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Methods. 2015 Mar;75:61-8. doi: 10.1016/j.ymeth.2015.01.017. Epub 2015 Feb 7.

Structured illumination microscopy and correlative microscopy to study autophagy.

Author information

1
Cellular Microbiology and Physics of Infection, Center for Infection and Immunity of Lille, Institut Pasteur de Lille, F-59021 Lille, France; CNRS UMR8204, F-59021 Lille, France; University of Lille-Nord de France, F-59021 Lille, France. Electronic address: ligeon@immunology.uzh.ch.
2
INSERM U1019, F-59021 Lille, France; BioImaging Center Lille-Nord de France, IFR142, Institut Pasteur de Lille, F-59021 Lille, France.
3
INSERM U1019, F-59021 Lille, France; CNRS UMR8161, F-59021 Lille, France.
4
Cellular Microbiology and Physics of Infection, Center for Infection and Immunity of Lille, Institut Pasteur de Lille, F-59021 Lille, France; BioImaging Center Lille-Nord de France, IFR142, Institut Pasteur de Lille, F-59021 Lille, France.
5
Cellular Microbiology and Physics of Infection, Center for Infection and Immunity of Lille, Institut Pasteur de Lille, F-59021 Lille, France; CNRS UMR8204, F-59021 Lille, France; University of Lille-Nord de France, F-59021 Lille, France; INSERM U1019, F-59021 Lille, France; BioImaging Center Lille-Nord de France, IFR142, Institut Pasteur de Lille, F-59021 Lille, France.

Abstract

Autophagy is a predominant eukaryotic mechanism for the engulfment of "portions" of cytoplasm allowing their degradation to recycle metabolites. The autophagy is ubiquitous among the life kingdom revealing the importance of this pathway that appears more complex than previously thought. Several reviews have already addressed how to monitor this pathway and have highlighted the existence of new routes such as the LC3-associated phagocytosis (LAP) and the non-canonical autophagy. The principal difference between autophagosomes and LAP vacuoles is that the former has two limiting membranes positives for LC3 whereas the latter has one. Herein, we propose to emphasize the use of correlative light electron microscopy (CLEM) to answer some autophagy's related questions. The structured illumination microscopy (SIM) relatively easy to implement allows to better observe the Atg proteins recruitment and localization during the autophagy process. While LC3 recruitment is performed using light microscopy the ultrastructural morphological analysis of LC3-vacuoles is ascertained by electron microscopy. Hence, these combined and correlated approaches allow to tackle the LAP vs. autophagosome issue.

KEYWORDS:

Autophagy; Correlative light-electron microscopy; LC3-associated phagocytosis; Structured illumination microscopy

PMID:
25667106
DOI:
10.1016/j.ymeth.2015.01.017
[Indexed for MEDLINE]

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