Name: In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice (Video) | JoVE Journal
Uploaded: 2017-11-22T13:00:00.000+00:00
Duration: 8 min 40 s
Description: Learn robust methods to detect mitophagy in human cells, C. elegans, and mice using in vitro and in vivo techniques. These approaches advance research on neurodegenerative diseases and mitochondrial dysfunction.

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5 minutes

We thank Dr. Atsushi Miyawaki and Dr. Richard J. Youle for sharing the mt-Keima plasmid and mt-Keima integrated Hela cells. We thank Raghavendra A. Shamanna and Dr. Deborah L. Croteau for the critical reading of the manuscript. This research was supported by the Intramural Research Program of the NIH (VAB), National Institute on Ageing, as well as a 2014-2015 and a 2016-2017 NIA intra-laboratory grant (EFF, VAB). EFF was supported by HELSE SOR-OST RHF (Project No: 2017056) and the Research Council of Norway (Project No: 262175).
AUTHOR CONTRIBUTIONS:
EFF designed the manuscript and prepared the draft; KP, NS, EMF, RDS, JSK, SAC, YH, and ED wrote different sections of the paper; NT, JP, HN, and VAB revised the manuscript and provided expertise.

<ol><li>Fang, E. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nuclear+DNA+damage+signalling+to+mitochondria+in+ageing%5BTitle%5D)+AND+%22Nat+Rev+Mol+Cell+Biol%22%5BJournal%5D)">Nuclear DNA damage signalling to mitochondria in ageing.</a> Nat Rev Mol Cell Biol. 17 (5), 308-321 (2016).</li>
<li>Scheibye-Knudsen, M., Fang, E. F., Croteau, D. L., Wilson, D. M. 3rd, Bohr, V. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Protecting+the+mitochondrial+powerhouse%5BTitle%5D)+AND+%22Trends+Cell+Biol%22%5BJournal%5D)">Protecting the mitochondrial powerhouse.</a> Trends Cell Biol. 25 (3), 158-170 (2015).</li>
<li>Sun, N., Youle, R. J., Finkel, T. <a target="_blank" href="http://www.cell.com/molecular-cell/pdf/S1097-2765(16)00081-2.pdf">The Mitochondrial Basis of Aging.</a> Mol Cell. 61 (5), 654-666 (2016).</li>
<li>Shadel, G. S., Horvath, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mitochondrial+ROS+signaling+in+organismal+homeostasis%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Mitochondrial ROS signaling in organismal homeostasis.</a> Cell. 163 (3), 560-569 (2015).</li>
<li>Palikaras, K., Lionaki, E., Tavernarakis, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Coordination+of+mitophagy+and+mitochondrial+biogenesis+during+ageing+in+C.+elegans%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Coordination of mitophagy and mitochondrial biogenesis during ageing in C. elegans.</a> Nature. 521 (7553), 525-528 (2015).</li>
<li>Lazarou, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+ubiquitin+kinase+PINK1+recruits+autophagy+receptors+to+induce+mitophagy%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">The ubiquitin kinase PINK1 recruits autophagy receptors to induce mitophagy.</a> Nature. 524 (7565), 309-314 (2015).</li>
<li>Kerr, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mitophagy+and+Alzheimer%27s+Disease%3A+Cellular+and+Molecular+Mechanisms%5BTitle%5D)+AND+%22Trends+neurosci%22%5BJournal%5D)">Mitophagy and Alzheimer's Disease: Cellular and Molecular Mechanisms.</a> Trends neurosci. 40 (3), 151-166 (2017).</li>
<li>Fivenson, E. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mitophagy+in+neurodegeneration+and+aging%5BTitle%5D)+AND+%22Neurochem+Int%22%5BJournal%5D)">Mitophagy in neurodegeneration and aging.</a> Neurochem Int. , (2017).</li>
<li>Menzies, F. M., Fleming, A., Rubinsztein, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Compromised+autophagy+and+neurodegenerative+diseases%5BTitle%5D)+AND+%22Nat+Rev+Neurosci%22%5BJournal%5D)">Compromised autophagy and neurodegenerative diseases.</a> Nat Rev Neurosci. 16 (6), 345-357 (2015).</li>
<li>Rubinsztein, D. C., Shpilka, T., Elazar, Z. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/22240478">Mechanisms of autophagosome biogenesis.</a> Curr Biol. 22 (1), R29-R34 (2012).</li>
<li>Kerr, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mitophagy+and+Alzheimer%27s+disease%3A+cellular+and+molecular+mechanisms%5BTitle%5D)+AND+%22Trends+neurosci%22%5BJournal%5D)">Mitophagy and Alzheimer's disease: cellular and molecular mechanisms.</a> Trends neurosci. 40 (3), 151-166 (2017).</li>
<li>Menzies, F. M., Moreau, K., Puri, C., Renna, M., Rubinsztein, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Measurement+of+autophagic+activity+in+mammalian+cells%5BTitle%5D)+AND+%22Curr+Protoc+Cell+Biol%22%5BJournal%5D)">Measurement of autophagic activity in mammalian cells.</a> Curr Protoc Cell Biol. , Chapter 15, Unit 15 16(2012).</li>
<li>Katayama, H., Kogure, T., Mizushima, N., Yoshimori, T., Miyawaki, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+sensitive+and+quantitative+technique+for+detecting+autophagic+events+based+on+lysosomal+delivery%5BTitle%5D)+AND+%22Chem+Biol%22%5BJournal%5D)">A sensitive and quantitative technique for detecting autophagic events based on lysosomal delivery.</a> Chem Biol. 18 (8), 1042-1052 (2011).</li>
<li>Sun, N., et al. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/26549682">Measuring In Vivo Mitophagy.</a> Mol Cell. 60 (4), 685-696 (2015).</li>
<li>Fang, E. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Defective+mitophagy+in+XPA+via+PARP-1+hyperactivation+and+NAD%28%2B%29%2FSIRT1+reduction%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Defective mitophagy in XPA via PARP-1 hyperactivation and NAD(+)/SIRT1 reduction.</a> Cell. 157 (4), 882-896 (2014).</li>
<li>Diot, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+novel+quantitative+assay+of+mitophagy%3A+Combining+high+content+fluorescence+microscopy+and+mitochondrial+DNA+load+to+quantify+mitophagy+and+identify+novel+pharmacological+tools+against+pathogenic+heteroplasmic+mtDNA%5BTitle%5D)+AND+%22Pharmacol+Res%22%5BJournal%5D)">A novel quantitative assay of mitophagy: Combining high content fluorescence microscopy and mitochondrial DNA load to quantify mitophagy and identify novel pharmacological tools against pathogenic heteroplasmic mtDNA.</a> Pharmacol Res. 100, 24-35 (2015).</li>
<li>Schiavi, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Iron-Starvation-Induced+Mitophagy+Mediates+Lifespan+Extension+upon+Mitochondrial+Stress+in+C.+elegans%5BTitle%5D)+AND+%22Curr+Biol%22%5BJournal%5D)">Iron-Starvation-Induced Mitophagy Mediates Lifespan Extension upon Mitochondrial Stress in C. elegans.</a> Curr Biol. 25 (14), 1810-1822 (2015).</li>
<li>Rosado, C. J., Mijaljica, D., Hatzinisiriou, I., Prescott, M., Devenish, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rosella%3A+a+fluorescent+pH-biosensor+for+reporting+vacuolar+turnover+of+cytosol+and+organelles+in+yeast%5BTitle%5D)+AND+%22Autophagy%22%5BJournal%5D)">Rosella: a fluorescent pH-biosensor for reporting vacuolar turnover of cytosol and organelles in yeast.</a> Autophagy. 4 (2), 205-213 (2008).</li>
<li>Sargsyan, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rapid+parallel+measurements+of+macroautophagy+and+mitophagy+in+mammalian+cells+using+a+single+fluorescent+biosensor%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">Rapid parallel measurements of macroautophagy and mitophagy in mammalian cells using a single fluorescent biosensor.</a> Sci Rep. 5, 12397(2015).</li>
<li>Altun, Z. F., Hall, D. H. Introduction to C. elegans anatomy. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aAltun%2C+ZF+%22WormAtlas%22">WormAtlas</a>. , (2009).</li>
<li>Chaudhuri, J., Parihar, M., Pires-daSilva, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+introduction+to+worm+lab%3A+from+culturing+worms+to+mutagenesis%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">An introduction to worm lab: from culturing worms to mutagenesis.</a> J Vis Exp. (47), e2293(2011).</li>
<li>Palikaras, K., Tavernarakis, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vivo+Mitophagy+Monitoring+in+Caenorhabditis+elegans+to+Determine+Mitochondrial+Homeostasis%5BTitle%5D)+AND+%22Bio+Protoc%22%5BJournal%5D)">In vivo Mitophagy Monitoring in Caenorhabditis elegans to Determine Mitochondrial Homeostasis.</a> Bio Protoc. 7 (7), e2215(2017).</li>
<li>Palikaras, K., Tavernarakis, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Assessing+Mitochondrial+Selective+Autophagy+in+the+Nematode+Caenorhabditis+elegans%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">Assessing Mitochondrial Selective Autophagy in the Nematode Caenorhabditis elegans.</a> Methods Mol Biol. 1567, 349-361 (2017).</li>
<li>Sun, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+fluorescence-based+imaging+method+to+measure+in+vitro+and+in+vivo+mitophagy+using+mt-Keima%5BTitle%5D)+AND+%22Nat+Protoc%22%5BJournal%5D)">A fluorescence-based imaging method to measure in vitro and in vivo mitophagy using mt-Keima.</a> Nat Protoc. 12 (8), 1576-1587 (2017).</li>
<li>Eiyama, A., Okamoto, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Assays+for+Mitophagy+in+Yeast%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">Assays for Mitophagy in Yeast.</a> Methods Mol Biol. 1567, 337-347 (2017).</li>
<li>McWilliams, T. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((mito-QC+illuminates+mitophagy+and+mitochondrial+architecture+in+vivo%5BTitle%5D)+AND+%22J+Cell+Biol%22%5BJournal%5D)">mito-QC illuminates mitophagy and mitochondrial architecture in vivo.</a> J Cell Biol. 214 (3), 333-345 (2016).</li>
</ol>

The Bohr laboratory has CRADA arrangements with ChromaDex and GlaxoSmithKline.

Accurate measurement of mitophagy is technically demanding. Here, we have presented several robust techniques which allow for both qualitative detection of mitophagy and quantification of mitophagy levels in the most common laboratory experimental models.
To acquire replicable data, an experimental design with at least three biological repeats is necessary. All researchers involved in experimentation and analysis must be blinded to experimental group identities. Furthermore, imaging fields mus...

Mitophagy is essential for mitochondrial maintenance. Mitochondria intersect multiple cell signaling pathways and are universal sub-cellular organelles responsible for cellular energy production, cell metabolism, and calcium homeostasis1,2,3,4. Mitochondria constantly experience challenges from endogenous and exogenous sources, such as reactive oxygen species (ROS) and mitochondrial toxicants, respectively, which lead to the generation of &#34;aged&#34; and dysfunctional mitochondria. Accumulation of damaged mitochondria decreases the efficiency of ATP production while increasing the amount of harmful ROS, and has been linked to age-related diseases such as metabolic diseases, AD, and PD1,5,6. To prevent mitochondria induced cellular dysfunction, cells need to specifically recognize damaged mitochondria and efficiently remove them through a cellular process termed mitochondrial autophagy (mitophagy). This demonstrated importance of mitophagy in health and disease illustrates the need for accurate and efficient methods to detect mitophagy both in vitro and in vivo.
Mitophagy is a multiple-step process involving many proteins and protein complexes5,7,8. In brief, a damaged mitochondrion is first recognized and engulfed by a double-membraned phagophore, which can originate from the plasma membrane, endoplasmic reticulum, Golgi complex, nucleus, or mitochondrion itself9,10. The spherical phagophore elongates and eventually seals the mitochondria inside, constituting the mitochondrial autophagosome (mitophagosome). The mitophagosome then fuses with the lysosome for degradation, forming an autolysosome in which the damaged mitochondrion is degraded and recycled7,8. Major autophagic proteins also involved in mitophagy include: Autophagy Related 7 (ATG7) and Beclin1 (initiation), Microtubule-Associated Protein 1A/1B-Light Chain 3 (LC3-II) (LGG-1 in C. elegans) and p62 (components of phagophore), and lysosomal-associated membrane glycoprotein 2 (LAMP2)6,7. In addition, there are several essential proteins unique to mitophagy, including PTEN-induced Putative Kinase 1 (PINK-1), Parkin1, Nuclear Dot Protein 52 kDa (NDP52), optineurin, BCL2 Interacting Protein 3 Like (NIX/BNIP3L) (DCT-1 in C. elegans), among others5,6,11.
A common method to detect changes in levels of autophagy is by the ratio of LC3-II/LC3-I or LC3-II/actin. However, this method is nonspecific, as an increase in this ratio may reflect either an increased initiation or an impaired fusion of mitophagosome to lysosome12. Another method is to evaluate the colocalization between an autophagy marker (e.g., LC3) and a mitochondrial protein (e.g., Translocase of Outer Mitochondrial Membrane 20 (TOMM20, which could be degraded by proteasomes)). However, this can only indicate changes in total mitophagy levels and cannot distinguish the step(s) at which blockage occurs. This can be clarified by using lysosomal inhibitors (e.g., E64d+Pepstatin A, termed EP) in parallel to cause the accumulation of mitophagosomes. The difference between the number of mitophagosomes at baseline and the number of mitophagosomes following treatment with EP can indicate mitophagy. These limitations have prompted the development of novel mitophagy detection techniques. In view of the increasing relevance of mitophagy in a wide spectrum of human diseases, we present several robust mitophagy detection techniques which may be useful for researchers. We cover both in vitro and in vivo techniques and recommend combining multiple techniques to verify changes of mitophagy.

<table><tbody><tr><td>mt-Keima mouse</td><td>Jackson Laboratory</td><td></td><td></td></tr><tr><td>Lipofectamine 2000 DNA transfection reagent</td><td>Thermofisher</td><td>#11668027</td><td></td></tr><tr><td>Opti-MEM medium (Gibco)</td><td>Thermofisher</td><td>#31985062</td><td>serum-free medium</td></tr><tr><td>mtKemia plasmid: pCHAC-mt-mKeima</td><td>addgene</td><td>#72342</td><td></td></tr><tr><td>COXII antibody (mouse)</td><td>abcam</td><td>#ab110258</td><td></td></tr><tr><td>LAMP2 antibody (rabbit)</td><td>NOVUS</td><td>#CD107b</td><td></td></tr><tr><td>goat-anti-rabbit with wavelength 568 nm of red fluorescent protein (RFP)</td><td>Thermofisher</td><td>#Z25306</td><td>Alexa Fluor 568 dye</td></tr><tr><td>goat-anti-mouse with wavelength 488 nm of green fluorescent protein (GFP)</td><td>Thermofisher</td><td>#Z25002</td><td>Alexa Fluor 488 dye</td></tr><tr><td>prolong gold antifade with DAPI</td><td>Invitrogen</td><td>#P36931</td><td></td></tr><tr><td>6-well plate</td><td>SIGMA&lt;br/&gt; Corning Costar</td><td>#CLS3516</td><td></td></tr><tr><td>4-well chamber slide</td><td>THermofisher, Nunc Lab-Tek</td><td>#171080</td><td></td></tr><tr><td>Nunc F 96-well plate</td><td>Thermofisher</td><td>#152038</td><td></td></tr><tr><td>LC3B antibody rabbit</td><td>NOVUS</td><td>#NB100-2220</td><td></td></tr><tr><td>DNA antibody</td><td>Progen Biotechnik</td><td>#anti-DNA mouse monoclonal, AC-30-10</td><td></td></tr><tr><td>DAPI</td><td>Thermofisher</td><td>#D1306</td><td>antifade mounting medium with DAPI</td></tr><tr><td>IN Cell analyzer (fluorescent reader )</td><td>GE Healthcare Life Sciences</td><td>#IN Cell analyzer 2200</td><td></td></tr><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&amp;ndash;Dulbecco&#039;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[p&lt;em&gt;myo-3&lt;/em&gt; TOMM-20::Rosella]</td><td>Material inquiry to Tavernarakis Nektarios</td><td></td><td>Maintain transgenic animals at 20 &amp;deg;C</td></tr><tr><td>IR: N2; Ex[p&lt;em&gt;dct-1&lt;/em&gt; DCT-1::GFP; p&lt;em&gt;myo-3&lt;/em&gt; DsRed::LGG-1]</td><td>Material inquiry to Tavernarakis Nektarios</td><td></td><td></td></tr><tr><td>p&lt;em&gt;myo-3&lt;/em&gt; TOMM-20::Rosella</td><td>Material inquiry to Tavernarakis Nektarios</td><td></td><td></td></tr><tr><td>p&lt;em&gt;dct-1&lt;/em&gt; DCT-1::GFP</td><td>Material inquiry to Tavernarakis Nektarios</td><td></td><td></td></tr><tr><td>p&lt;em&gt;myo-3&lt;/em&gt; DsRed::LGG-1</td><td>Material inquiry to Tavernarakis Nektarios</td><td></td><td></td></tr><tr><td>Paraquat solution</td><td>see supplementary data for preparation</td><td></td><td></td></tr><tr><td>M9 buffer</td><td>see supplementary data for preparation</td><td></td><td></td></tr><tr><td>M9-levamisole buffer</td><td>see supplementary data for preparation</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>see supplementary data for preparation</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 &amp;deg;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 &amp;ndash; BLX-E365</td><td>UV light source; 356 nm</td></tr></tbody></table>

in vitro and in vivo detection of mitophagy in human cells, c. elegans, and mice

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a wide variety of disorders including metabolic diseases, premature aging syndromes, and neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD). Maintenance of mitochondrial health depends on mitochondrial biogenesis and the efficient clearance of dysfunctional mitochondria through mitophagy. Experimental methods to accurately detect autophagy/mitophagy, especially in animal models, have been challenging to develop. Recent progress towards the understanding of the molecular mechanisms of mitophagy has enabled the development of novel mitophagy detection techniques. Here, we introduce several versatile techniques to monitor mitophagy in human cells, Caenorhabditis elegans (e.g., Rosella and DCT-1/ LGG-1 strains), and mice (mt-Keima). A combination of these mitophagy detection techniques, including cross-species evaluation, will improve the accuracy of mitophagy measurements and lead to a better understanding of the role of mitophagy in health and disease.

Detection of Mitophagy in Human Cells:
Using the procedure presented here, human HeLa cells were transfected with mt-Keima plasmid. Healthy cells demonstrated a well-organized mitochondrial network (GFP, 488 nm) with few incidences of mitophagy (RFP, 561 nm). However, cells pretreated with a mitochondrial uncoupler FCCP (30 µM for 3 h) exhibited a profound increase in mitophagy incidence (Figure 1A). M...

Mitophagy, the process of clearing damaged mitochondria, is necessary for mitochondrial homeostasis and health maintenance. This article presents some of the latest mitophagy detection methods in human cells, Caenorhabditis elegans, and mice.

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a ...

in-vitro-vivo-detection-mitophagy-human-cells-c-elegans

Research

JoVE Journal

Medicine

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.

An In Vivo Assay to Detect Mitophagy in Transgenic Nematodes

A protocol is described for in vivo detection of effects of mitochondrial inhibitors in the model organism Caenorhabditis elegans and for identification of potential enhancing compounds. This protocol can be used to screen drug libraries for compounds modulating mitochondrial function.

A Screenable In Vivo Assay for Mitochondrial Modulators Using Transgenic Bioluminescent Caenorhabditis elegans

We herein describe a phagocytosis assay using the dispersed embryonic cells of Drosophila. It enables us to easily and precisely quantify in vivo phagocytosis levels, and to identify new molecules required for the phagocytosis of apoptotic cells.

Phagocytosis Assay for Apoptotic Cells in Drosophila Embryos

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.

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

A protocol for imaging the dynamic microtubules in vivo using fluorescently labeled End binding protein has been presented. We described the methods to label, image, and analyze the dynamic microtubules in the Posterior lateral microtubule (PLM) neuron of C. elegans.

In vivo Assessment of Microtubule Dynamics and Orientation in Caenorhabditis elegans Neurons

Exploring mitophagy through electron microscopy, genetic sensors, and immunofluorescence requires costly equipment, skilled personnel, and a significant time investment. Here, we demonstrate the efficacy of a commercial fluorescence dye kit in quantifying the mitophagy process in both Caenorhabditis elegans and a liver cancer cell line.

Detection of Mitophagy in Caenorhabditis elegans and Mammalian Cells Using Organelle-Specific Dyes

This protocol outlines two methods for the quantitative analysis of mitophagy in pancreatic β-cells: first, a combination of cell-permeable mitochondria-specific dyes, and second, a genetically encoded mitophagy reporter. These two techniques are complementary and can be deployed based on specific needs, allowing for flexibility and precision in quantitatively addressing mitochondrial quality control.

Complementary Approaches to Interrogate Mitophagy Flux in Pancreatic &#946;-Cells

This protocol provides a standardized approach for imaging mitochondrial morphology in multiple tissues of C. elegans during aging.

Imaging and Quantifying Mitochondrial Morphology in C. elegans During Aging

A method is described to propagate human retinoblastoma tumors in mice. Tumor cells are directly injected into the eyes of immune deficient mice. Secondary tumors have been successfully established using both cells directly harvested from human tumors and cultured tumorspheres.

Establishment and Propagation of Human Retinoblastoma Tumors in Immune Deficient Mice

We here describe how to perform multi-electrode array recordings of human epileptic cortical tissue. Epileptic tissue resection, slice preparation and multi-electrode array recordings of interictal and ictal events are demonstrated in detail.

Multi-electrode Array Recordings of Human Epileptic Postoperative Cortical Tissue

Learn robust methods to detect mitophagy in human cells, C. elegans, and mice using in vitro and in vivo techniques. These approaches advance research on neurodegenerative diseases and mitochondrial dysfunction.

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

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