Keima-Red for mitophagy detection in living cells

Mitophagy is the selective degradation of old or depolarised mitochondria by autophagy and contributes to maintaining a healthy population of mitochondria. Since damaged mitochondria lead to collapse cell homoeostasis, mitophagy is believed to be protective against diseases related to mitochondrial dysfunction such as in neurodegenerative disorders.

Parkin, an ubiquitin ligase known as the gene responsible for Parkinson’s disease, plays an important role in the autophagic elimination of mitophagy. When mitochondria are depolarised 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 recognised and executed by the autophagic process.

Emission wavelength character of Keima-Red under various pH condition

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. The ratio of fluorescent intensity in each excitation condition is an indicator of mitophagy in living cells. CoralHue® dimeric Keima-Red (dKeima-Red) and CoralHue® monomeric Keima-Red (mKeima-Red) are red fluorescent proteins with extremely large Stokes shift. They absorb light maximally at 440 nm and emit red light at 616 nm and 620 nm, respectively. There are no other fluorescent proteins with this unique fluorescence. Due of this characteristic, they are excited by a very short wavelength but emit a long wavelength. The large Stokes shift property of Keima-Red allows effective applications such as for single wavelength excitation simultaneous multi-color imaging and single laser line FCCS.

Mitochondrial targeting-mKeima-Red for mitophagy detection

When MT-mKeima-Red, which is the Keima tagged with a mitochondrial-localised signal peptide sequence, and Parkin is expressed in target cells, mitophagy is detected and is visualised through the difference in the fluorescent wavelengths observed before and after drug treatment. Mitophagy is induced by the administration of CCCP and oligomycin, drugs that affect mitochondrial membrane potential and is observed with 550nm/440nm ratio images (A”, B”, C”).

Through mitophagy induction, the mitochondria-localised MT-mKeima-Red is displayed in red in ratio images showing its localisation in an acidic environment (B-B”). The neutraliser NH4Cl is then administered which forces the entire cell into a neutral environment and the image turns to blue (C-C”). The result matched findings in which the progression of mitophagy causes mitochondria to be engulfed in lysosomes when in an acidic environment.

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