bims-resufa 2020-03-22 papers

Issue of 2020–03–22
three papers selected by
Vera Strogolova, Marquette University

Methods Cell Biol. 2020 ;pii: S0091-679X(19)30161-X. [Epub ahead of print]155 545-555

Method for live-cell super-resolution imaging of mitochondrial cristae and quantification of submitochondrial membrane potentials.

Dane M Wolf, Mayuko Segawa, Orian S Shirihai, Marc Liesa.

The emergence of diffraction-unlimited live-cell imaging technologies has enabled the examination of mitochondrial form and function in unprecedented detail. We recently developed an approach for visualizing the inner mitochondrial membrane and determined that cristae membranes possess distinct mitochondrial membrane potentials, representing unique bioenergetic subdomains within the same organelle. Here, we outline a methodology for resolving cristae and inner boundary membranes using the LSM880 with Airyscan. Furthermore, we demonstrate how to analyze TMRE fluorescence intensity using the Nernst equation to calculate membrane potentials of individual cristae. Altogether, using these new techniques to study the electrochemical properties of the cristae can help to gain deeper insight into the still elusive nature of the mitochondrion.

Keywords: Airyscan; Cristae; Live-cell imaging; Mitochondrial membrane potential; Super-resolution imaging

DOI: https://doi.org/10.1016/bs.mcb.2019.12.006

Methods Cell Biol. 2020 ;pii: S0091-679X(19)30157-8. [Epub ahead of print]155 181-197

Analyzing electron transport chain supercomplexes.

Rebeca Acín-Pérez, Pablo Hernansanz-Agustín, José Antonio Enríquez.

This review focuses on three independent and complementary approaches to obtain information on the combined function of respiratory complexes when present in different structural situations, either as individual complexes or when superassembled with other complexes. We review the utility of in-gel activity after blue native electrophoresis, integrated oxygen consumption of supercomplexes containing complex IV, and spectrophotometric activity measurements.

Keywords: Blue native gel electrophoresis; Electron transport chain; Mitochondria respiration; Oxidative phosphorylation; Supercomplexes

DOI: https://doi.org/10.1016/bs.mcb.2019.12.002

Methods Cell Biol. 2020 ;pii: S0091-679X(19)30119-0. [Epub ahead of print]155 3-31

Isolation of mitochondria from cells and tissues.

Pin-Chao Liao, Christian Bergamini, Romana Fato, Liza A Pon, Francesco Pallotti.

Isolated mitochondria are useful to study fundamental processes including mitochondrial respiration, metabolic activity, protein import, membrane fusion, protein complex assembly, as well as interactions of mitochondria with the cytoskeleton, nuclear encoded mRNAs, and other organelles. In addition, studies of the mitochondrial proteome, phosphoproteome, and lipidome are dependent on preparation of highly purified mitochondria (Boldogh, Vojtov, Karmon, & Pon, 1998; Cui, Conte, Fox, Zara, & Winge, 2014; Marc et al., 2002; Meeusen, McCaffery, & Nunnari, 2004; Reinders et al., 2007; Schneiter et al., 1999; Stuart & Koehler, 2007). Most methods to isolate mitochondria rely on differential centrifugation, a two-step centrifugation carried out at low speed to remove intact cells, cell and tissue debris, and nuclei from whole cell extracts followed by high speed centrifugation to concentrate mitochondria and separate them from other organelles. However, methods to disrupt cells and tissue vary. Moreover, density gradient centrifugation or affinity purification of the organelle are used to further purify mitochondria or to separate different populations of the organelle. Here, we describe protocols to isolate mitochondria from different cells and tissues as well as approaches to assess the purity and integrity of isolated organelles.

Keywords: Affinity purification; Mitochondria; Subcellular fractionation; Yeast

DOI: https://doi.org/10.1016/bs.mcb.2019.10.002