Signal-Sustained Imaging of Mitophagy with an Enzyme-Activatable Metabolic Lipid Labeling Probe

• Published in: Autophagy Vol. 20; no. 11; pp. 2556 - 2570
• Main Authors: Zou, Xiaoxue, Wen, Shixiong, Xu, Lichun, Gao, Lei, Wang, Xunxiang, Hu, Xiao, Han, Jiahuai, Han, Shoufa
• Format: Journal Article
• Language: English
• Published: United States Taylor & Francis 01-11-2024
• Subjects: Enzyme activation; fluorescence-on; metabolic lipid labeling; mitophagy imaging; ph-independent fluorescence; mitophagy imaging; metabolic lipid labeling; fluorescence-on; Enzyme activation; ph-independent fluorescence
• ISSN: 1554-8627; 1554-8635; 1554-8635
• DOI: 10.1080/15548627.2024.2367192

Imaging of mitophagy is of significance as aberrant mitophagy is engaged in multiple diseases. Mitophagy has been imaged with synthetic or biotic pH sensors by reporting pH acidification en route delivery into lysosomes. To circumvent uncertainty of acidity-dependent signals, we herein report an
e
nzyme-activatable probe
c
ovalently
a
ttached on
m
itochondrial inner membrane (ECAM) for signal-persist mitophagy imaging. ECAM is operated via ΔΨm-driven accumulation of Mito-proGreen in mitochondria and covalent linking of the trapped probe with azidophospholipids metabolically incorporated into the mitochondrial inner membrane. Upon mitophagy, ECAM is delivered into lysosomes and hydrolyzed by LNPEP/leucyl aminopeptidase, yielding turn-on green fluorescence that is immune to lysosomal acidity changes and stably retained in fixed cells. With ECAM, phorbol-12-myristate-13-acetate (PMA) was identified as a highly potent inducer of mitophagy. Overcoming signal susceptibility of pH probes and liability of ΔΨm probes to dissipation from stressed mitochondria, ECAM offers an attractive tool to study mitophagy and mitophagy-inducing therapeutic agents.
Abbreviations: Baf-A1, bafilomycin A
1
; CCCP, carbonyl cyanide m-chlorophenylhydrazone; DBCO, dibenzocyclooctyne; ECAM, enzyme-activated probe covalently attached on mitochondrial inner membrane; GFP, green fluorescent protein; LAMP2, lysosomal associated membrane protein 2; LNPEP/LAP, leucyl and cystinyl aminopeptidase; PMA, phorbol-12-myristate-13-acetate; ΔΨm, mitochondrial transmembrane potential; RFP, red fluorescent protein; TPP, triphenylphosphonium.