Journal: The Journal of biological chemistry

Article Title: Labeling and measuring stressed mitochondria using a PINK1-based ratiometric fluorescent sensor.

doi: 10.1016/j.jbc.2021.101279

Figure Lengend Snippet: Figure 1. Development of a novel sensor to detect mitochondrial stress. A, schematic representation of mito-Pain F. Numbers above the construct indicate amino acid residues. B, a schematic diagram of the measurement of mitochondrial stress by mito-Pain. C, the T2A peptide sequence did not interfere with the localization of PINK1-GFP. HeLa cells stably expressing mito-Pain F or PINK1-GFP were cultured with CCCP for 24 h before fixation. The cells were stained with an antibody against Tom20 and observed by fluorescence microscopy. The scale bar represents 5 μm. The inset scale bar represents 1 μm. D, mito-Pain F is efficiently cleaved at the T2A site. HeLa cells and HeLa cells expressing mito-Pain F or PINK1-GFP were treated with CCCP or DMSO for 24 h and analyzed by immunoblotting using antibodies against PINK1, GFP, and RFP. Asterisks indicate nonspecific bands. E, PINK1-GFP, but not RFP-Omp25, of mito-Pain F was significantly increased under mitochondrial depolarization. HeLa cells stably expressing mito-Pain F were cultured with CCCP or DMSO for 24 h and then analyzed by flow cytometry. Cells for fluorescence microscopy were subjected to the same treatment and then fixed. The data represent the mean (left bottom panel: GFP-RFP fluorescence ratio) (n = 9). The main scale bar represents 5 μm. The inset scale bar represents 1 μm. CCCP, carbonyl cyanide m-chlorophenyl hydrazone; DMSO, dimethyl sulfoxide; mito-Pain, mitochondrial PINK1 accumulation index; mito-Pain F, mitochondrial PINK1 accumulation index (containing full-length PINK1); MTS, mitochondrial targeting sequence; OMS, outer mitochondrial membrane localization signal; PINK1, PTEN-induced putative kinase 1; TMD, transmembrane domain.

Article Snippet: The PB1 domain (PKCζ) (amino acid residues 15–98) was amplified from HeLa total cDNA, Omp25 (amino acid residues 107–146) was amplified from HEK293 total cDNA, Parkin (human) was amplified from YFP-Parkin (Addgene; plasmid #23955), and Rpn4 (amino acid residues 178–327) was amplified from Saccharomyces cerevisiae (BY4741) genomic DNA.

Techniques: Construct, Sequencing, Stable Transfection, Expressing, Cell Culture, Staining, Microscopy, Western Blot, Cytometry, Membrane