Integrated High-Content Quantification of Intracellular ROS Levels and Mitochondrial Morphofunction

Abstract

Oxidative stress arises from an imbalance between the production of reactive oxygen species (ROS) and their removal by cellular antioxidant systems. Especially under pathological conditions, mitochondria constitute a relevant source of cellular ROS. These organelles harbor the electron transport chain, bringing electrons in close vicinity to molecular oxygen. Although a full understanding is still lacking, intracellular ROS generation and mitochondrial function are also linked to changes in mitochondrial morphology. To study the intricate relationships between the different factors that govern cellular redox balance in living cells, we have developed a high-content microscopy-based strategy for simultaneous quantification of intracellular ROS levels and mitochondrial morphofunction. Here, we summarize the principles of intracellular ROS generation and removal, and we explain the major considerations for performing quantitative microscopy analyses of ROS and mitochondrial morphofunction in living cells. Next, we describe our workflow and finally, we illustrate that a multiparametric readout enables the unambiguous classification of chemically perturbed cells as well as laminopathy patient cells. Principles of intracellular ROS generation and removalReactive oxygen species (ROS) are small, short-lived derivatives of molecular oxygen (O 2 ) of radical and non-radical nature (Halliwell and Gutteridge 2007 • ), nitrate radical (NO 3 • ) and many other nitrogen derivatives. ROS were originally described as molecular constituents of the defense system of phagocytic cells, but it has become clear that besides their damaging properties, they also function as signaling molecules and mediate a variety of other cellular responses including cell proliferation, differentiation, gene expression and migration (Lambeth 2004;Bartz and Piantadosi 2010). Intracellular ROS metabolismROS can be generated at various sites in the cell (Fig. 1A). This can be either deliberately, e.g. by NADPH oxidases (NOX), or as a byproduct, e.g. during normal cellular respiration in mitochondria (Babior 1999;Turrens 2003;Murphy 2009). The NOX family of NADPH oxidases (NOX1, NOX2, NOX3, NOX4, NOX5, DUOX1 and DUOX2) are proteins that transport electrons (e -) from NADPH across biological membranes (plasma or endomembranes) (Bedard and Krause 2007;Dupre-Crochet et al. 2013). The activation mechanisms and tissue distribution of the isoforms differ but they all use O 2 as e --acceptor, producing O 2•-. Through ROS generation, they play a role in many cellular processes including host defense, regulation of gene expression and cell differentiation (Bedard and Krause 2007). Despite their sometimes significant contribution to the global ROS pools, NOX are not the predominant source of intracellular ROS. Mitochondria are considered the major culprit, in particular under pathological conditions. Mitochondrial ROS are generated as a byproduct of the oxidative phosphorylation (OXPHOS, cf. below). Irrespective of its source, ROS production generally sta...

Mitochondrial Morphofunction in Mammalian Cells

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“…H 2 DCFDA), a reporter of cellular oxidant levels (365). The acquired images are then processed ( Fig.…”

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