Measuring Mitochondrial Transmembrane Potential by TMRE Staining

Crowley, Lisa C.; Christensen, Melinda E.; Waterhouse, Nigel J.

doi:10.1101/pdb.prot087361

Cited by 129 publications

(83 citation statements)

References 9 publications

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“…In S. cerevisiae , LSO/Erg3 is not required for viability in media containing ergosterol but mutants fail to grow on non-fermentable substrates such as glycerol and ethanol, suggesting that this enzyme is needed for respiration (31, 61). To assess the role of LSO in mitochondrial functions in L. major , we first examined the mitochondrial membrane potential (ΔΨ m ) after labeling cells with tetramethylrhodamine ethyl ester (TMRE) (62). Compared to WT and add-back parasites, lso – mutants had 30-50% higher ΔΨ m in the early stationary stage but did not show significant difference in log phase or late log phase (Fig.…”

Section: Resultsmentioning

confidence: 99%

Lathosterol oxidase (sterol C5-desaturase) deletion confers resistance to amphotericin B and sensitivity to acidic stress inLeishmania major

Frankfater²,

Hsu³

et al. 2020

Preprint

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8Lathosterol oxidase (LSO) catalyzes the formation of C5-C6 double bond in the synthesis 1 9 3 1 expression. Together, these findings suggest that the C5-C6 double bond is vital for the structure 3 2 of sterol core, and while the loss of LSO can lead to amphotericin B resistance, it also makes 3 3Leishmania parasites vulnerable to biologically relevant stress. 3 4 3 IMPORTANCE 3 5 Sterols are essential membrane components in eukaryotes and sterol synthesis inhibitors 3 6 can have potent effects against pathogenic fungi and trypanosomatids. Understanding the roles of 3 7 sterols will facilitate the development of new drugs and counter drug resistance. Lathosterol 3 8 oxidase (aka sterol C5-desaturase) is required for the formation of C5-C6 double bond in the 3 9 sterol core structure in mammals, fungi, protozoans, plants and algae. Functions of this C5-C6 4 0 double bond are not well understood. In this study, we generated and characterized a lathosterol 4 1 oxidase-null mutant in Leishmania major. Our data suggest that the C5-C6 double bond is vital 4 2 for the structure and membrane-stabilizing functions of leishmanial sterols. In addition, our 4 3 results imply that while mutations in lathosterol oxidase can confer resistance to amphotericin B, 4 4an important antifungal and antiprotozoal agent, the alteration in sterol structure leads to 4 5 significant defects in stress response that could be exploited for drug development. 4 6 4

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Section: Resultsmentioning

confidence: 99%

Lathosterol oxidase (sterol C5-desaturase) deletion confers resistance to amphotericin B and sensitivity to acidic stress inLeishmania major

Frankfater²,

Hsu³

et al. 2020

Preprint

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“…A mitochondria’s transmembrane potential is closely linked to its functions (Zorova et al, 2018) and can be visualized by the fluorescent, potentiometric probe TMRE. This live probe emits more fluorescence as the membrane potential becomes more negative (Crowley et al, 2016). To determine whether IGF1R attenuation affected mitochondrial membrane potential, we treated wild type larvae with NVP-AEW541, a selective inhibitor of IGF1R phosphorylation (Chablais and Jazwinska, 2010), and labeled the hair cells’ mitochondria with TMRE.…”

Section: Resultsmentioning

confidence: 99%

Pregnancy-associated plasma protein-aa supports hair cell survival by regulating mitochondrial function

Alassaf

Daykin

Mathiaparanam

et al. 2019

Preprint

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19To support cell survival, mitochondria must balance energy production with oxidative stress. Inner 20 ear hair cells are particularly vulnerable to oxidative stress; thus require tight mitochondrial regulation. 21 We identified a novel molecular regulator of the hair cells' mitochondria and survival: Pregnancy-22 associated plasma protein-aa (Pappaa). Hair cells in zebrafish pappaa mutants exhibit mitochondrial 23 defects, including elevated mitochondrial calcium, transmembrane potential, and reactive oxygen 24 species (ROS) production and reduced antioxidant expression. In pappaa mutants, hair cell death is 25 enhanced by stimulation of mitochondrial calcium or ROS production and suppressed by a 26 mitochondrial ROS scavenger. As a secreted metalloprotease, Pappaa stimulates extracellular insulin-27 like growth factor 1 (IGF1) bioavailability. We found that the pappaa mutants' enhanced hair cell loss 28 can be suppressed by stimulation of IGF1 availability and that Pappaa-IGF1 signaling acts post-29 developmentally to support hair cell survival. These results reveal Pappaa as an extracellular regulator 30 of hair cell survival and essential mitochondrial function. 31 32Without a sufficient regenerative capacity, a nervous system's form and function critically depends 33 on the molecular and cellular mechanisms that support its cells' longevity. Neural cell survival is 34 inherently challenged by the nervous system's high energy demand, which is required to support basic 35 functions, including maintaining membrane potential, propagating electrical signals, and coordinating 36 the release and uptake of neurotransmitters (Halliwell, 2006; Kann and Kovács, 2007; Howarth et al., 37 2012). Metabolic energy is primarily supplied by mitochondrial oxidative phosphorylation (Kann and 38 Kovács, 2007). Although this process is essential to cell survival, a cytotoxic consequence is the 39 generation of reactive oxygen species (ROS). Oxidative stress caused by ROS accumulation damages 40 vital cell components including DNA, proteins, and lipids (Schieber and Chandel, 2014). Neural cells 41 are particularly vulnerable to oxidative stress due not only to their energy demand and thereby ROS 42 production, but also to their relatively insufficient antioxidant capacity (Halliwell, 1992). This 43 heightened susceptibility to oxidative stress-mediated cell death is believed to underlie aging and 44 neurodegenerative disorders, including Alzheimer's disease (AD), Parkinson's disease (PD), and 45 Amyotrophic lateral sclerosis (ALS) (Perry et al., 2002; Barber et al., 2006; Mattson and Magnus, 46 2006; Blesa et al., 2015). 47 Hair cells of the inner ear are a population of neural cells that are particularly susceptible to 48 oxidative stress-induced death (Gonzalez-Gonzalez, 2017) These specialized sensory cells relay sound 49 and balance information to the central nervous system. Hair cell death or damage, which is irreversible 50 in mammals, is the primary cause of hearing loss, and is exacerbated by ag...

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“…To assess mitochondrial mass, lung cells were stained with Mitotracker Green FM (Invitrogen) (25). To assess mitochondrial potential, cells were stained with tetramethylrhodamine ester (TMRE; Life Tech) and processed as described previously (26). To evaluate glucose uptake, 100 l of 5 mM 2-NBDG (Cayman Chemical) was administered by tail vein injection.…”

Section: Discussionmentioning

confidence: 99%

Interleukin-22 (IL-22) Binding Protein Constrains IL-22 Activity, Host Defense, and Oxidative Phosphorylation Genes during Pneumococcal Pneumonia

et al. 2019

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Streptococcus pneumoniae is the most common cause of community-acquired pneumonia worldwide, and interleukin-22 (IL-22) helps contain pneumococcal burden in lungs and extrapulmonary tissues. Administration of IL-22 increases hepatic complement 3 and complement deposition on bacteria and improves phagocytosis by neutrophils. The effects of IL-22 can be tempered by a secreted natural antagonist, known as IL-22 binding protein (IL-22BP), encoded by Il22ra2. To date, the degree to which IL-22BP controls IL-22 in pulmonary infection is not well defined. Here, we show that Il22ra2 inhibits IL-22 during S. pneumoniae lung infection and that Il22ra2 deficiency favors downregulation of oxidative phosphorylation (OXPHOS) genes in an IL-22-dependent manner. Il22ra2−/− mice are more resistant to S. pneumoniae infection, have increased IL-22 in lung tissues, and sustain longer survival upon infection than control mice. Transcriptome sequencing (RNA-seq) analysis of infected Il22ra2−/− mouse lungs revealed downregulation of genes involved in OXPHOS. Downregulation of this metabolic process is necessary for increased glycolysis, a crucial step for transitioning to a proinflammatory phenotype, in particular macrophages and dendritic cells (DCs). Accordingly, we saw that macrophages from Il22ra2−/− mice displayed reduced OXPHOS gene expression upon infection with S. pneumoniae, changes that were IL-22 dependent. Furthermore, we showed that macrophages express IL-22 receptor subunit alpha-1 (IL-22Ra1) during pneumococcal infection and that Il22ra2−/− macrophages rely more on the glycolytic pathway than wild-type (WT) controls. Together, these data indicate that IL-22BP deficiency enhances IL-22 signaling in the lung, thus contributing to resistance to pneumococcal pneumonia by downregulating OXPHOS genes and increasing glycolysis in macrophages.

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Measuring Mitochondrial Transmembrane Potential by TMRE Staining

Cited by 129 publications

References 9 publications

Lathosterol oxidase (sterol C5-desaturase) deletion confers resistance to amphotericin B and sensitivity to acidic stress inLeishmania major

Lathosterol oxidase (sterol C5-desaturase) deletion confers resistance to amphotericin B and sensitivity to acidic stress inLeishmania major

Pregnancy-associated plasma protein-aa supports hair cell survival by regulating mitochondrial function

Interleukin-22 (IL-22) Binding Protein Constrains IL-22 Activity, Host Defense, and Oxidative Phosphorylation Genes during Pneumococcal Pneumonia

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