an in vivo assay to detect mitophagy in transgenic nematodes

An In Vivo Assay to Detect Mitophagy in Transgenic Nematodes

On day one, pick L4 larvae of transgenic animals expressing both DCT-1::GFP and DsRed::LGG-1 in body wall muscle cells and place them onto an OP50-seeded nematode growth media plate. Place 5 to 10 worms onto each 3.5-centimeter plate using at least three plates. When finished, incubate the nematodes at 20 degrees Celsius.
Next, on day 5, synchronize the nematodes by selecting 15 to 20 L4 transgenic larvae and transferring them onto fresh OP50-seeded plates. Use at least 5 plates for each experimental condition. On day seven, prepare some of the vehicle plates for mitophagy-affecting drugs of interest and some to use as positive controls.
Next, expose E. coli-seeded plates to UV light for 15 minutes at an intensity of 222 microwatts per centimeter squared to ensure the mitophagy-inducing compounds on the plates are not metabolized by bacteria. Now, add the compound of interest in 10 microliters to the top of the seeded plates, and add an equivalent amount of compound vehicle to the vehicle plates as a negative control.
For positive control of mitophagy induction, add paraquat, a mitochondrial toxicant. Gently swirl the plates until the solution cuts the entire surface, and allow the plates to dry with the lids closed at room temperature for at least one hour. Once dry, transfer 10 to 20 2-day-old adult transgenic animals to the plates. Incubate them at 20 degrees Celsius for two days.
On day 9, prepare 2% agarose pads, and add 1 droplet of 20 millimolar levamisole in M9 per pad. Immobilize the transgenic animals for imaging by placing them in the M9-levamisole drop. Then, gently place a coverslip on the top of the drop. Place the sample on a confocal microscope stage and image the single body wall muscle cells by taking Z-Stack images under 63 times magnification.

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Universidad de Cartagena UDEC

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Immunology

Antibody-Based Technologies

Antibody-Based Imaging and Detection Methods

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1. Detection of Mitophagy in C. elegans
NOTE: The nematode C. elegans provides a platform to assay mitophagy at the organismal level. Two strains can be used to monitor mitophagy: (1) mitochondria-targeted Rosella (mtRosella) or (2) mitophagy receptor DCT-1 fused with GFP along with autophagosomal marker LGG-1 fused with Discosoma sp. red fluorescent protein (DsRed).
<ol>
	<li>Mitophagy monitoring using Rosella biosensor 
	NOTE: Rosella is a molecular biosensor that combines a pH-insensitive DsRed fused to a pH-sensitive GFP variant. The Rosella biosensor takes advantage of the pH differences between the acidic lysosome (pH ~4.5) and other cellular compartments. This biosensor has been used to successfully monitor mitophagy in Saccharomyces cerevisiae and several mammalian cell lines, such as HeLa, HEK293, and HCT116. We adapted this versatile dual-fluorescent reporter and generated transgenic C. elegans nematodes expressing mitochondria-targeted Rosella (mtRosella) in body-wall muscle cells. Mitophagy induction is indicated by a reduced GFP/DsRed ratio of mtRosella fluorescence.
	<ol>
		<li>Day 1: Pick L4 larvae of worms expressing mitochondria-targeted Rosella (mtRosella) in body-wall muscle cells onto a Nematode Growth Media (NGM) plate seeded with E. coli (OP50) using a dissection microscope. Place 10-20 worms/plate on at least three 3.5 cm plates. Incubate the nematodes at the standard temperature of 20 &#176;C. 
		NOTE: See reference for the worm anatomy, including identification of L4 larvae, and the way to make a pick which is used to transfer worms from one location to another.</li>
		<li>Day 5: Synchronize nematodes by selecting 15-20 L4 transgenic larvae and transferring them onto fresh OP50 seeded NGM plate. Use at least five plates per experimental condition.</li>
		<li>Day 7: Prepare vehicle plates for mitophagy-affecting drugs of interest or positive controls.
		<ol>
			<li>Kill E. coli (OP50) bacteria seeded by exposing NGM plates for 15 min with 222 &#181;W/cm2 (intensity) of UV light to ensure that mitophagy-inducing compounds are not metabolized by bacteria. Plates must be exposed with 2,000 J/m2. 
			&#8203;NOTE: Seconds of exposure needed = 2,000/((intensity in &#181;W/cm2)*100).</li>
			<li>Add compound(s) of interest to the top of seeded NGM plates. Add an equivalent amount of compound vehicle (solvent used to dissolve compound, such as dimethyl sulfoxide (DMSO)) to the vehicle plates as a negative control. For positive control of mitophagy induction, a mitochondrial toxicant, paraquat (8 mM final concentration) was used. Add a solution containing 10 &#181;L of 8 M paraquat stock with 190 &#181;L of ddH2O on the top of the agar plate for the treatment group. For the vehicle group, add a solution containing 10 &#181;L of DMSO with 190 &#181;L of ddH2O on the top of the agar plate. 
			NOTE: The paraquat-working solution may be kept for 1 month at 4 &#176;C.</li>
			<li>Gently swirl the plates until the drug or vehicle coats the entire surface. Allow the plates to dry with the lids closed at room temperature for at least 1 h before transferring the worms. 
			NOTE: Drug plates can also be prepared by adding drugs in the liquid NGM before it solidifies.</li>
		</ol>
		</li>
		<li>Day 7: Transfer 10-20 of 2-day-old adult transgenic animals prepared in step 2.1.2 to plates containing either paraquat, compound(s) of interest, or vehicle (DMSO). Incubate the transgenic animals at 20 &#176;C for 2 days.</li>
		<li>Day 9: Prepare 2% agarose pads. Then add one droplet (10 &#181;L) of 20 mM levamisole in M9 per pad. Immobilize the transgenic animals for imaging, by placing them in the M9-levamisole drop. Gently place a coverslip on the top.</li>
		<li>Capture single transgenic animals using a camera attached to an epifluorescence microscope. Acquire fluorescent images of whole transgenic nematodes expressing mtRosella in body-wall muscle cells at 10X magnification. Save the collected images.</li>
		<li>Process the acquired images with ImageJ software. Analyze the body wall muscle cells located in the head of the worm to avoid autofluorescence in the intestinal tissue. Measure the average pixel intensity value and total area for each fluorescent image only of the head region of each animal. To analyze the specific area of interest:
		<ol>
			<li>Select &#39;split channel&#39; under the &#39;image&#39; and &#39;color&#39; drop-down menu to convert images .</li>
			<li>Utilize the &#39;freehand selection&#39; tool to capture the fluorescent area of interest.</li>
			<li>Select the &#39;measurement&#39; command under the &#39;analyze&#39; drop-down menu and perform pixel intensity analysis.</li>
			<li>Pixel intensity should be normalized by dividing intensity by the total area analyzed. Upon normalization, calculate the GFP to DsRed ratio.</li>
		</ol>
		</li>
		<li>Use statistical analysis software to either conduct student t-test (comparison between two groups) or ANOVA (comparison among multiple groups) for statistical analysis with p &#60; 0.05 as significant.</li>
	</ol>
	</li>
	<li>Assessing mitophagy using co-localization between DCT-1 and LGG-1 
	NOTE: DCT-1 is an outer mitochondrial membrane protein that acts as a mitophagy receptor in response to stress conditions, similar to its putative orthologues BNIP3 and NIX/BNIP3L in mammals. Thus, mitophagy initiation is assessed by co-localization between DCT-1 mitophagy receptor and autophagosomal membrane protein LGG-1 (homolog of the mammalian LC3).
	<ol>
		<li>Day 1: Pick L4 larvae of transgenic animals expressing both DCT-1::GFP and DsRed::LGG-1 in body-wall muscle cells5 onto an OP50 seeded NGM plate. Place 5-10 worms/3.5 cm plate, and use at least three plates. Incubate the nematodes at 20 &#176;C. 
		NOTE: The transgenes are located on separate plasmids and they are not integrated in the genome. Pick up nematodes carrying both rol-6(su1006) and pmyo-2 GFP contransformation markers. Only transgenic worms with both contransformation markers express both DCT-1::GFP and DsRed::LGG-1 in body-wall muscle cells.</li>
		<li>Follow the steps from 1.1.2-1.1.5.</li>
		<li>Image single body-wall muscle cells using a camera attached to a confocal microscope. Detect the borders of entire body-wall muscle cells and take z-stack images under 63x magnification. Higher magnification can also be used.</li>
		<li>Open and process images acquired with the confocal software. Initiation of mitophagy is indicated by co-localization of the mitophagy receptor (DCT-1::GFP) to the autophagosomal marker (DsRed::LGG-1). Manually measure co-localization events in each stack of body-wall muscle cell. It is essential to keep all microscope and camera settings (lens and magnifier, filters, exposure time, resolution, laser intensity, gain, etc.) consistent throughout experiments. All settings should be noted.</li>
		<li>Use statistical analysis software to either conduct student t-test (comparison between two groups) or ANOVA (comparison among multiple groups) for statistical analysis with p &#60; 0.05 as significant. For two-way comparisons use the Student&#39;s t-test (p &#60; 0.05 is considered significant). For multiple comparisons use the single factor (ANOVA) variance analysis, corrected by the post hoc Bonferroni test. We recommend analyzing at least 50-70 animals or 30-50 body-wall muscle cells for each experimental condition. Repeat the experiment at least three times.</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Eclipse TE-2000e confocal microscope</td>
			<td>Nikon</td>
			<td>#TE-2000e</td>
			<td></td>
		</tr>
		<tr>
			<td>Colocalization software</td>
			<td>Volocity</td>
			<td>#Volocity 6.3</td>
			<td>alternative Zeiss ZEN 2012 software</td>
		</tr>
		<tr>
			<td>IN Cell Investigator Software</td>
			<td>GE Healthcare Life Sciences</td>
			<td>#28408974</td>
			<td></td>
		</tr>
		<tr>
			<td>cell culture medium</td>
			<td>Thermofisher</td>
			<td>#DMEM&#8211;Dulbecco&#39;s Modified Eagle Medium</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Thermofisher</td>
			<td>#15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Sigma-Aldrich</td>
			<td>#12003C-1000ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture Incubator</td>
			<td>Thermofisher</td>
			<td>#Thermo Forma 3110 CO2 Water Jacketed Incubator</td>
			<td></td>
		</tr>
		<tr>
			<td>epifluorescence microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td>Zeiss Axio Imager Z2</td>
		</tr>
		<tr>
			<td>camera</td>
			<td>Olympus</td>
			<td></td>
			<td>Olympus DP71</td>
		</tr>
		<tr>
			<td>confocal microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td>Zeiss Axio Observer Z1</td>
		</tr>
		<tr>
			<td>confocal software</td>
			<td>Zeiss</td>
			<td></td>
			<td>ZEN 2012</td>
		</tr>
		<tr>
			<td>image analysis software</td>
			<td>Image J</td>
			<td>colocalization analysis, etc</td>
			<td>https://imagej.nih.gov/ij/</td>
		</tr>
		<tr>
			<td>statistical analysis software</td>
			<td>GraphPad Software Inc., San Diego, USA</td>
			<td></td>
			<td>GraphPad Prism software package</td>
		</tr>
		<tr>
			<td>material to make a worm pick</td>
			<td>Surepure Chemetals</td>
			<td>#4655</td>
			<td>The pick is made of 30 gauge 90% platinum 10% iridium wire</td>
		</tr>
		<tr>
			<td>IR: N2;Ex[pmyo-3 TOMM-20::Rosella]</td>
			<td>Material inquiry to Tavernarakis Nektarios</td>
			<td></td>
			<td>Maintain transgenic animals at 20 &#176;C</td>
		</tr>
		<tr>
			<td>IR: N2; Ex[pdct-1 DCT-1::GFP; pmyo-3 DsRed::LGG-1]</td>
			<td>Material inquiry to Tavernarakis Nektarios</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pmyo-3 TOMM-20::Rosella</td>
			<td>Material inquiry to Tavernarakis Nektarios</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pdct-1 DCT-1::GFP</td>
			<td>Material inquiry to Tavernarakis Nektarios</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pmyo-3 DsRed::LGG-1</td>
			<td>Material inquiry to Tavernarakis Nektarios</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Paraquat solution</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>M9 buffer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>M9-levamisole buffer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Microscope Slides and Coverslips</td>
			<td>Fisher Scientific</td>
			<td>#B9992000</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical forceps</td>
			<td>STERIS Animal Health</td>
			<td>19 Piece Canine Spay Pack Economy</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical scissors</td>
			<td>STERIS Animal Health</td>
			<td>19 Piece Canine Spay Pack Economy</td>
			<td></td>
		</tr>
		<tr>
			<td>1x PBS</td>
			<td>Thermofisher</td>
			<td>#AM9625</td>
			<td>10x PBS needs to be diluted to 1x PBS by using ddH2O</td>
		</tr>
		<tr>
			<td>shaker</td>
			<td>Fisher Scientific</td>
			<td>#11-676-178</td>
			<td>Thermo Scientific MaxQ HP Tabletop Orbital Small Open Air Platform Shaker Package A</td>
		</tr>
		<tr>
			<td>2% agarose pad</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Vibroslice blades</td>
			<td>World precision instruments</td>
			<td>#BLADES-2</td>
			<td>single-edge blade</td>
		</tr>
		<tr>
			<td>metal plate</td>
			<td>MSC</td>
			<td>#78803988</td>
			<td>0.012 in thick x 6 in wide x 12 in long, 430 Stainless Steel Sheet</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td></td>
			<td></td>
			<td>detergent</td>
		</tr>
		<tr>
			<td>Methyl viologen dichloride hydrate</td>
			<td>Sigma-Aldrich</td>
			<td>#856177</td>
			<td>paraquat</td>
		</tr>
		<tr>
			<td>Incubator for nematodes</td>
			<td>AQUALYTIC</td>
			<td></td>
			<td>Incubator to maintain 20 &#176;C</td>
		</tr>
		<tr>
			<td>Dissecting stereomicroscope</td>
			<td>Olympus</td>
			<td>SMZ645</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Zeiss</td>
			<td>AxioObserver Z1</td>
			<td>For nematodes (step 2)</td>
		</tr>
		<tr>
			<td>epifluorescence microscope</td>
			<td>Zeiss</td>
			<td>AxioImager Z2</td>
			<td>For nematodes (step 2)</td>
		</tr>
		<tr>
			<td>UV crosslinker</td>
			<td>Vilber Lourmat</td>
			<td>BIO-LINK &#8211; BLX-E365</td>
			<td>UV light source; 356 nm</td>
		</tr>
	</tbody>
</table>

Source: Fang, E. F., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/56301">In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice.</a> J. Vis. Exp. (2017).
This video demonstrates an in vivo assay to study mitophagy in Caenorhabditis elegans. Upon exposing the nematodes to a mitophagy-inducing drug, the occurrence of mitophagy is assessed by co-localization between DCT-1, a mitophagy receptor on the damaged mitochondria, and the autophagosomal membrane protein LGG-1.

1. Detection of Mitophagy in C. elegans
NOTE: The nematode C. elegans provides a platform to assay mitophagy at the organismal level. Two strains can be ...

Take transgenic nematodes, with body-wall muscle cells expressing green fluorophore-tagged DCT-1, an outer mitochondrial membrane protein, and a red fluorophore-tagged LGG-1, a phagophore membrane protein.
Place the worms on top of a growth medium containing a mitophagy-inducing drug.
The drug damages the cellular mitochondria, initiating mitophagy or selective clearance of damaged mitochondria.
DCT-1 on damaged mitochondria undergoes ubiquitination and phosphorylation, facilitating interaction with LGG-1 on the phagophore membrane.
The phagophore expands around the mitochondria to form an autophagosome targeted for lysosomal degradation.
Place the nematodes in an anesthetic on a microscope slide to immobilize them, and place a coverslip.
Under a fluorescence microscope, visualize yellow spots within the muscle cells, indicating the co-localization of DCT-1 emitting green fluorescence and LGG-1 displaying red fluorescence. This confirms the occurrence of mitophagy.

32 Views. Test in vivo do wykrywania mitofagii u nicieni transgenicznych

Video: Test in vivo do wykrywania mitofagii u nicieni transgenicznych - Experiment

Sensitive Measurement of Mitophagy by Flow Cytometry Using the pH-dependent Fluorescent Reporter mt-Keima

Mitophagy is a process of selective removal of damaged or unnecessary mitochondria using autophagy machinery. Close links have been found between defective mitophagy and various human diseases, including neurodegenerative diseases, cancer, and metabolic diseases. In addition, recent studies have shown that mitophagy is involved in normal cellular processes, such as differentiation and development. However, the precise role of and molecular mechanisms underlying mitophagy require further study. Therefore, it is critical to develop a robust and convenient method for measuring changes in mitophagy activity. Here, we describe a detailed protocol for quantitatively assessing mitophagy activity through flow cytometry using the mitochondria-targeted fluorescent protein Keima (mt-Keima). This flow cytometry assay can analyze mitophagy activity more rapidly and sensitively than conventional microscopy- or immunoblotting-based methods. This protocol can be applied to analyze mitophagy activity in various cell types.

Mitophagy, the selective degradation of mitochondria, has been implicated in mitochondrial homeostasis and is deregulated in various human diseases. However, convenient experimental methods for measuring mitophagy activity are very limited. Here, we provide a sensitive assay for measuring mitophagy activity using flow cytometry.

Mitophagy is a process of selective removal of damaged or unnecessary mitochondria using autophagy machinery. Close links have been found between ...

JoVE Journal

Biology

Protection of H9c2 Myocardial Cells from Oxidative Stress by Crocetin via PINK1/Parkin Pathway-Mediated Mitophagy

This study aimed to explore the oxidative stress-protective effect of crocetin on H2O2-mediated H9c2 myocardial cells through in vitro experiments, and further explore whether its mechanism is related to the impact of mitophagy. This study also aimed to demonstrate the therapeutic effect of safflower acid on oxidative stress in cardiomyocytes and explore whether its mechanism is related to the effect of mitophagy. Here, an H2O2-based oxidative stress model was constructed and assessed the degree of oxidative stress injury of cardiomyocytes by detecting the levels of lactate dehydrogenase (LDH), creatine kinase (CK), malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH Px). Reactive oxygen species (ROS)-detecting fluorescent dye DCFH-DA, JC-1 dye, and TUNEL dye were employed to assess mitochondrial damage and apoptosis. Autophagic flux was measured by transfecting Ad-mCherry-GFP-LC3B adenovirus. Mitophagy-related proteins were then detected via western blotting and immunofluorescence. However, crocetin (0.1-10 &#181;M) could significantly improve cell viability and reduce apoptosis and oxidative stress damage caused by H2O2. In cells with excessive autophagic activation, crocetin could also reduce autophagy flow and the expression of mitophagy-related proteins PINK1 and Parkin, and reverse the transfer of Parkin to mitochondria. Crocetin could reduce H2O2-mediated oxidative stress damage and the apoptosis of H9c2 cells, and its mechanism was closely related to mitophagy.

Protection of H9c2 Myocardial Cells from Oxidative Stress by Crocetin via PINK1/Parkin Pathway-Mediated Mitophagy

Based on in vitro experiments, this study revealed the mechanism of crocetin in repairing oxidative stress damage of cardiomyocytes by influencing mitophagy, in which the PINK1/Parkin signaling pathway plays an important role.

This study aimed to explore the oxidative stress-protective effect of crocetin on H2O2-mediated H9c2 myocardial cells through in vitro experiments, and ...

Medicine

Visualizing Mitophagy with Fluorescent Dyes for Mitochondria and Lysosome

Mitochondria, being the powerhouses of the cell, play important roles in bioenergetics, free radical generation, calcium homeostasis, and apoptosis. Mitophagy is the primary mechanism of mitochondrial quality control and is generally studied using microscopic observation, however in vivo mitophagy assays are difficult to perform. Evaluating mitophagy by imaging live organelles is an alternative and necessary method for mitochondrial research. This protocol describes the procedures for using the cell-permeant green-fluorescent mitochondria dye MitoTracker Green and the red-fluorescent lysosome dye LysoTracker Red in live cells, including the loading of the dyes, visualization of the mitochondria and the lysosome, and expected outcomes. Detailed steps for the evaluation of mitophagy in live cells, as well as technical notes about microscope software settings, are also provided. This method can help researchers observe mitophagy using live-cell fluorescent microscopy. In addition, it can be used to quantify mitochondria and lysosomes and assess mitochondrial morphology.

Mitophagy is the primary mechanism of mitochondrial quality control. However, the evaluation of mitophagy in vivo is hindered by the lack of reliable quantitative assays. Presented here is a protocol for the observation of mitophagy in living cells using a cell-permeant green-fluorescent mitochondria dye and a red-fluorescent lysosome dye.

Mitochondria, being the powerhouses of the cell, play important roles in bioenergetics, free radical generation, calcium homeostasis, and apoptosis. ...

An In Vivo Assay to Detect Mitophagy in Transgenic Nematodes

Transkrypcja

An In Vivo Assay to Detect Mitophagy in Transgenic Nematodes