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[
Bio Protoc,
2016]
The rate of oxygen consumption is a vital marker indicating cellular function during lifetime under normal or metabolically challenged conditions. It is used broadly to study mitochondrial function (Artal-Sanz and Tavernarakis, 2009; Palikaras et al., 2015; Ryu et al., 2016) or investigate factors mediating the switch from oxidative phosphorylation to aerobic glycolysis (Chen et al., 2015; Vander Heiden et al., 2009). In this protocol, we describe a method for the determination of oxygen consumption rates in the nematode Caenorhabditis elegans.
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[
Bio Protoc,
2017]
Perturbation of mitochondrial function is a major hallmark of several pathological conditions and ageing, underlining the essential role of fine-tuned mitochondrial activity (Lopez-Otin et al., 2013). Mitochondrial selective autophagy, known as mitophagy, mediates the removal of dysfunctional and/or superfluous organelles, preserving cellular and organismal homeostasis (Palikaras and Tavernarakis, 2014; Pickrell and Youle, 2015; Scheibye-Knudsen et al., 2015). In this protocol, we describe a method for assessing mitophagy in the nematode Caenorhabditis elegans.
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[
Bio Protoc,
2016]
Eukaryotic cells heavily depend on adenosine triphosphate (ATP) generated by oxidative phosphorylation (OXPHOS) within mitochondria. ATP is the major energy currency molecule, which fuels cell to carry out numerous processes, including growth, differentiation, transportation and cell death among others (Khakh and Burnstock, 2009). Therefore, ATP levels can serve as a metabolic gauge for cellular homeostasis and survival (Artal-Sanz and Tavernarakis, 2009; Gomes et al., 2011; Palikaras et al., 2015). In this protocol, we describe a method for the determination of intracellular ATP levels using a bioluminescence approach in the nematode Caenorhabditis elegans.
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[
Methods Enzymol,
2005]
RNA interference (RNAi) was first discovered in the nematode Caenorhabditis elegans (Fire et al., 1998; Guo and Kemphues, 1995). The completion of the C. elegans genome in 1998 coupled with the advent of RNAi techniques to knock down gene function ushered in a new age in the field of functional genomics. There are four methods for double-stranded RNA (dsRNA) delivery in C. elegans: (1) injection of dsRNA into any region of the animal (Fire et al., 1998), (2) feeding with bacteria producing dsRNA (Timmons et al., 2001), (3) soaking in dsRNA (Tabara et al., 1998), and (4) in vivo production of dsRNA from transgenic promoters (Tavernarakis et al., 2000). In this chapter, we discuss the molecular genetic mechanisms, techniques, and applications of RNAi in C. elegans.
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[
Mech Ageing Dev,
2007]
An explanation is offered for the increased lifespan of Caenorhabditis elegans when mRNA translation is inhibited due to loss of the initiation factor IFE-2 [Hansen, M., Taubert, T., Crawford, D., Libina, N., Lee, S.-J., Kenyon, C., 2007. Lifespan extension by conditions that inhibit translation in Caenorhabditis elegans. Ageing Cell 6, 95-110; Pan, K.Z., Palter, J.E., Rogers, A.N., Olsen, A., Chen, D., Lithgow, G.J., Kapahi, P., 2007. Inhibition of mRNA translation extends lifespan in Caenorhabditis elegans. Ageing Cell 6, 111-119; Syntichaki, P., Troulinaki, K., Tavernarakis, N., 2007. eIF4E function in somatic cells modulates ageing in Caenorhabditis elegans. Nature 445, 922-926]. It is suggested that the general reduction of protein synthesis, due to the decreased frequency of mRNA translation, also lowers the cellular load of erroneously synthesized polypeptides which the constitutive protein homeostatic apparatus (proteases and chaperones proteins) normally eliminates. This situation results in "spare" proteolytic and chaperone function which can then deal with those proteins modified post-synthetically, e.g. by oxidation and/or glycation, which are thought to contribute to the senescent phenotype. This increased availability of proteolytic and chaperone functions may thereby contribute to the observed increase in organism stress resistance and lifespan.
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[
J Cell Biol,
2006]
Necrotic cell death is defined by distinctive morphological characteristics that are displayed by dying cells (Walker, N.I., B.V. Harmon, G.C. Gobe, and J.F. Kerr. 1988. Methods Achiev. Exp. Pathol. 13:18-54). The cellular events that transpire during necrosis to generate these necrotic traits are poorly understood. Recent studies in the nematode Caenorhabditis elegans show that cytoplasmic acidification develops during necrosis and is required for cell death (Syntichaki, P., C. Samara, and N. Tavernarakis. 2005. Curr. Biol. 15:1249-1254). However, the origin of cytoplasmic acidification remains elusive. We show that the alkalization of endosomal and lysosomal compartments ameliorates necrotic cell death triggered by diverse stimuli. In addition, mutations in genes that result in altered lysosomal biogenesis and function markedly affect neuronal necrosis. We used a genetically encoded fluorescent marker to follow lysosome fate during neurodegeneration in vivo. Strikingly, we found that lysosomes fuse and localize exclusively around a swollen nucleus. In the advanced stages of cell death, the nucleus condenses and migrates toward the periphery of the cell, whereas green fluorescent protein-labeled lysosomal membranes fade, indicating lysosomal rupture. Our findings demonstrate a prominent role for lysosomes in cellular destruction during necrotic cell death, which is likely conserved in metazoans.
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[
MicroPubl Biol,
2021]
MEC-4 and UNC-8 are subunits of the DEG/ENaC family of voltage-independent Na+ channels in C. elegans (Driscoll and Chalfie 1991, Canessa, Horisberger et al. 1993, Waldmann, Champigny et al. 1996, Waldmann, Champigny et al. 1997, de Weille, Bassilana et al. 1998, Waldmann and Lazdunski 1998). While MEC-4 is expressed in body touch neurons where it mediates the transduction of gentle touch sensation (Driscoll and Chalfie 1991, O'Hagan, Chalfie et al. 2005), UNC-8 is primarily expressed in motoneurons where it is involved in synaptic remodeling during development (Tavernarakis, Shreffler et al. 1997, Miller-Fleming, Petersen et al. 2016). Both MEC-4 and UNC-8 can be hyperactivated by genetic mutations that hinder channel closing, called (d) mutations (Driscoll and Chalfie 1991, Shreffler, Magardino et al. 1995, Goodman, Ernstrom et al. 2002, Wang, Matthewman et al. 2013). C. elegans neurons and Xenopus oocytes expressing these hyperactive variants of MEC-4 and UNC-8 undergo cell death due to uncontrolled flux of ions into the cell. Cell death in Xenopus oocytes and in cultured C. elegans neurons can be prevented by incubation with the DEG/ENaC channel blocker amiloride (Goodman, Ernstrom et al. 2002, Suzuki, Kerr et al. 2003, Wang, Matthewman et al. 2013).