Mito-kaede photoactivation and chase experiment for mitophagy: optimizing flux measurement via fluid exchange system
Hiroyuki Morinaga,
Yoh Sugawara,
Yoshinori Kitagawa,
Jingyuan Chen,
Nobuo Yasuda,
Hiroki Ogata,
Yoshihiro Yamaguchi,
Masao Kaneki,
Joseph A Jeevendra Martyn,
Shingo Yasuhara
Affiliations
Hiroyuki Morinaga
Department of Anesthesiology, Critical Care & Pain Medicine, Massachusetts General Hospital, Shriners Hospitals for Children, & Harvard Medical School
Yoh Sugawara
Department of Anesthesiology, Critical Care & Pain Medicine, Massachusetts General Hospital, Shriners Hospitals for Children, & Harvard Medical School
Yoshinori Kitagawa
Department of Anesthesiology, Critical Care & Pain Medicine, Massachusetts General Hospital, Shriners Hospitals for Children, & Harvard Medical School
Jingyuan Chen
Department of Anesthesiology, Critical Care & Pain Medicine, Massachusetts General Hospital, Shriners Hospitals for Children, & Harvard Medical School
Nobuo Yasuda
Okayama Healthcare Professional University
Hiroki Ogata
Department of Anesthesiology, Critical Care & Pain Medicine, Massachusetts General Hospital, Shriners Hospitals for Children, & Harvard Medical School
Yoshihiro Yamaguchi
Department of Trauma & Critical Care Medicine, Kyorin University,Faculty of Medicine
Masao Kaneki
Department of Anesthesiology, Critical Care & Pain Medicine, Massachusetts General Hospital, Shriners Hospitals for Children, & Harvard Medical School
Joseph A Jeevendra Martyn
Department of Anesthesiology, Critical Care & Pain Medicine, Massachusetts General Hospital, Shriners Hospitals for Children, & Harvard Medical School
Shingo Yasuhara
Department of Anesthesiology, Critical Care & Pain Medicine, Massachusetts General Hospital, Shriners Hospitals for Children, & Harvard Medical School
Modulating autophagy and mitophagy, vital cellular quality control systems, offer therapeutic potential for critical illnesses. However, limited drug screening options hinder progress. We present a novel assay using the photoswitchable fluorescent reporter, mito-Kaede, to quantify mitophagy flux. Mito-Kaede's superior UV-induced photoconversion and brightness post-conversion make it ideal for prolonged mitochondrial dynamics tracking. Its specificity in responding to mitophagy, confirmed by parkin-knockout cells, adds value. When coupled with a custom fluid exchange system, enabling efficient medium changes, precise mitophagy observations become feasible. This mitophagy assay, alongside our methodological insights, can decipher mitophagy's role in pathology and supports drug screening efforts.
