small fluorescent probes for the analysis of autophagy kinetics

Hajime Tajima Sakurai; Hidefumi Iwashita; Satoko Arakawa; Alifu Yikelamu; Mizuki Kusaba; Satoshi Kofuji; Hiroshi Nishina; Munetaka Ishiyama; Yuichiro Ueno; Shigeomi Shimizu

doi:10.1016/j.isci.2023.107218

Development of small fluorescent probes for the analysis of autophagy kinetics

iScience. 2023 Jun 28;26(7):107218. doi: 10.1016/j.isci.2023.107218. eCollection 2023 Jul 21.

Authors

Affiliations

¹ Department of Pathological Cell Biology, Medical Research Institute, Tokyo Medical and Dental University, TMDU, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan.
² Department of Biochemistry and Molecular Biology, Graduate School of Science, University of Hyogo, Harima Science Garden City, Hyogo 678-1205, Japan.
³ Dojindo Laboratories, Tabaru 2025-5, Mashiki-machi, Kumamoto 861-2202, Japan.
⁴ Department of Chemistry, Faculty of Science, Fukuoka University, Jonan-Ku, Fukuoka 814-0180, Japan.
⁵ Department of Developmental and Regenerative Biology, Medical Research Institute, Tokyo Medical and Dental University, TMDU, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan.

Abstract

Autophagy is a dynamic process that degrades subcellular constituents, and its activity is measured by autophagic flux. The tandem proteins RFP-GFP-LC3 and GFP-LC3-RFP-LC3ΔG, which enable the visualization of autophagic vacuoles of different stages by differences in their fluorescent color, are useful tools to monitor autophagic flux, but they require plasmid transfection. In this study, we hence aimed to develop a new method to monitor autophagic flux using small cell-permeable fluorescent probes. We previously developed two green-fluorescent probes, DALGreen and DAPGreen, which detect autolysosomes and multistep autophagic vacuoles, respectively. We here developed a red-fluorescent autophagic probe, named DAPRed, which recognizes various autophagic vacuoles. By the combinatorial use of these green- and red-fluorescent probes, we were able to readily detect autophagic flux. Furthermore, these probes were useful not only for the visualization of canonical autophagy but also for alternative autophagy. DAPRed was also applicable for the detection of autophagy in living organisms.

Keywords: Biological sciences tools; Biotechnology; Cell biology; Optical imaging.