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Fluorescence Methods to Measure Pexophagy

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Selective Autophagy

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2845))

Abstract

We outline our approach for studying the selective autophagy of peroxisomes (pexophagy), using fluorescence microscopy in tissue cell culture models. Ratiometric reporters, which specifically localize to peroxisomes, allow a quantitative assessment of pexophagy in fixed and live cells, as well as whole organisms. We discuss chemical and physiological inducers of pexophagy and any overlap with the induction of mitophagy.

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References

  1. Islinger M, Grille S, Fahimi HD, Schrader M (2012) The peroxisome: an update on mysteries. Histochem Cell Biol 137(5):547–574. https://doi.org/10.1007/s00418-012-0941-4

    Article  CAS  PubMed  Google Scholar 

  2. Wanders RJA et al (2023) The physiological functions of human peroxisomes. Physiol Rev 103(1):957–1024. https://doi.org/10.1152/physrev.00051.2021

    Article  CAS  PubMed  Google Scholar 

  3. Tang D, Kroemer G (2020) Peroxisome: the new player in ferroptosis. Signal Transduct Target Ther 5(1):273. https://doi.org/10.1038/s41392-020-00404-3

    Article  PubMed  PubMed Central  Google Scholar 

  4. Zou Y et al (2020) Plasticity of ether lipids promotes ferroptosis susceptibility and evasion. Nature 585(7826):603–608. https://doi.org/10.1038/s41586-020-2732-8

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Jo DS et al (2020) Loss of HSPA9 induces peroxisomal degradation by increasing pexophagy. Autophagy 16(11):1989–2003. https://doi.org/10.1080/15548627.2020.1712812

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Marcassa E et al (2018) Dual role of USP30 in controlling basal pexophagy and mitophagy. EMBO Rep 19:e45595. https://doi.org/10.15252/embr.201745595

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Riccio V et al (2019) Deubiquitinating enzyme USP30 maintains basal peroxisome abundance by regulating pexophagy. J Cell Biol 218(3):798–807. https://doi.org/10.1083/jcb.201804172

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Waterham HR, Ferdinandusse S, Wanders RJ (2016) Human disorders of peroxisome metabolism and biogenesis. Biochim Biophys Acta 1863(5):922–933. https://doi.org/10.1016/j.bbamcr.2015.11.015

    Article  CAS  PubMed  Google Scholar 

  9. Nazarko TY (2017) Pexophagy is responsible for 65% of cases of peroxisome biogenesis disorders. Autophagy 13(5):991–994. https://doi.org/10.1080/15548627.2017.1291480

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. McWilliams TG et al (2016) Mito-QC illuminates mitophagy and mitochondrial architecture in vivo. J Cell Biol 214(3):333–345. https://doi.org/10.1083/jcb.201603039

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Katayama H et al (2011) A sensitive and quantitative technique for detecting autophagic events based on lysosomal delivery. Chem Biol 18(8):1042–1052. https://doi.org/10.1016/j.chembiol.2011.05.013

    Article  CAS  PubMed  Google Scholar 

  12. Sun N et al (2015) Measuring in vivo mitophagy. Mol Cell 60(4):685–696. https://doi.org/10.1016/j.molcel.2015.10.009

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Lee JJ et al (2018) Basal mitophagy is widespread in Drosophila but minimally affected by loss of Pink1 or parkin. J Cell Biol 217(5):1613–1622. https://doi.org/10.1083/jcb.201801044

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Barone FG et al. (2023) Whole organism and tissue specific analysis of pexophagy in Drosophila. bioRxiv:2023.2011.2017.567516. https://doi.org/10.1101/2023.11.17.567516

  15. Skowyra ML, Feng P, Rapoport TA (2023) Towards solving the mystery of peroxisomal matrix protein import. Trends Cell Biol. https://doi.org/10.1016/j.tcb.2023.08.005

  16. Kimura S, Noda T, Yoshimori T (2007) Dissection of the autophagosome maturation process by a novel reporter protein, tandem fluorescent-tagged LC3. Autophagy 3(5):452–460. https://doi.org/10.4161/auto.4451

    Article  CAS  PubMed  Google Scholar 

  17. Koch A et al (2005) A role for Fis1 in both mitochondrial and peroxisomal fission in mammalian cells. Mol Biol Cell 16(11):5077–5086. https://doi.org/10.1091/mbc.e05-02-0159

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Deosaran E et al (2013) NBR1 acts as an autophagy receptor for peroxisomes. J Cell Sci 126(Pt 4):939–952. https://doi.org/10.1242/jcs.114819

    Article  CAS  PubMed  Google Scholar 

  19. Nazarko TY et al (2014) Peroxisomal Atg37 binds Atg30 or palmitoyl-CoA to regulate phagophore formation during pexophagy. J Cell Biol 204(4):541–557. https://doi.org/10.1083/jcb.201307050

    Article  PubMed  PubMed Central  Google Scholar 

  20. Dolese DA et al (2022) Degradative tubular lysosomes link pexophagy to starvation and early aging in C. elegans. Autophagy 18(7):1522–1533. https://doi.org/10.1080/15548627.2021.1990647

    Article  CAS  PubMed  Google Scholar 

  21. Wilhelm LP et al (2022) BNIP3L/NIX regulates both mitophagy and pexophagy. EMBO J:e111115. https://doi.org/10.15252/embj.2022111115

  22. Liang JR, Lingeman E, Ahmed S, Corn JE (2018) Atlastins remodel the endoplasmic reticulum for selective autophagy. J Cell Biol 217(10):3354–3367. https://doi.org/10.1083/jcb.201804185

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Barone FG, Urbe S, Clague MJ (2023) Segregation of pathways leading to pexophagy. Life Sci Alliance 6(5). https://doi.org/10.26508/lsa.202201825

  24. Lee RM et al (2024) Believing is seeing—the deceptive influence of bias in quantitative microscopy. J Cell Sci 137(1). https://doi.org/10.1242/jcs.261567

  25. Schindelin J et al (2012) Fiji: an open-source platform for biological-image analysis. Nat Methods 9(7):676–682. https://doi.org/10.1038/nmeth.2019

    Article  CAS  PubMed  Google Scholar 

  26. Montava-Garriga L, Singh F, Ball G, Ganley IG (2020) Semi-automated quantitation of mitophagy in cells and tissues. Mech Ageing Dev 185:111196. https://doi.org/10.1016/j.mad.2019.111196

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. An H, Harper JW (2018) Systematic analysis of ribophagy in human cells reveals bystander flux during selective autophagy. Nat Cell Biol 20(2):135–143. https://doi.org/10.1038/s41556-017-0007-x

    Article  CAS  PubMed  Google Scholar 

  28. Li H et al (2021) The peroxisome-autophagy redox connection: a double-edged sword? Front Cell Dev Biol 9:814047. https://doi.org/10.3389/fcell.2021.814047

    Article  PubMed  PubMed Central  Google Scholar 

  29. Narendra D, Tanaka A, Suen DF, Youle RJ (2008) Parkin is recruited selectively to impaired mitochondria and promotes their autophagy. J Cell Biol 183(5):795–803. https://doi.org/10.1083/jcb.200809125

    Article  PubMed  PubMed Central  Google Scholar 

  30. Novak I et al (2010) Nix is a selective autophagy receptor for mitochondrial clearance. EMBO Rep 11(1):45–51. https://doi.org/10.1038/embor.2009.256

    Article  CAS  PubMed  Google Scholar 

  31. Ney PA (2015) Mitochondrial autophagy: origins, significance, and role of BNIP3 and NIX. Biochim Biophys Acta 1853(10 Pt B):2775–2783. https://doi.org/10.1016/j.bbamcr.2015.02.022

    Article  CAS  PubMed  Google Scholar 

  32. McWilliams TG et al (2018) Basal mitophagy occurs independently of PINK1 in mouse tissues of high metabolic demand. Cell Metab 27(2):439–449. e435. https://doi.org/10.1016/j.cmet.2017.12.008

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Elcocks H et al (2023) FBXL4 ubiquitin ligase deficiency promotes mitophagy by elevating NIX levels. EMBO J:e112799. https://doi.org/10.15252/embj.2022112799

  34. Nguyen-Dien GT et al (2022) FBXL4 suppresses mitophagy by restricting the accumulation of NIX and BNIP3 mitophagy receptors. bioRxiv:2022.2010.2012.511867 https://doi.org/10.1101/2022.10.12.511867

  35. Cao Y et al (2023) A mitochondrial SCF-FBXL4 ubiquitin E3 ligase complex degrades BNIP3 and NIX to restrain mitophagy and prevent mitochondrial disease. EMBO J:e113033. https://doi.org/10.15252/embj.2022113033

  36. Costello JL, Passmore JB, Islinger M, Schrader M (2018) Multi-localized proteins: the peroxisome-mitochondria connection. Subcell Biochem 89:383–415. https://doi.org/10.1007/978-981-13-2233-4_17

    Article  CAS  PubMed  Google Scholar 

  37. Liang JR et al (2015) USP30 deubiquitylates mitochondrial Parkin substrates and restricts apoptotic cell death. EMBO Rep 16(5):618–627. https://doi.org/10.15252/embr.201439820

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Bingol B, Sheng M (2016) Mechanisms of mitophagy: PINK1, parkin, USP30 and beyond. Free Radic Biol Med 100:210–222. https://doi.org/10.1016/j.freeradbiomed.2016.04.015

    Article  CAS  PubMed  Google Scholar 

  39. Rusilowicz-Jones EV et al (2022) Benchmarking a highly selective USP30 inhibitor for enhancement of mitophagy and pexophagy. Life Sci Alliance 5(2). https://doi.org/10.26508/lsa.202101287

  40. Rusilowicz-Jones EV et al (2020) USP30 sets a trigger threshold for PINK1-PARKIN amplification of mitochondrial ubiquitylation. Life Sci Alliance 3(8). https://doi.org/10.26508/lsa.202000768

  41. Rasmussen NL, Kournoutis A, Lamark T, Johansen T (2022) NBR1: the archetypal selective autophagy receptor. J Cell Biol 221(11). https://doi.org/10.1083/jcb.202208092

  42. Kluge AF et al (2018) Novel highly selective inhibitors of ubiquitin specific protease 30 (USP30) accelerate mitophagy. Bioorg Med Chem Lett 28(15):2655–2659. https://doi.org/10.1016/j.bmcl.2018.05.013

    Article  CAS  PubMed  Google Scholar 

  43. Phu L et al (2020) Dynamic regulation of mitochondrial import by the ubiquitin system. Mol Cell 77(5):1107–1123 e1110. https://doi.org/10.1016/j.molcel.2020.02.012

    Article  CAS  PubMed  Google Scholar 

  44. Fang TZ et al (2023) Knockout or inhibition of USP30 protects dopaminergic neurons in a Parkinson’s disease mouse model. Nat Commun 14(1):7295. https://doi.org/10.1038/s41467-023-42876-1

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgments

FB has received PhD funding from the Wellcome Trust (102172/Z/13Z). MC is a Royal Society Industry Fellow INF\R2\212031.

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Authors and Affiliations

  1. Biochemistry, Cell and Systems Biology, Institute of Systems, Molecular and Integrative Biology, University of Liverpool, Liverpool, UK

    Francesco G. Barone, Sylvie Urbé & Michael J. Clague

  2. Biochemistry Section, Surgical Neurology Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, USA

    Francesco G. Barone

Authors
  1. Francesco G. Barone
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  2. Sylvie Urbé
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  3. Michael J. Clague
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Corresponding author

Correspondence to Michael J. Clague .

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Editors and Affiliations

  1. School of Life Sciences, University of Warwick, West Midlands, UK

    Ioannis P. Nezis

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© 2024 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

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Barone, F.G., Urbé, S., Clague, M.J. (2024). Fluorescence Methods to Measure Pexophagy. In: Nezis, I.P. (eds) Selective Autophagy. Methods in Molecular Biology, vol 2845. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-4067-8_11

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  • DOI: https://doi.org/10.1007/978-1-0716-4067-8_11

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-4066-1

  • Online ISBN: 978-1-0716-4067-8

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