The x axis represents mitochondrial position, and the y axis is time (moving from top to bottom).
The first frame of each time-lapse imaging series is shown above a kymograph generated from the movie. ( F) Mitochondrial movement in representative axons transfected with mito-DsRed along with indicated constructs and dissected from Parkin +/+ ( Left) or Parkin −/− ( Right) animals. n = 40–50 cells for each condition from three independent transfections per genotype. ( D and E) YFP intensity in cytoplasmic and mitochondrial compartments ( D) and average number of mitochondrial fragments ( E) in cells coexpressing MiroS156E with either YFP-ParkinWT or YFP-ParkinC431F. ( C) Average number of small (0.2–5 μm in diameter) and rounded (circularity ∼0.5–1) mitochondria in cells expressing either GFP or YFP-Parkin in PINK1 +/+ and PINK1 −/− genetic backgrounds. Overexpression of Myc-MiroS156E, and to a lesser extent Myc-Miro, increased YFP intensity exclusively in the mitochondrial compartment. ( B) The average median gray value of YFP-Parkin in PINK1 +/+ (gray bars) and PINK1 −/− (white bars) cells in the cytoplasmic and the mitochondrial compartments plotted for each condition. YFP fluorescence was used to estimate Parkin levels and localization.
( A) Rat embryonic fibroblasts were transfected with the indicated constructs and stained with α-Tom20 to label mitochondria and α-Myc to label Myc-Miro. Miro PINK1 Parkin mitochondrial transport mitophagy.Ĭellular effects upon mimicking Miro phosphorylation on S156. We propose that the status of Miro phosphorylation influences the decision to undergo Parkin-dependent mitochondrial arrest, which, in the context of PINK1 action on other substrates, can restrict mitochondrial dynamics before mitophagy. The effects of the T298E/T299E phosphomimetic were dominant over S156E substitution. In contrast, mimicking phosphorylation of Miro on T298/T299 inhibited PINK1-induced Miro ubiquitination, Parkin recruitment, and Parkin-dependent mitochondrial arrest. Although Miro S156E promoted Parkin recruitment it was insufficient to trigger mitophagy in the absence of broader PINK1 action. We show that mimicking PINK1 phosphorylation of Miro on S156 promoted the interaction of Parkin with Miro, stimulated Miro ubiquitination and degradation, recruited Parkin to the mitochondria, and via Parkin arrested axonal transport of mitochondria. PINK1, however, has other mitochondrial substrates, including Miro (also called RhoT1 and -2), although the significance of those substrates is less clear. PINK1 activates Parkin by phosphorylating both Parkin and ubiquitin. Before the onset of mitophagy, the pathway blocks mitochondrial motility by causing Miro degradation. The PTEN-induced putative kinase 1 (PINK1)/Parkin pathway can tag damaged mitochondria and trigger their degradation by mitophagy.