Development of small fluorescent probes for the analysis of autophagy kinetics
July 6, 2023
Abstract
Highlights
Development of a red fluorescent probe for monitoring autophagy
Combinational use of the probes allows visualization of autophagy kinetics
The probes also detect the alternative autophagic compartments
Zebrafish stained with DAPRed represent autophagic activity in vivo
Summary
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.
Development of a red fluorescent probe for monitoring autophagy
Combinational use of the probes allows visualization of autophagy kinetics
The probes also detect the alternative autophagic compartments
Zebrafish stained with DAPRed represent autophagic activity in vivo
Summary
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.
Journal Article
JOURNAL：iScience
TITLE：Development of small fluorescent probes for the analysis of autophagy kinetics
DOI：https://doi.org/10.1016/j.isci.2023.107218
TITLE：Development of small fluorescent probes for the analysis of autophagy kinetics
DOI：https://doi.org/10.1016/j.isci.2023.107218
Correspondence to
Shigeomi Shimizu, M.D., Ph.D., Professor
Department of Pathological Cell Biology,
Medical Research Institute,
Tokyo Medical and Dental University (TMDU)
E-mail：shimizu.pcb(at)mri.tmd.ac.jp
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