Mitochondrial Dynamics and Activity in Legionella-Infected Cells
The study of Legionella pneumophila interactions with host mitochondria during infection has been historically limited by the techniques available to analyze and quantify mitochondrial dynamics and activity in living cells. Recently, new, powerful techniques such as high-content microscopy or mitochondrial respiration assays (Seahorse) have been developed to quantitatively analyze mitochondrial parameters. Here we present state-of-the-art methods adapted to analyze mitochondrial dynamics and activity during Legionella infection of living human primary macrophages.
Key wordsMitochondrial dynamics Legionella pneumophila High-content microscopy Seahorse Mitochondrial respiration assay Mitochondrial fission Mitochondrial morphology MitoTracker dyes Bioenergetics Living-cell assays
Work in C.B. laboratory is financed by Institut Pasteur and ANR (grant no. ANR-10-LABX-62-IBEID). P.E. is funded by the Fondation pour la Recherche Médicale (FRM) project DEQ20120323697. P.B. and O.R.S. received financial support from the European Community (ERC-STG INTRACELLTB no. 260901, MM4TB no. 260872), the ANR (grant no. ANR-10-EQPX-04-01), and the Région Nord Pas de Calais (convention no. 12000080). We thank Nathalie Aulner and the Imagopole-CiTech (part of France-BioImaging supported by ANR grant no. ANR-10-INSB-04-01, Conseil de la Region Ile-de-France, FRM), and the Technology Core of the Center for Translational Science (CRT) at Institut Pasteur, for technical support during the setup of the methods presented here.
- 7.Labbé K, Murley A, Nunnari J (2014) Determinants and functions of mitochondrial behavior. Annu Rev Cell Dev Biol 30:357–391. https://doi.org/10.1146/annurev-cellbio-101011-155756CrossRefPubMedGoogle Scholar
- 11.Leonard AP, Cameron RB, Speiser JL et al (2015) Quantitative analysis of mitochondrial morphology and membrane potential in living cells using high-content imaging, machine learning, and morphological binning. Biochim Biophys Acta 1853:348–360. https://doi.org/10.1016/j.bbamcr.2014.11.002CrossRefPubMedGoogle Scholar
- 15.Tiaden A, Spirig T, Weber SS et al (2007) The Legionella pneumophila response regulator LqsR promotes host cell interactions as an element of the virulence regulatory network controlled by RpoS and LetA. Cell Microbiol 9:2903–2920. https://doi.org/10.1111/j.1462-5822.2007.01005.xCrossRefPubMedGoogle Scholar