Name: Protocol for Measuring Mitophagy in Caenorhabditis elegans
Uploaded: 2023-05-19T13:40:00
Description: Watch this Scientific Journal Video about Protocol for Measuring Mitophagy in Caenorhabditis elegans at JoVE.com

protocol for measuring mitophagy in caenorhabditis elegans

Cuantificación de la mitofagia en C. elegans y una línea celular de cáncer de hígado

Mitochondria are essential for all aerobic animals, including humans. They convert the chemical energy of biomolecules to adenosine triphosphate (ATP) via oxidative phosphorylation1, synthesize heme2, degrade fatty acids through &#946; oxidation3, regulate calcium4 and iron5 homeostasis, control cell death by apoptosis6, and generate reactive oxygen species (ROS), which play a vital role in redox homeostasis7. Two complementary and opposite processes maintain the integrity and proper function of the mitochondria: the synthesis of new mitochondrial components (biogenesis) and the selective removal of damaged ones through mitochondrial autophagy (i.e., mitophagy)8.
Several mitophagy pathways are mediated by enzymes, such as PINK1/Parkin, and receptors, including FUNDC1, FKBP8, and BNIP/NIX9,10. Notably, the selective degradation of mitochondrial components can occur independently of the autophagosome machinery (i.e., through mitochondrial-derived vesicles)11. However, the endpoints of the different selective mitophagy pathways are similar&#160;(i.e.,mitochondrial degradation by lysosomal enzymes)12,13. For this reason, various methods for identifying and measuring mitophagy rely on the colocalization of mitochondrial and lysosomal markers14,15,16,17 and decreased levels of mitochondrial proteins/mitochondrial DNA18.
Below is a concise description of the existing experimental methodologies for measuring mitophagy in animal cells using fluorescence microscopy, emphasizing the mitophagy endpoint phase.
Mitophagy biosensors 
Mitochondrial degradation occurs within the acidic environment of the lysosome19. Therefore, mitochondrial components, including proteins, experience a shift from a neutral to an acidic pH at the endpoint of the mitophagy process. This pattern underpins the mechanism of action of several mitophagy biosensors, including mito-Rosella18 and tandem mCherry-GFP-FIS114. These sensors contain a pH-sensitive green fluorescence protein (GFP) and a pH-insensitive red fluorescence protein (RFP). Therefore, at the endpoint of mitophagy, the green-to-red fluorescence ratio drops significantly due to the quenching of the GFP fluorophore. The major limitations of these sensors are (1) possible F&#246;rster resonance energy transfer (FRET) between the fluorophores; (2) the differential maturation rate of GFP and RFP; (3) dissociation between the GFP and RFP due to proteolytic cleavage of the polypeptide that connects them; (4) fluorescence-emission overlap; and (5) differential fluorophore brightness and quenching15,16.
A sensor that overcomes some of these limitations is the Keima mitochondrial sensor17. The mt-Keima sensor (derived from the coral protein Keima) displays a single emission peak (620 nm). However, its excitation peaks are pH-sensitive. As a result, there is a transition from a green excitation (440 nm) to a red one (586 nm) when shifting from a high pH to an acidic pH16,17. A more recent mitophagy sensor, Mito-SRAI, has advanced the field by allowing for measurements in fixed biological samples20. However, despite the many advantages of genetic sensors, such as the ability to express them in specific tissues/cells and target them to distinct mitochondrial compartments, they also have limitations. One limitation is that the genetic sensors need to be expressed in cells or animals, which can be time-consuming and resource-intensive.
Additionally, the expression of the sensors within mitochondria themselves may influence the mitochondrial function. For example, expressing mitochondrial GFP (mtGFP) in the C. elegans worm body wall muscles expands the mitochondrial network21. This phenotype depends on the function of the stress-activated transcription factor ATFS-1, which plays an essential role in the activation of unfolded protein response in mitochondria (UPRmt)21. Therefore, although genetically encoded mitochondria/mitophagy biosensors are extremely useful for monitoring mitochondria homeostasis in vivo, they may affect the very process they are designed to measure.
Mitochondria/lysosome-specific antibodies and dyes 
Another strategy for testing mitochondrial/lysosome colocalization is to use antibodies against mitochondrial/lysosomal proteins, such as the mitochondrial outer membrane protein TOM20 and lysosomal-associated membrane protein 1 (LAMP1)22. In most cases, secondary antibodies that are conjugated to a fluorophore are used to detect the fluorescence signal via microscopy. Another strategy is to combine genetic constructs with mitochondrial/lysosomal dyes, such as expressing a LAMP1::GFP fusion construct in cells while staining them with a red mitochondrial dye (e.g., Mitotracker Red)16. These methodologies, while effective, require specific antibodies and often involve working with fixed specimens or generating cells/transgenic animals expressing fluorescently labeled mitochondria/lysosomes.
Here, we outline the utilization of a commercial lysosome/mitochondria/nuclear staining kit for assessing the mitophagy-activating properties of synthetic diamine O,O (octane-1,8-diyl)bis(hydroxylamine), hereafter referred to as VL-85023, in C. elegans worms and the human cancer cell line Hep-3B (Figure 1). The staining kit contains a mixture of lysosomal/mitochondrial/nuclear-targeted dyes that specifically stain these organelles23. We previously used this kit to demonstrate the mitophagy activity of 1,8 diaminooctane (hereafter referred to as VL-004) in C. elegans23. Importantly, we validated the staining kit results with the mito-Rosella biosensor and qPCR measurements of the mitochondrial:nuclear DNA content23. This staining kit offers the following advantages. First, there is no need to generate transgenic animals or cells expressing a mitochondrial biosensor. Therefore, we can study unmodified wild-type animals or cells and, thus, save much time, money, and labor. Moreover, as mentioned, expressing mitochondrial biosensors can change the mitochondrial function. Second, the kit is cost-effective, easy to use, and fast. Third, although we demonstrate the method in C. elegans and human cells, it could be modified for other cell types and organisms.
With that said, like any method, the staining kit protocol has drawbacks. For example, the incubation of the worms with the reagent is carried out in the absence of food (we have seen that even dead bacteria significantly decrease the staining efficiency). Although the incubation time is relatively short, it is possible that even in this time frame, homeostatic responses may be altered, including mitophagy. In addition, the binding of the dyes to the ER/mitochondrial/nuclear proteins and other biomolecules may affect the activities of these organelles. Moreover, unlike mitophagy measurement with genetic sensors, we work with worms and cells that have undergone chemical fixation. Therefore, it is impossible to continue monitoring the same worms/cells at different times. Hence, we recommend combining different methodologies to validate the function of mitophagy in a particular physiological process. Below, we present new data demonstrating that VL-850 induces robust mitophagy in C. elegans worms and Hep-3B cells. Therefore, these data further support the hypothesis that VL-850 extends the lifespan of C. elegans and protects C. elegans from oxidative damage through the induction of healthy mitophagy. We have used the proton ionophore carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), which is a potent mitophagy inducer24, as a positive control.

Autor destacado: Detección de mitofagia en Caenorhabditis elegans y células de mamíferos utilizando colorantes específicos de orgánulos  

Detección de mitofagia en Caenorhabditis elegans y células de mamíferos utilizando colorantes específicos de orgánulos

Degradation of damaged mitochondria by mitophagy is essential for the function of the mitochondrial network. Unfortunately, this process declines with age. We develop and use drugs that activate mitophagy, thereby promoting healthy aging to understand the function of mitophagy in aging and age-related diseases, including Alzheimer's.One of the biggest challenges is to measure the mitophagy process without affecting it. Recent studies have shown that GFP expression in the mitochondria of muscle cells induces stress that is MT UPR. Therefore, using dyes in non-transgenic animals or cells is essential and complements genetic technologies.Our protocol has three main advantages. First, there is no need to generate transgenic animals or cells expressing mitophagy biosensors. Second, no need for expensive infrastructure and expertise such as in case of electron microscopy.And third, the organelle dye cocktail is commercially available and the protocol is simple and fast. The new mitophagy-activating compound VL-850 induces robust mitophagy, and in this way protects from oxidative stress and promote lifespan. Now the big question is to determine the underlying mechanism of action.We explored the mechanism at multiple levels, from the cellular to the organismal using C.elegans and mammalian cells.

Watch this Scientific Journal Video about Protocol for Measuring Mitophagy in Caenorhabditis elegans at JoVE.com

Protocol for Measuring Mitophagy in Caenorhabditis elegans  

Protocolo para la medición de la mitofagia en Caenorhabditis elegans

Para comenzar, tome los hermafroditas C.Elegans adultos jóvenes sincronizados en un tubo de microcentrífuga. Agregue 200 microlitros de tampón M9, que contiene Poloxamer 188, Pluronic F127 y dos microlitros del reactivo del kit de tinción al pellet de gusano. Cubra los tubos con papel de aluminio para proteger los tintes de la luz.Deje que la mezcla gire a 20 RPM durante una hora a temperatura ambiente. Luego gire los gusanos a 500 g durante un minuto. Después de eso, retire la solución de tinción sin alterar el pellet de gusano.Lave los gusanos con tampón M9 tres veces y transfiéralos a una placa de agar NGM sembrada que contenga el tratamiento adecuado. Lave los gusanos de las placas en tubos de microcentrífuga nuevos con un mililitro de tampón M9. Después, fije los gusanos con formaldehído al 1% en hielo durante 30 minutos.Y lave los gusanos con un mililitro de tampón M9 tres veces para eliminar cualquier formaldehído residual. Después de peletizar los gusanos, aspirar la cantidad máxima de sobrenadante, manteniendo el pellet intacto en 10 microlitros de M9. Luego, para preparar 2.5% de agarosa, pese 0.125 gramos de agarosa en un tubo de ensayo de vidrio de borosilicato de 10 mililitros. Agregue cinco mililitros de tampón M9 y disuelva la agarosa calentando suavemente el tubo con un mechero bunsen.Transfiera la agarosa derretida a un baño seco a 75 grados centígrados. Agregue 100 microlitros de agarosa derretida en un vidrio de cubierta de microscopio glosser de cubierta con una punta de un mililitro. Inmediatamente, coloca otro tobogán perpendicularmente sobre la gota de agarosa formando una forma de cruz.Después de dos minutos, separe suavemente las diapositivas empujando el vidrio de la cubierta superior dejando la almohadilla de agarosa en la diapositiva de la cubierta inferior. Transfiera los gusanos a la almohadilla de agarosa con una pipeta de vidrio Pasteur. Use una mecha hecha de una toallita de laboratorio para eliminar el exceso de líquido.Luego cubra los gusanos con un cubreobjetos más pequeño y aplique esmalte de uñas transparente a la periferia para evitar la evaporación. Coloque la corredera en una caja oscura para protegerla de la luz. Use un microscopio confocal para obtener imágenes de los gusanos dentro de las 24 horas en las longitudes de onda apropiadas usando una lente de aumento de 60 veces.A continuación, abra el software de imágenes y haga clic derecho en el área gris. En la ventana emergente que aparece, seleccione adquisición. Almohadilla completa Ti2.Adquisición de ND y tablas de búsqueda. En la almohadilla completa Ti2, seleccione 60 veces y ajuste la perilla de enfoque fino del microscopio para enfocar los gusanos. A continuación, seleccione disco giratorio y elija la opción de agrupación de 16 bits-no.Para cada filtro de fluorescencia, ajuste el tiempo de exposición a 500 milisegundos y 20 milisegundos para campo brillante. Una vez establecidos estos parámetros, seleccione ejecutar ahora y espere a que la imagen de salida se genere como un archivo ND2.

Research

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Biology

Protocol for Measuring Mitophagy in Caenorhabditis elegans

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Mitochondria are essential for various biological functions, including energy production, lipid metabolism, calcium homeostasis, heme biosynthesis, ...

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