Mitophagy is the selective degradation of old or depolarized mitochondria by autophagy and contributes maintaining a healthy population of mitochondria. Since damaged mitochondria lead to collapse cell homeostasis, mitophagy is believed to protect against diseases related to mitochondrial dysfunction such as neurodegenerative disorders.
Parkin, an ubiquitin ligase known as the gene responsible for Parkinson’s disease, plays an important role in autophagic elimination of mitophagy. When mitochondria are depolarized and dysfunctional, PTEN-induced putative kinase protein 1 (PINK1) accumulates on the outer membrane, and recruits Parkin on the damaged mitochondria. The outer membrane on the mitochondria is then ubiquitinated through the ubiquitin ligase activity of Parkin. Finally, the poly- ubiquitinated mitochondria are selectively recognized and executed by autophagic process.
The fluorescent protein Keima has an excitation spectrum that changes according to pH. A short wavelength (440 nm) is predominant for excitation in a neutral environment, whereas a long wavelength (586 nm) is predominant in an acidic environment. When observing a ratio (586 nm/440 nm) image obtained from image data by using these two excitation wavelengths, Keima in a neutral environment has a low ratio value and is displayed in blue. However, in an acidic environment, Keima has a high ratio value and is displayed in red.
When MT-mKeima-Red, which is the Keima tagged with a mitochondria targeting signal peptide sequence, and Parkin is expressed in target cells, mitophagy is detected and visualized through the difference in fluorescent wavelengths observed before and after drug treatment.
Mitophagy is induced by administration of CCCP and oligomycin, drugs that affect mitochondrial membrane potential, and is observed with 550 nm/440 nm ratio images (A”, B”, C”). Through mitophagy induction, the mitochondria-localized MT-mKeima-Red is displayed in red in ratio images, showing its localization in an acidic environment (B-B”). The neutralizer NH4Cl is then administered, which forces the entire cell into a neutral environment, and the image returns to blue (C-C”).
This result matches findings in which the progression of mitophagy causes mitochondria to be engulfed in lysosomes when in an acidic environment.
pMitophagy Keima-Red mPark2 enables the co-expression of MT-mKeima-Red (mitochondria-targeted monomeric Keira-Red) and Parkin (Park2) from the internal ribosome entry site (IRES), as parts of a bicistronic transcript in mammalian cells. This allows the efficient transfection, and the easier imaging and quantitative analysis. The system is also applicable to the HCA (High content analysis) with IN Cell Analyzer.
The stable system that can be used in drug discovery screening.
Mitophagy is visualized as red in the Ratio (550/440) column. The red color represents high and purple color represents low ratio of Ex.550/Ex. 440.
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