Reticulocyte mitophagy Monitoring mitochondrial clearance in a mammalian model

Mitochondria are the primary site of energy production in animal cells. In mitochondria, the flow of electrons through the electron transport chain creates a potential difference across the inner membrane, which is utilized for ATP production. However, due to inherent inefficiencies in electron transport, reactive oxygen species are also produced, which damage mitochondrial proteins and nucleic acids, and impair mitochondrial function.(1) Decreased mitochondrial function causes increased reactive oxygen species generation, a decline in cellular function, and potentially cell death.(2) Therefore, to maintain cellular homeostasis, mechanisms have evolved to selectively eliminate defective mitochondria.(3) Mitochondria are constantly undergoing cycles of fission and fusion, and this process appears to have a role in mitochondrial quality control. Following fission, daughter mitochondria are produced, which can differ in their membrane polarization. Depolarized mitochondria are less likely to undergo subsequent fusion, and more likely to undergo autophagic clearance.(4) As would be predicted, given the potential for cytochrome c release, depolarization is a powerful stimulus for mitochondrial clearance. Depolarization causes recruitment of the E3 ubiquitin ligase Parkin to mitochondria, which is required for their subsequent engulfment by autophagosomes.(5) Macroautophagy pathways also appear to have a role, as hepatocytes deficient for the E1-like enzyme Atg7 accumulate abnormal mitochondria.(6) Finally, recent studies in a developmental model have yielded insight into this process. Newly formed erythrocytes, also known as reticulocytes, eliminate their entire cohort of mitochondria during development.(7) This process depends on the mitochondrial protein NIX, is partially dependent on autophagy, and is independent of mitochondrial depolarization.(8-10) Here we describe the use of reticulocytes to study mitochondrial clearance.