Immunofluorescence-Based Measurement of Autophagosome Formation During Mitophagy

Benjamin S. Padman, Michael Lazarou

Research output: Chapter in Book/Conference paperChapterpeer-review

1 Citation (Scopus)

Abstract

Damaged, dysfunctional, or excess mitochondria are removed from cells via a selective form of macroautophagy termed mitophagy. The clearance of mitochondria during mitophagy is mediated by double-membrane vesicles called autophagosomes, which encapsulate mitochondria that have been tagged for mitophagic removal before delivering them to lysosomes for degradation. A variety of different mitophagy pathways exist that differ in their mechanisms of initiation but share a common pathway of autophagosome formation. Autophagosome biogenesis is regulated by a number of autophagy factors which translocate from the cytosol to spatially defined focal points (foci) on the mitochondrial surface after mitophagy has been initiated. The functional analysis of autophagosome biogenesis requires the use of microscopy-based techniques which assess the recruitment of autophagy factors to mitophagic foci representing autophagosome formation sites. Here, we describe a routine method for the quantitative 3D analysis of mitophagic foci in PINK1/Parkin mitophagy immunofluorescence samples through the application of object-based image analysis (OBIA) to 3D confocal imaging datasets. The approach enables unbiased high-throughput characterisation of autophagosome biogenesis during mitophagy.

Original languageEnglish
Title of host publicationMethods in Molecular Biology
EditorsHelin Norberg, Erik Norberg
PublisherHumana Press Inc.
Chapter13
Pages207-226
Number of pages20
ISBN (Electronic)978-1-0716-2071-7
ISBN (Print) 978-1-0716-2070-0
DOIs
Publication statusPublished - 1 Jan 2022

Publication series

NameMethods in Molecular Biology
Volume2445
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029

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