Name: development of a mobile mitochondrial physiology laboratory for measuring mitochondrial energetics in the field
Uploaded: 2021-08-27T15:00:00
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The authors acknowledge Mark Nelms and John Tennant from the Electrical and Computer Engineering department of the Samuel Ginn College of Engineering at Auburn University for helping with the structural and electrical outfitting of the AU MitoMobile. Additionally, the authors acknowledge the funding to outfit the AU MitoMobile and research from an Auburn University Presidential Awards for Interdisciplinary Research (PAIR) grant.

The following sections describe the mitochondrial laboratory methods. All animal handling and tissue collection procedures were approved by the Auburn University Institutional Animal Care and Use Committee (#2019-3582).
1. Description of buffers used for data collection
NOTE: These buffers can be prepared in a stationary laboratory and moved to the mobile laboratory prior to the field trip (unless otherwise noted below).
<ol>
	<li>Prepare the skeletal muscle mitoc...

<ol>
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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Bioenergetics 3. Third edition. , (2002).</li><li>Brand, M. D., Nicholls, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+mitochondrial+dysfunction+in+cells.">Assessing mitochondrial dysfunction in cells.</a> Biochemical Journal. 435 (2), 297-312 (2011).</li><li>Mowry, A. V., Donoviel, Z. S., Kavazis, A. N., Hood, W. 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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mitohormetic+response+to+pro-oxidant+exposure+in+the+house+mouse.">A mitohormetic response to pro-oxidant exposure in the house mouse.</a> American Journal of Physiology-Regulatory, Integrative and Comparative Physiology. 314 (1), 122-134 (2018).</li><li>Boutagy, N. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+mitochondria+from+minimal+quantities+of+mouse+skeletal+muscle+for+high+throughput+microplate+respiratory+measurements.">Isolation of mitochondria from minimal quantities of mouse skeletal muscle for high throughput microplate respiratory measurements.</a> Journal of Visualized Experiments: JoVE. (105), e53217 (2015).</li><li>Djafarzadeh, S., Jakob, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+intact+mitochondria+from+skeletal+muscle+by+differential+centrifugation+for+high-resolution+respirometry+measurements.">Isolation of intact mitochondria from skeletal muscle by differential centrifugation for high-resolution respirometry measurements.</a> Journal of Visualized Experiments: JoVE. (121), e55251 (2017).</li><li>Garcia-Cazarin, M. L., Snider, N. N., Andrade, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+isolation+from+skeletal+muscle.">Mitochondrial isolation from skeletal muscle.</a> Journal of Visualized Experiments: JoVE. (49), e2452 (2011).</li><li>Pravdic, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complex+I+and+ATP+synthase+mediate+membrane+depolarization+and+matrix+acidification+by+isoflurane+in+mitochondria.">Complex I and ATP synthase mediate membrane depolarization and matrix acidification by isoflurane in mitochondria.</a> European Journal of Pharmacology. 690 (1-3), 149-157 (2012).</li><li>Brooks, S. P., Lampi, B. J., Bihun, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+euthanasia+methods+on+rat+liver+metabolism.">The influence of euthanasia methods on rat liver metabolism.</a> Journal of the American Association for Laboratory Animal Science. 38 (6), 19-24 (1999).</li><li>Overmyer, K. A., Thonusin, C., Qi, N. R., Burant, C. F., Evans, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+anesthesia+and+euthanasia+on+metabolomics+of+mammalian+tissues:+studies+in+a+C57BL/6J+mouse+model.">Impact of anesthesia and euthanasia on metabolomics of mammalian tissues: studies in a C57BL/6J mouse model.</a> PLoS One. 10 (2), 0117232 (2015).</li><li>Kuzmiak, S., Glancy, B., Sweazea, K. L., Willis, W. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+function+in+sparrow+pectoralis+muscle.">Mitochondrial function in sparrow pectoralis muscle.</a> Journal of Experimental Biology. 215 (12), 2039-2050 (2012).</li><li>Bradford, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+and+sensitive+method+for+the+quantitation+of+microgram+quantities+of+protein+utilizing+the+principle+of+protein-dye+binding.">A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.</a> Analytical Biochemistry. 72 (1-2), 248-254 (1976).</li><li>Figueiredo, P. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+lifelong+sedentary+behavior+on+mitochondrial+function+of+mice+skeletal+muscle.">Impact of lifelong sedentary behavior on mitochondrial function of mice skeletal muscle.</a> J Gerontol A Biol Sci Med Sci. 64 (9), 927-939 (2009).</li><li>Scheibye-Knudsen, M., Quistorff, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+mitochondrial+respiration+by+inorganic+phosphate;+comparing+permeabilized+muscle+fibers+and+isolated+mitochondria+prepared+from+type-1+and+type-2+rat+skeletal+muscle.">Regulation of mitochondrial respiration by inorganic phosphate; comparing permeabilized muscle fibers and isolated mitochondria prepared from type-1 and type-2 rat skeletal muscle.</a> European Journal of Applied Physiology. 105 (2), 279-287 (2009).</li><li>Kuznetsov, A. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+mitochondrial+function+in+situ+in+permeabilized+muscle+fibers,+tissues+and+cells.">Analysis of mitochondrial function in situ in permeabilized muscle fibers, tissues and cells.</a> Nature Protocols. 3 (6), 965-976 (2008).</li><li>Hughey, C. C., Hittel, D. S., Johnsen, V. L., Shearer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Respirometric+oxidative+phosphorylation+assessment+in+saponin-permeabilized+cardiac+fibers.">Respirometric oxidative phosphorylation assessment in saponin-permeabilized cardiac fibers.</a> Journal of Visualized Experiments: JoVE. (48), e2431 (2011).</li><li>Gaviraghi, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+permeabilization+as+a+new+method+for+assessment+of+mitochondrial+function+in+insect+tissues.">Mechanical permeabilization as a new method for assessment of mitochondrial function in insect tissues.</a> Mitochondrial Medicine. Vol. 2: Assessing Mitochonndria. , 67-85 (2021).</li><li>Hedges, C. P., Wilkinson, R. T., Devaux, J. B. L., Hickey, A. J. 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The mobile mitochondrial physiology laboratory enables researchers to isolate mitochondria and measure mitochondrial respiration rates within 2 h of tissue collection at remote field sites. The results presented herein suggest that measurements of mitochondrial respiration made in the AU MitoMobile are comparable to measurements made in a university research laboratory. Specifically, the values for state 3, state 4, and RCR for wild-derived Mus musculus presented here are comparable with previously published res...

To date, studies designed to measure mitochondrial energetics have been limited to laboratory animals or animals captured near established physiology laboratories, which precluded scientists from performing mitochondrial bioenergetic studies in tissues collected from animals during such activities as migration, diving, and hibernation1,2,3,4,5,6. While many investigators have successfully measured the basal and peak metabolic rates and daily energy expenditures of wild animals7,8, the capacity of researchers to measure the performance of mitochondria has remained limited (but see1,4,9). This is partly due to the need for fresh tissue for isolating mitochondria and a laboratory facility to perform the isolations within about 2 h of obtaining the fresh tissue. Once the mitochondria have been isolated, the mitochondrial respiration measurements should also be completed within ~1 h.
Isolated mitochondrial respiration rates are usually performed by measuring oxygen concentration in a sealed container connected to a Clark electrode. The theory behind this method is founded on the basic observation that oxygen is the last electron acceptor of mitochondrial respiration during oxidative phosphorylation. Therefore, as oxygen concentration falls during an experiment, it is assumed that adenosine triphosphate (ATP) production occurs10. Consumed oxygen is a proxy for produced ATP. Researchers can create specific experimental conditions using different substrates and initiate adenosine diphosphate (ADP)-stimulated respiration (state 3) by adding predetermined amounts of ADP to the chamber. Following the phosphorylation of the exogenous ADP to ATP, the oxygen consumption rate decreases, and state 4 is reached and can be measured. Furthermore, the addition of specific inhibitors allows information regarding leak respiration and uncoupled respiration to be obtained10. The ratio of state 3 to state 4 determines the respiratory control ratio (RCR), which is the indicator of overall mitochondrial coupling10,11. Lower values of RCR indicate overall mitochondrial dysfunction, whereas higher RCR values suggest a greater extent of mitochondrial coupling10.
As previously stated, the collection of biological material, mitochondrial isolation, and measurement of respiration rates must be completed within 2 h of obtaining tissue. To accomplish this task without transporting animals over large distances to established laboratories, a mobile mitochondrial physiology laboratory was constructed to be taken to field locations where these data can be collected. A 2018 Jayco Redhawk recreational vehicle was converted into a mobile molecular physiology laboratory and named the Auburn University (AU) MitoMobile (Figure 1A). A recreational vehicle was selected because of the built-in refrigerator, freezer, water storage tank and plumbing, electricity powered by 12-volt batteries, gas generator, propane tank, and self-leveling system. Further, the recreational vehicle provides the capability of staying at remote sites overnight for data collection. The front of the vehicle was not altered and provides the driving and sleeping quarters (Figure 1B). Previously installed bedroom amenities (bed, TV, and cabinet) in the rear of the vehicle and the stovetop were removed.
Custom-made stainless-steel shelving and a custom quartz countertop supported by 80/20 aluminum framing were installed in place of the bedroom amenities and stovetop (Figure 1C). The laboratory benches provide adequate space for data collection (Figure 1D). Power consumption of each piece of equipment (i.e., refrigerated centrifuge, mitochondrial respiration chambers, plate readers, computers, homogenizers, scales, portable ultra-freezer, and other general laboratory supplies) was taken into consideration. To support the large voltage and current demands of the centrifuge, the electrical system was upgraded to that of aircraft-grade equipment. An external compartment in the rear of the vehicle was converted into a liquid nitrogen storage bay, which meets the United States Department of Transportation&#39;s guidelines for liquid nitrogen storage and transport. This storage unit was constructed with stainless steel and has proper venting to keep any expanding nitrogen gas from leaking into the passenger compartment of the vehicle.
To confirm that the mobile laboratory can be used in mitochondrial bioenergetic studies, mitochondria were isolated, and mitochondrial respiration rates from wild-derived house mice (Mus musculus) hindlimb skeletal muscle were measured. Because Mus musculus is a model organism, the mitochondrial respiration rates of this species are well-established12,13,14. Although previous studies have documented mitochondrial isolation via differential centrifugation15,16,17, a brief overview of the methods used in the mobile mitochondrial physiology laboratory methods is described below.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.7 mL centrifuge tubes</td>
			<td>VWR</td>
			<td>87003-294</td>
			<td></td>
		</tr>
		<tr>
			<td>2.0 mL centrifuge tubes</td>
			<td>VWR</td>
			<td>87003-298</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL centrifuge tubes</td>
			<td>VWR</td>
			<td>21009-681</td>
			<td>Nalgene Oak Ridge Centrifuge Tube</td>
		</tr>
		<tr>
			<td>ADP</td>
			<td>VWR</td>
			<td>97061-104</td>
			<td></td>
		</tr>
		<tr>
			<td>ATP</td>
			<td>VWR</td>
			<td>700009-070</td>
			<td></td>
		</tr>
		<tr>
			<td>Bradford</td>
			<td>VWR</td>
			<td>7065-020</td>
			<td></td>
		</tr>
		<tr>
			<td>Clear 96 well plate</td>
			<td>VWR</td>
			<td>82050-760</td>
			<td>Greiner Bio-One</td>
		</tr>
		<tr>
			<td>Dounce homogenizer</td>
			<td>VWR</td>
			<td>22877-284</td>
			<td>Corning</td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>VWR</td>
			<td>EM-4100</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter paper</td>
			<td></td>
			<td></td>
			<td>Included with Hansatech OxyGraph</td>
		</tr>
		<tr>
			<td>Free-fatty acid BSA</td>
			<td>VWR</td>
			<td>89423-672</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>VWR</td>
			<td>BDH8005-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamate</td>
			<td>VWR</td>
			<td>A12919</td>
			<td></td>
		</tr>
		<tr>
			<td>Hamilton Syringes</td>
			<td>VWR</td>
			<td>60373-985</td>
			<td>Gaslight 1700 Series Syringes</td>
		</tr>
		<tr>
			<td>Hansatech OxyGraph</td>
			<td>Hansatech Instruments Ltd</td>
			<td></td>
			<td>No Catalog Number, but can be found under Products --&#62; Electrode Control Units</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>VWR</td>
			<td>97062-350</td>
			<td></td>
		</tr>
		<tr>
			<td>Malate</td>
			<td>VWR</td>
			<td>97062-140</td>
			<td></td>
		</tr>
		<tr>
			<td>Mannitol</td>
			<td>VWR</td>
			<td>97061-052</td>
			<td></td>
		</tr>
		<tr>
			<td>Membrane</td>
			<td></td>
			<td></td>
			<td>Included with Hansatech OxyGraph</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>VWR</td>
			<td>97063-152</td>
			<td></td>
		</tr>
		<tr>
			<td>MOPS</td>
			<td>VWR</td>
			<td>80503-004</td>
			<td></td>
		</tr>
		<tr>
			<td>Policeman</td>
			<td>VWR</td>
			<td>470104-462</td>
			<td></td>
		</tr>
		<tr>
			<td>Polytron</td>
			<td>Thomas Scientific</td>
			<td>11090044</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride (KCl)</td>
			<td>VWR</td>
			<td>97061-566</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease</td>
			<td>VWR</td>
			<td>97062-366</td>
			<td>Trypsin is commonly used; however, other proteases can be used.</td>
		</tr>
		<tr>
			<td>Pyruvic acid</td>
			<td>VWR</td>
			<td>97061-448</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Dithionite</td>
			<td>VWR</td>
			<td>AA33381-22</td>
			<td></td>
		</tr>
		<tr>
			<td>Succinate</td>
			<td>VWR</td>
			<td>89230-086</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>VWR</td>
			<td>BDH0308-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-Base</td>
			<td>VWR</td>
			<td>97061-794</td>
			<td></td>
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		<tr>
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development of a mobile mitochondrial physiology laboratory for measuring mitochondrial energetics in the field

Mitochondrial energetics is a central theme in animal biochemistry and physiology, with researchers using mitochondrial respiration as a metric to investigate metabolic capability. To obtain the measures of mitochondrial respiration, fresh biological samples must be used, and the entire laboratory procedure must be completed within approximately 2 h. Furthermore, multiple pieces of specialized equipment are required to perform these laboratory assays. This creates a challenge for measuring mitochondrial respiration in the tissues of wild animals living far from physiology laboratories as live tissue cannot be preserved for very long after collection in the field. Moreover, transporting live animals over long distances induces stress, which can alter mitochondrial energetics.
This manuscript introduces the Auburn University (AU) MitoMobile, a mobile mitochondrial physiology laboratory that can be taken into the field and used on-site to measure mitochondrial metabolism in tissues collected from wild animals. The basic features of the mobile laboratory and the step-by-step methods for measuring isolated mitochondrial respiration rates are presented. Additionally, the data presented validate the success of outfitting the mobile mitochondrial physiology laboratory and making mitochondrial respiration measurements. The novelty of the mobile laboratory lies in the ability to drive to the field and perform mitochondrial measurements on the tissues of animals captured on site.

The current manuscript investigated the mitochondrial respiration of wild-derived Mus musculus (n = 7, male = 5, female = 2; age = 1.30 &#177; 0.2 years) in a mobile mitochondrial physiology laboratory (Figure 1). To measure skeletal muscle mitochondrial respiration, the entire hindlimb, thus aerobic and anaerobic muscle, was used for mitochondrial isolation (Figure 2). Examples of raw mitochondrial respiration data are shown in Fig...

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Development of a Mobile Mitochondrial Physiology Laboratory for Measuring Mitochondrial Energetics in the Field

We designed and constructed a mobile laboratory to measure respiration rates in isolated mitochondria of wild animals captured at field locations. Here, we describe the design and outfitting of a mobile mitochondrial laboratory and the associated laboratory protocols.

Mitochondrial energetics is a central theme in animal biochemistry and physiology, with researchers using mitochondrial respiration as a metric to ...

The development of a mobile mitochondrial laboratory allows physiologists to take their work into the field, and as a result, they can evaluate a fascinating diversity of energetic challenges that animals display. Captivity is stressful for many animals, so with a mobile lab, we can now take the lab to the animals rather than bring the animals back to the laboratory. With the ability to isolate and measure mitochondrial respiration in the field, techniques that were primarily used by biomedicine can now be used in ecological and evolutionary context.Begin by parking the mobile laboratory on flat ground, then turn on the generator, and level the vehicle. Extend the slide before setting up the equipment. Proceed to set up and calibrate the mitochondrial respiration chambers to the desired temperature of experiments and the current barometric pressure as per the manufacturer's instructions.Use a cut five milliliter pipette tip to transfer the minced tissue to a 50 milliliter centrifuge tube, and homogenize the tissue with a blade at 50%power for five seconds. To digest the tissue, add five milligrams of protease per gram of the wet muscle for seven minutes, mixing the solution every 30 seconds. Terminate the reaction by adding an equal volume of the isolation buffer containing BSA.Next, centrifuge the homogenate at 500 G for 10 minutes, then transfer the supernatant using a cut five milliliter pipette tip through the double layered cheesecloth into a clean 50 milliliter centrifuge tube, then centrifuge the filtered supernatant at 3, 500 G for 10 minutes to precipitate a brown mitochondrial pellet. After discarding the supernatant, add the same volume of the isolation buffer containing BSA to the centrifuge tube, and resuspend the mitochondrial pellet with a flexible scraper by gently working the mitochondrial pellet off the walls of the centrifuge tube, then centrifuge the tube at 3, 500 G for 10 minutes. Resuspend the pellet in the isolation buffer without BSA to repeat the centrifugation step as described earlier.After the second centrifugation, resuspend the mitochondrial pellet in the resuspension buffer with a clean, flexible scraper, and transfer the resuspended mitochondria to a Dounce homogenizer with a cut one milliliter pipette tip. With the homogenizer, carefully homogenize the mitochondrial suspension with four to five passes. Use a fresh cut one milliliter pipette tip to transfer the homogenized mitochondrial suspension in a labeled two milliliter microcentrifuge tube.For the mitochondrial respiration measurements of the complex one substrates, add 945 microliters of the respiration buffer to the mitochondrial respiration chamber. Ensure that the stirrer is spinning when the buffer temperature is maintained at 37 degrees Celsius. Seal the chamber with the top, and then start recording the data collection.After the oxygen concentration has stabilized, add 20 microliters of the mitochondrial suspension to the chamber, and place the lid. In the software, denote that the mitochondria were added to the chamber. Add 10 microliters each of one molar glutamate, 200 millimolar malate, and 200 millimolar pyruvate to the chamber with the individual syringes, and wait until the signal stabilizes before indicating the added substrates in the software.Use a separate syringe to add five microliters of adenosine diphosphate, or ADP, to the chamber. Denote the added ADP in the software, and observe the rapid oxygen consumption of state three. After state three respiration and mitochondrial oxygen consumption has slowed for four minutes to indicate state four respiration, terminate the recording and save the data file.For the complex two substrates, repeat the procedure by adding 963 microliters of the respiration buffer to the chamber. After adding 20 microliters of the mitochondrial suspension, sequentially add two microliters of four micrograms per microliter rotenone and 10 microliters of 500 millimolar succinate to the chamber using the separate syringes. When the signal stabilizes, denote the substrate addition in the software.With the addition of five microliters of ADP, observe the rapid oxygen consumption of state three and indicate the addition of ADP in the software. After state three respiration and mitochondrial oxygen consumption has slowed for four minutes to indicate state four respiration, terminate the recording and save the data file. The representative results indicate the raw mitochondrial respiration data.The steep slope of step three in the complex one substrates represents the high maximal respiration rate. The successful isolation of the mitochondria from the hind limb skeletal muscle was observed by the sharp turn and the stabilization of a new slope of state four. A similar pattern could be observed for complex two driven mitochondrial respiration.The poor functioning of the mitochondria resulted in the coupling of the mitochondria for the complex one driven mitochondrial respiration. However, another typical example of poor mitochondria functioning showed the uncoupled complex two mitochondrial respiration with a flat line after the addition of ADP, and no turn to produce the state four data. The numerical values of state three, state four, and the respiratory control ratio, or RCR, for both complex one and complex two were determined from the respiration measurement data During collection, additional tissues can be flash frozen in order to use them in the future for measuring mitochondrial density or oxidative damage.In addition, after the procedure, isolated mitochondria can also be frozen to measure the activities of electron transport and chain complexes. The capacity to isolate and measure mitochondrial respiration in the field will pave the way to improve our understanding of the role of mitochondria in the evolution of complex life.

Research

JoVE Journal

Biology

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