Advertisement

Autophagy pp 601-610 | Cite as

Imaging the Dynamics of Mitophagy in Live Cells

  • Andrew S. Moore
  • Erika L. F. Holzbaur
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1880)

Abstract

Investigating the precise spatiotemporal dynamics of mitophagy can provide insights into how mitochondrial quality control is regulated in different tissues and organisms. Here, we outline live imaging assays to quantitatively assess mitophagy dynamics in real time. This protocol describes both chemical and optogenetic techniques to induce mitochondrial damage with high spatial and temporal control. Using these assays, mitochondria can be tracked from before they sustain damage up to their engulfment by autophagosomes and acidification by lysosomes.

Key words

Mitophagy Parkin LC3 OPTN Autophagy Mitochondria Ubiquitin Lysosome 

References

  1. 1.
    Nguyen T, Padman B, Lazarou M (2016) Deciphering the molecular signals of PINK1/Parkin mitophagy. Trends Cell Biol 26:733CrossRefGoogle Scholar
  2. 2.
    Wong Y, Holzbaur E (2014) Optineurin is an autophagy receptor for damaged mitochondria in parkin-mediated mitophagy that is disrupted by an ALS-linked mutation. Proc Natl Acad Sci U S A 111:E4439–E4448CrossRefGoogle Scholar
  3. 3.
    Lazarou M et al (2015) The ubiquitin kinase PINK1 recruits autophagy receptors to induce mitophagy. Nature 524:309–314CrossRefGoogle Scholar
  4. 4.
    Moore A, Holzbaur E (2016) Dynamic recruitment and activation of ALS-associated TBK1 with its target optineurin are required for efficient mitophagy. Proc Natl Acad Sci U S A 113:E3349–E3358CrossRefGoogle Scholar
  5. 5.
    Wang Y, Nartiss Y, Steipe B, McQuibban AG, Kim PK (2012) ROS-induced mitochondrial depolarization initiates PARK2/PARKIN-dependent mitochondrial degradation by autophagy. Autophagy 8:1462–1476 http://www.tandfonline.com/doi/abs/10.4161/auto.21211CrossRefGoogle Scholar
  6. 6.
    Pankiv S et al (2007) p62/SQSTM1 binds directly to Atg8/LC3 to facilitate degradation of ubiquitinated protein aggregates by autophagy. J Biol Chem 282:24131CrossRefGoogle Scholar
  7. 7.
    Schindelin J, Arganda-Carreras I, Frise E, Kaynig V (2012) Fiji: an open-source platform for biological-image analysis. Nat Methods 9(7):676–682 https://www.nature.com/articles/nmeth.2019CrossRefGoogle Scholar
  8. 8.
    Grimm JB, English BP, Chen J, Slaughter JP, Zhang Z (2015) A general method to improve fluorophores for live-cell and single-molecule microscopy. Nat Methods 12(3):244–250 https://www.nature.com/articles/nmeth.3256CrossRefGoogle Scholar
  9. 9.
    Perry S, Norman J, Barbieri J, Brown E, Gelbard H (2011) Mitochondrial membrane potential probes and the proton gradient: a practical usage guide. BioTechniques 50:98–115CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of PhysiologyUniversity of Pennsylvania Perelman School of MedicinePhiladelphiaUSA

Personalised recommendations