M.E.H. acknowledges the Deutsche Forschungsgemeinschaft (DFG), project number 413985647, for financial support. The authors thank Dr. Michael Kiebler, Ludwig-Maximilians University, Munich, for his&#160;generous and extensive support. We are grateful to Walter Neupert for his scientific input, helpful discussions, and ongoing inspiration.&#160;J.F. thanks the Graduate School Life Science Munich (LSM) for support.

1. Buffers and stock solutions
<ol>
	<li>Make a 1 M 3-morpholinopropane-1-sulfonic acid (MOPS) solution in deionized water, pH 7.4. Store at 4 °C.</li>
	<li>Prepare 500 mM ethylenediaminetetraacetic acid (EDTA) in deionized water, pH 8.0. Store at room temperature.</li>
	<li>Prepare 2.4 M sorbitol in deionized water. Store at room temperature after autoclaving.</li>
	<li>Prepare 2.5 M sucrose in deionized water. Store at room temperature after autoclaving.</li>
	<li>Prepare 200 mM phenylmethylsulfonyl fluoride (PMSF) in isopropanol. Store at −20 °C. 
	CAUTION: PMSF is toxic if swallowed and causes severe skin burns and eye damage. Do not breathe in the dust, and wear protective gloves and goggles.</li>
	<li>Prepare 72% trichloroacetic acid (TCA) in deionized water. Store at 4 °C. 
	CAUTION: TCA may cause respiratory irritation, severe skin burns, and eye damage. Do not breathe in the dust, and wear protective gloves and goggles.</li>
	<li>Prepare the SM buffer: 20 mM MOPS and 0.6 M sorbitol, pH 7.4.</li>
	<li>Prepare the swelling buffer: 20 mM MOPS, 0.5 mM EDTA, 1 mM PMSF, and protease inhibitor cocktail, pH 7.4.</li>
	<li>Prepare the sucrose gradient buffers: 0.8 M, 0.96 M, 1.02 M, 1.13 M, and 1.3 M sucrose in 20 mM MOPS, and 0.5 mM EDTA, pH 7.4. 
	NOTE: All the buffers can be stored at 4 °C; however, it is recommended to store the sucrose-containing buffers at −20 °C to avoid fungal growth. The protease inhibitors have to be added freshly prior to use.</li>
</ol>
2. Generation of submitochondrial vesicles
<ol>
	<li>Isolate crude yeast mitochondria freshly according to established protocols34,35. 
	NOTE: For this experiment, mitochondria were isolated from Saccharomyces cerevisiae. The generation of gradient pure mitochondria devoid of other organelles is not necessary.</li>
	<li>Resuspend 10 mg of freshly isolated mitochondria in 1.6 mL SM buffer (4 °C) by pipetting (Figure 2, step 1).</li>
	<li>Transfer the mitochondrial suspension to a pre-cooled 100 mL Erlenmeyer flask.</li>
	<li>Slowly add 16 mL of swelling buffer using a 20 mL glass pipette. Apply constant mild stirring on ice during the addition. This osmotic treatment results in water uptake into the matrix space. The swelling of the mitochondrial inner membrane will disrupt the outer membrane. However, both membranes will stay in contact at the contact sites9(Figure 2, step 2).</li>
	<li>Incubate the samples under constant mild stirring for 30 min on ice.</li>
	<li>Slowly add 5 mL of 2.5 M sucrose solution using a 5 mL glass pipette to increase the sucrose concentration in the samples to approximately 0.55 M. This osmotic treatment results in a water efflux from the matrix36. The shrinkage of the inner membrane is intended to maximize its distance to the residual fragments of the outer membrane. This decreases the probability of generating artificial hybrid vesicles of inner and outer membranes through sonication (Figure 2, step 3).</li>
	<li>Incubate the samples under mild stirring for 15 min on ice.</li>
	<li>Subject the mitochondria to sonication to generate submitochondrial membrane vesicles (Figure 2, step 4).
	<ol>
		<li>Transfer the mitochondrial suspension to a pre-cooled rosette cell.</li>
		<li>Sonicate the mitochondrial suspension at a 10% amplitude for 30 s while cooling the rosette cell in an ice bath.</li>
		<li>Rest the suspension for 30 s in an ice bath.</li>
		<li>Repeat step 2.8.2 and step 2.8.3 three more times. 
		NOTE: The sonication conditions will vary according to the sonicator used. Optimization will be necessary for individual machines. Sonication that is too harsh will lead to the artificial formation of vesicles consisting of mitochondrial inner and outer membranes.</li>
	</ol>
	</li>
</ol>
3. Separation of submitochondrial vesicles
<ol>
	<li>Separate the generated vesicles from the remaining intact mitochondria by centrifugation at 20,000 x g for 20 min at 4 °C. The intact mitochondria will pellet while the vesicles will stay in the supernatant.</li>
	<li>Concentrate the vesicle mixture.
	<ol>
		<li>Transfer the supernatant to fresh ultracentrifugation tubes.</li>
		<li>Load a cushion of 0.3 mL of 2.5 M sucrose solution at the bottom of the tube using a 1 mL syringe equipped with a 0.8 mm x 120 mm cannula.</li>
		<li>Centrifuge at 118,000 x g for 100 min at 4 °C. 
		NOTE: Here, a swinging bucket rotor with a maximum radius of 153.1 mm was used. The centrifugation conditions strongly depend on the rotor and will have to be adapted when another rotor is used.</li>
	</ol>
	</li>
	<li>The vesicles will appear as a disc on the top of the sucrose cushion after the centrifugation. Discard approximately 90% of the supernatant. Now, harvest the concentrated vesicles by resuspending them in the remaining buffer including the 2.5 M sucrose by pipetting up and down. Transfer the suspension to an ice-cold Dounce homogenizer.</li>
	<li>Homogenize the suspension with at least 10 strokes using a polytetrafluoroethylene potter.</li>
	<li>Prepare the sucrose gradient.
	<ol>
		<li>For an 11 mL step gradient with five steps (1.3 M, 1.13 M, 1.02 M, 0.96 M, and 0.8 M sucrose in 20 mM MOPS and 0.5 mM EDTA, pH 7.4), each layer has 2.2 mL of sucrose solution. 
		NOTE: A refractometer is recommended to measure and adjust the sucrose concentrations; however, it is not essential. Apply 10 µL of the buffer to the refractometer, and detect the respective refractive indices. The calculation of sucrose concentration is as follows: 
		<img alt="Refractometry equation for sucrose concentration in diagram; evaluates refractive index change." src="/files/ftp_upload/65444/65444eq01v2.jpg" style="margin: auto;" data-altupdatedat="1747911167000" data-alt="Equation 1"> 
		1.3333 represents the refractive index of water. 0.048403 is a sucrose specific conversion index obtained from the linear scaling of molar refractivity to sucrose concentration in aqueous solutions 37.</li>
		<li>Add the highest sucrose concentration to the centrifugation tube, and put the tube at −20 °C. Wait until the layer is completely frozen before applying the next one. Proceed accordingly until the last layer is added, and store the gradients at −20 °C. 
		NOTE: Remember to transfer the frozen gradients to 4 °C when starting the experiment to allow the gradients to thaw. This will take approximately 3 h. For the centrifugation here, a swinging bucket rotor with a capacity of 13.2 mL was used. When a different rotor is used, the gradient step volumes must be adapted accordingly.</li>
	</ol>
	</li>
	<li>Measure the sucrose concentration of the samples using a refractometer. If there is not access to a refractometer, one could alternatively assume that the sucrose concentration is 2 M. This supposed sucrose concentration will then be the basis for the next step.</li>
	<li>Adjust the sucrose concentration to 0.6 M by the addition of an appropriate amount of 20 mM MOPS, 0.5 mM EDTA, 1 mM PMSF, and protease inhibitor cocktail, pH 7.4. If the sucrose concentration is estimated as 2 M instead of being measured, it is recommended to test whether the concentration is low enough after adjustment. Apply a small aliquot of the sample (ca. 50 µL) on top of 200 µL of 0.8 M sucrose in a test tube. The sample must stay on top of the 0.8 M sucrose.</li>
	<li>Load the samples (approximately 1 mL) on top of the sucrose gradient (keep 10% for later reference). Careful pipetting is important to avoid the disturbance of the gradient (Figure 2, step 5).</li>
	<li>Separate the vesicles by centrifugation at 200,000 x g and 4 °C for 12 h. If possible, set the centrifuge to slow acceleration and deceleration to avoid the disturbance of the gradient (Figure 2, step 6). 
	NOTE: Here, a swinging bucket rotor with a maximum radius of 153.1 mm was used. The centrifugation conditions strongly depend on the rotor and will have to be adapted when another rotor is used.</li>
	<li>Harvest the gradient from top to bottom in 700 µL fractions using a 1 mL pipette. This will result in 17 fractions, which allows for a sufficient resolution.</li>
</ol>
4. Analysis of submitochondrial vesicles
<ol>
	<li>Concentrate the proteins by subjecting each fraction to two sequentially performed TCA precipitations38 to prepare the SDS-PAGE samples.
	<ol>
		<li>Add 200 µL of 72% TCA to the individual fractions, and mix until the solution is homogeneous. Incubate the fractions for 30 min on ice, and pellet the precipitated proteins by centrifugation at 20,000 x g and 4 °C for 20 min. Discard the supernatant, add 500 µL of 28 % TCA solution, mix well, and repeat the centrifugation step.</li>
		<li>Wash the pellets with 1 mL of acetone (−20°C), and centrifuge for 10 min at 20,000 x g and 4 °C. Discard the supernatant, and let the pellets air dry. Resuspend the pellets in 60 µL of SDS sample buffer, and incubate the samples for 5 min at 95 °C. 
		NOTE: A large amount of sucrose will remain, particularly in the high-density fractions, after the first TCA precipitation. This will be removed through the additional TCA precipitation.</li>
	</ol>
	</li>
	<li>Analyze 20 µL of each fraction by SDS-PAGE39 and immunoblotting40,41,42,43.</li>
</ol>

Isolation of Mitochondrial Contact Sites

The authors declare that there are no conflicts of interest.

Mitochondrial subfractionation is a complicated experiment with several highly complex steps. Therefore, we aimed to further improve and, to a certain degree, simplify our established method32. Here, the challenges were the requirement for complicated and highly specialized equipment, which are often individual constructions, and the enormous time and energy consumption. To this end, we tried to remove the pumps and individual constructions used for casting and harvesting the linear gradient and c...

Mitochondria perform a variety of different functions in eukaryotes, with the most well-known being the production of ATP through oxidative phosphorylation. Other functions include the production of iron-sulfur clusters, lipid synthesis, and in higher eukaryotes, Ca2+ signaling, and the induction of apoptosis1,2,3,4. These functions are inseparably linked to their complex ultrastructure.
The mitochondrial ultrastructure was first described by electron microscopy5. It was shown that mitochondria are rather complex organelles consisting of two membranes: the mitochondrial outer membrane and the mitochondrial inner membrane. Thus, two aqueous compartments are formed by these membranes: the intermembrane space and the matrix. The mitochondrial inner membrane can be even further divided into different sections. The inner boundary membrane stays in close proximity to the outer membrane, and the cristae form invaginations. So-called crista junctions connect the inner boundary membrane and the cristae (Figure 1). Furthermore, electron micrographs of osmotically shrunken mitochondria reveal that sites exist at which the mitochondrial membranes are tightly connected6,7. These so-called contact sites are formed by protein complexes spanning the two membranes (Figure 1). It is thought that these interaction sites are essential for cell viability due to their importance for the regulation of mitochondrial dynamics and inheritance, as well as the transfer of metabolites and signals between the cytosol and the matrix8.
The MICOS complex in the mitochondrial inner membrane is probably the best characterized and the most versatile contact site-forming complex. MICOS was described in yeast in 2011, and it consists of six subunits9,10,11: Mic60, Mic27, Mic26, Mic19, Mic12, and Mic10. These form a complex of approximately 1.5 MDa that localizes to the crista junctions9,10,11. The deletion of either core subunit, Mic10 or Mic60, leads to the absence of this complex9,11, meaning these two subunits are essential for the stability of MICOS. Interestingly, MICOS forms not only one but multiple contact sites with various mitochondrial outer membrane proteins and complexes: the TOM complex11,12, the TOB/SAM complex9,12,13,14,15,16, the Fzo1-Ugo1 complex9, Por110, OM4510, and Miro17. This strongly indicates that the MICOS complex is involved in various mitochondrial processes, such as protein import, phospholipid metabolism, and the generation of the mitochondrial ultrastructure18. The latter function is probably the major function of&#160;MICOS, as the absence of the MICOS complex induced through the deletion of MIC10&#160;or MIC60&#160;leads to an abnormal mitochondrial ultrastructure that virtually completely lacks regular cristae. Instead, internal membrane vesicles without connection to the inner boundary membrane accumulate19, 20. Importantly, MICOS is conserved in form and function from yeast to&#160;human21. The association of mutations in MICOS subunits with severe human diseases also emphasizes its importance for higher eukaryotes22,23. Although MICOS is highly versatile, additional contact sites must exist (based on our unpublished observations). Indeed, several other contact sites have been identified, for instance, the mitochondrial fusion machineries Mgm1-Ugo1/Fzo124,25,26&#160;or Mdm31-Por1, which is involved in the biosynthesis of the mitochondrial-specific phospholipid cardiolipin27. Recently, we improved the method that led us to the identification of MICOS to identify Cqd1 as part of a novel contact site formed with the outer membrane complex Por1-Om1428. Interestingly, this contact site also seems to be involved in multiple processes such as mitochondrial membrane homeostasis, phospholipid metabolism, and the distribution of coenzyme Q28,29.
Here, we used a variation of the previously described fractionation of mitochondria9,30,31,32,33. Osmotic treatment of mitochondria leads to the disruption of the mitochondrial outer membrane and to a shrinkage of the matrix space, leaving the two membranes only in close proximity at contact sites. This allows for the generation of vesicles that consist exclusively of mitochondrial outer membrane or mitochondrial inner membrane or at contain contact sites of both membranes through mild sonication. Due to the mitochondrial inner membrane possessing a much higher protein-to-lipid ratio, mitochondrial inner membrane vesicles exhibit a higher density compared to mitochondrial outer membrane vesicles. The difference in density can be used to separate the membrane vesicles through sucrose buoyant density gradient centrifugation. Thus, the mitochondrial outer membrane vesicles accumulate at low sucrose concentrations, while the mitochondrial inner membrane vesicles are enriched at high sucrose concentrations. The vesicles containing contact sites concentrate at intermediate sucrose concentrations (Figure 2). The following protocol describes this improved method, which requires less specialized equipment, time, and energy compared to our previously established one32, in detail and provides a useful tool for the identification of possible contact site proteins.

<table><tbody><tr><td>13.2 mL, Open-Top Thinwall Ultra-Clear Tube, 14 x 89mm</td><td>Beckman Instruments, Germany</td><td>344059</td><td></td></tr><tr><td>50 mL, Open-Top Thickwall Polycarbonate Open-Top Tube, 29 x 104mm</td><td>Beckman Instruments, Germany</td><td>363647</td><td></td></tr><tr><td>A-25.50 Fixed-Angle Rotor- Aluminum, 8 x 50 mL, 25,000 rpm, 75,600 x g</td><td>Beckman Instruments, Germany</td><td>363055</td><td></td></tr><tr><td>Abbe refractometer</td><td>Zeiss, Germany</td><td></td><td>discontinued,&lt;br/&gt; any Abbe refractometer can be used</td></tr><tr><td>accu-jet pro Pipet Controller</td><td>Brandtech, USA</td><td>BR26320</td><td>discontinued,&lt;br/&gt; any pipet controller will suffice</td></tr><tr><td>Beaker 1000 mL</td><td>DWK Life Science, Germany</td><td>C118.1</td><td></td></tr><tr><td>Branson&amp;nbsp; Digital Sonifier W-250 D</td><td>Branson Ultrasonics, USA</td><td>FIS15-338-125</td><td></td></tr><tr><td>Branson Ultrasonic 3mm TAPERED MICROTIP</td><td>Branson Ultrasonics, USA</td><td>101-148-062</td><td></td></tr><tr><td>Branson Ultrasonics 200- and 400-Watt Sonifiers: Rosette Cooling Cell</td><td>Branson Ultrasonics, USA</td><td>15-338-70</td><td></td></tr><tr><td>Centrifuge Avanti JXN-26</td><td>Beckman Instruments, Germany</td><td>B37912</td><td></td></tr><tr><td>Centrifuge Optima XPN-100 ultra</td><td>Beckman Instruments, Germany</td><td>8043-30-0031</td><td></td></tr><tr><td>cOmplete Proteaseinhibtor-Cocktail</td><td>Roche, Switzerland</td><td>11697498001</td><td></td></tr><tr><td>D-Sorbit</td><td>Roth, Germany</td><td>6213</td><td></td></tr><tr><td>EDTA (Ethylendiamin-tetraacetic acid disodium salt dihydrate)</td><td>Roth, Germany</td><td>8043</td><td></td></tr><tr><td>Erlenmeyer flask, 100 mL</td><td>Roth, Germany</td><td>X747.1</td><td></td></tr><tr><td>graduated pipette, Kl. B, 25:0, 0.1</td><td>Hirschmann, Germany</td><td>1180170</td><td></td></tr><tr><td>graduated pipette, Kl. B, 5:0, 0.05</td><td>Hirschmann, Germany</td><td>1180153</td><td></td></tr><tr><td>ice bath</td><td>neoLab, Germany</td><td>&amp;nbsp;S12651</td><td></td></tr><tr><td>Magnetic stirrer RCT basic</td><td>IKA-Werke GmbH, Germany</td><td>Z645060GB-1EA</td><td></td></tr><tr><td>MOPS (3-(N-Morpholino)propanesulphonic acid)</td><td>Gerbu, Germany</td><td>1081</td><td></td></tr><tr><td>MyPipetman Select P1000</td><td>Gilson, USA</td><td>FP10006S</td><td></td></tr><tr><td>MyPipetman Select P20</td><td>Gilson, USA</td><td>FP10003S</td><td></td></tr><tr><td>MyPipetman Select P200</td><td>Gilson, USA</td><td>FP10005S</td><td></td></tr><tr><td>Omnifix 1 mL</td><td>Braun, Germany</td><td>4022495251879</td><td></td></tr><tr><td>Phenylmethylsulfonyl fluoride (PMSF)</td><td>Serva, Germany</td><td>32395.03</td><td></td></tr><tr><td>STERICAN cannula 21 Gx4 4/5 0.8x120 mm</td><td>Braun, Germany</td><td>4022495052414</td><td></td></tr><tr><td>stirring bar, 15 mm</td><td>VWR, USA</td><td>442-0366</td><td></td></tr><tr><td>Sucrose</td><td>Merck, Germany</td><td>S8501</td><td></td></tr><tr><td>SW 41 Ti Swinging-Bucket Rotor</td><td>Beckman Instruments, Germany</td><td>331362</td><td></td></tr><tr><td>Test tubes</td><td>Eppendorf, Germany</td><td>3810X</td><td></td></tr><tr><td>Tissue grinders, Potter-Elvehjem type, 2 mL glass vessel</td><td>VWR, USA</td><td>432-0200</td><td></td></tr><tr><td>Tissue grinders, Potter-Elvehjem type, 2 mL plunger with serrated tip</td><td>VWR, USA</td><td>432-0212</td><td></td></tr><tr><td>Trichloroacetic acid (TCA)</td><td>Sigma Aldrich, Germany</td><td>33731</td><td>discontinued,&lt;br/&gt; any TCA will suffice (CAS: 73-03-9)</td></tr><tr><td>TRIS</td><td>Roth, Germany</td><td>4855</td><td></td></tr></tbody></table>

an improved method to isolate mitochondrial contact sites

Mitochondria are present in virtually all eukaryotic cells and perform essential functions that go far beyond energy production, for instance, the synthesis of iron-sulfur clusters, lipids, or proteins, Ca2+ buffering, and the induction of apoptosis. Likewise, mitochondrial dysfunction results in severe human diseases such as cancer, diabetes, and neurodegeneration. In order to perform these functions, mitochondria have to communicate with the rest of the cell across their envelope, which consists of two membranes. Therefore, these two membranes have to interact constantly. Proteinaceous contact sites between the mitochondrial inner and outer membranes are essential in this respect. So far, several contact sites have been identified. In the method described here, Saccharomyces cerevisiae mitochondria are used to isolate contact sites and, thus, identify candidates that qualify for contact site proteins. We used this method to identify the mitochondrial contact site and cristae organizing system (MICOS) complex, one of the major contact site-forming complexes in the mitochondrial inner membrane, which is conserved from yeast to humans. Recently, we further improved this method to identify a novel contact site consisting of Cqd1 and the Por1-Om14 complex.

It is relatively easy to separate mitochondrial inner and outer membranes. However, the generation and separation of contact site-containing vesicles are much more difficult. In our opinion, two steps are critical and essential: the sonication conditions and the gradient used.
Usually, linear gradients are thought to have a better resolution compared to step gradients. However, their reproducible production is tedious and requires special equipment. Therefore, we established a method to genera...

Watch this Scientific Journal Video about Unveiling Mitochondrial Contact Sites and Architectural Insights at JoVE.com

Author Spotlight: Unveiling Mitochondrial Contact Sites and Architectural Insights 

Mitochondrial contact sites are protein complexes that interact with mitochondrial inner and outer membrane proteins. These sites are essential for the communication between the mitochondrial membranes and, thus, between the cytosol and the mitochondrial matrix. Here, we describe a method to identify candidates qualifying for this specific class of proteins.

An Improved Method to Isolate Mitochondrial Contact Sites

Mitochondria are present in virtually all eukaryotic cells and perform essential functions that go far beyond energy production, for instance, the ...

an-improved-method-to-isolate-mitochondrial-contact-sites

Research

JoVE Journal

Biochemistry

1.3K Views.  Ludwig-Maximilians University Munich. We willen de moleculaire functie van mitochondriale contactplaatsen begrijpen, waardoor de communicatie tussen de twee mitochondriale membranen mogelijk wordt. Dit is eigenlijk een voorwaarde om de functie voor mitochondriale biogenese en dus de levensvatbaarheid van cellen te begrijpen. We ontdekten een nieuwe contactplaats tussen het mitochondriale binnen- en buitenmembraan gecreëerd door Cqd1 en Om14-Por1.Onze gegevens suggereren dat deze site kan helpen bij het importeren van lipi...

Video: Auteur in de schijnwerpers: onthulling van mitochondriale contactsites en architecturale inzichten 

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

Mitochondria are essential organelles involved in numerous metabolic pathways in plants, most notably the production of adenosine triphosphate (ATP) from the oxidation of reduced compounds such as nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FADH2). The complete annotation of the Arabidopsis thaliana genome has established it as the most widely used plant model system, and thus the need to purify mitochondria from a variety of organs (leaf, root, or flower) is necessary to fully utilize the tools that are now available for Arabidopsis to study mitochondrial biology. Mitochondria are isolated by homogenization of the tissue using a variety of approaches, followed by a series of differential centrifugation steps producing a crude mitochondrial pellet that is further purified using continuous colloidal density gradient centrifugation. The colloidal density material is subsequently removed by multiple centrifugation steps. Starting from 100 g of fresh leaf tissue, 2 - 3 mg of mitochondria can be routinely obtained. Respiratory experiments on these mitochondria display typical rates of 100 - 250 nmol O2 min-1 mg total mitochondrial protein-1 (NADH-dependent rate) with the ability to use various substrates and inhibitors to determine which substrates are being oxidized and the capacity of the alternative and cytochrome terminal oxidases. This protocol describes an isolation method of mitochondria from Arabidopsis thaliana leaves using continuous colloidal density gradients and an efficient respiratory measurements of purified plant mitochondria.

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

As mitochondria are only a small percentage of the plant cell, they need to be purified for a range of studies. Mitochondria can be isolated from a variety of plant organs by homogenization, followed by differential and density gradient centrifugation to obtain a highly purified mitochondrial fraction.

Mitochondria are essential organelles involved in numerous metabolic pathways in plants, most notably the production of adenosine triphosphate (ATP) from ...

Rapid Isolation of the Mitoribosome from HEK Cells

The human mitochondria possess a dedicated set of ribosomes (mitoribosomes) that translate 13 essential protein components of the oxidative phosphorylation complexes encoded by the mitochondrial genome. Since all proteins synthesized by human mitoribosomes are integral membrane proteins, human mitoribosomes are tethered to the mitochondrial inner membrane during translation. Compared to the cytosolic ribosome the mitoribosome has a sedimentation coefficient of 55S, half the rRNA content, no 5S rRNA and 36 additional proteins. Therefore, a higher protein-to-RNA ratio and an atypical structure make the human mitoribosome substantially distinct from its cytosolic counterpart.
Despite the central importance of the mitoribosome to life, no protocols were available to purify the intact complex from human cell lines. Traditionally, mitoribosomes were isolated from mitochondria-rich animal tissues that required kilograms of starting material. We reasoned that mitochondria in dividing HEK293-derived human cells grown in nutrient-rich expression medium would have an active mitochondrial translation, and, therefore, could be a suitable source of material for the structural and biochemical studies of the mitoribosome. To investigate its structure, we developed a protocol for large-scale purification of intact mitoribosomes from HEK cells. Herein, we introduce nitrogen cavitation method as a faster, less labor-intensive and more efficient alternative to traditional mechanical shear-based methods for cell lysis. This resulted in preparations of the mitoribosome that allowed for its structural determination to high resolution, revealing the composition of the intact human mitoribosome and its assembly intermediates. Here, we follow up on this work and present an optimized and more cost-effective method requiring only ~1010 cultured HEK cells. The method can be employed to purify human mitoribosomal translating complexes, mutants, quality control assemblies and mitoribosomal subunits intermediates. The purification can be linearly scaled up tenfold if needed, and also applied to other types of cells.

Mitochondria have specialized ribosomes that diverged from their bacterial and cytoplasmic counterparts. Here we show how mitoribosomes can be obtained from their native compartment in HEK cells. The method involves isolation of mitochondria from suspension cells and consequent purification of mitoribosomes.

The human mitochondria possess a dedicated set of ribosomes (mitoribosomes) that translate 13 essential protein components of the oxidative ...

Mitochondrial Isolation from Skeletal Muscle

Mitochondria are organelles controlling the life and death of the cell. They participate in key metabolic reactions, synthesize most of the ATP, and regulate a number of signaling cascades2,3. Past and current researchers have isolated mitochondria from rat and mice tissues such as liver, brain and heart4,5. In recent years, many researchers have focused on studying mitochondrial function from skeletal muscles.
Here, we describe a method that we have used successfully for the isolation of mitochondria from skeletal muscles 6. Our procedure requires that all buffers and reagents are made fresh and need about 250-500 mg of skeletal muscle. We studied mitochondria isolated from rat and mouse gastrocnemius and diaphragm, and rat extraocular muscles. Mitochondrial protein concentration is measured with the Bradford assay. It is important that mitochondrial samples be kept ice-cold during preparation and that functional studies be performed within a relatively short time (~1 hr). Mitochondrial respiration is measured using polarography with a Clark-type electrode (Oxygraph system) at 37&deg;C7. Calibration of the oxygen electrode is a key step in this protocol and it must be performed daily. Isolated mitochondria (150 &mu;g) are added to 0.5 ml of experimental buffer (EB). State 2 respiration starts with addition of glutamate (5mM) and malate (2.5 mM). Then, adenosine diphosphate (ADP) (150 &mu;M) is added to start state 3. Oligomycin (1 &mu;M), an ATPase synthase blocker, is used to estimate state 4. Lastly, carbonyl cyanide p-[trifluoromethoxy]-phenyl-hydrazone (FCCP, 0.2 &mu;M) is added to measurestate 5, or uncoupled respiration 6. The respiratory control ratio (RCR), the ratio of state 3 to state 4, is calculated after each experiment. An RCR &ge;4 is considered as evidence of a viable mitochondria preparation.
In summary, we present a method for the isolation of viable mitochondria from skeletal muscles that can be used in biochemical (e.g., enzyme activity, immunodetection, proteomics) and functional studies (mitochondrial respiration).

This protocol describes a procedure to study the respiration of mitochondria isolated from skeletal muscles. This method was adapted from Scorrano et al. (2007). The mitochondrial isolation procedure requires about 2 hours. The mitochondrial respiration can be completed in about 1 hour.

Mitochondria are organelles controlling the life and death of the cell. They participate in key metabolic reactions, synthesize most of the ATP, and ...

Biology

Mitochondrial Fractionation in Small Tissue Samples for Supercomplex Analysis

Isolation of Mitochondria for Mitochondrial Supercomplex Analysis from Small Tissue and Cell Culture Samples

Over the last decades, the evidence accumulated about the existence of respiratory supercomplexes (SCs) has changed our understanding of the mitochondrial electron transport chain organization, giving rise to the proposal of the "plasticity model." This model postulates the coexistence of different proportions of SCs and complexes depending on the tissue or the cellular metabolic status. The dynamic nature of the assembly in SCs would allow cells to optimize the use of available fuels and the efficiency of electron transfer, minimizing reactive oxygen species generation and favoring the ability of cells to adapt to environmental changes. 
More recently, abnormalities in SC assembly have been reported in different diseases such as neurodegenerative disorders (Alzheimer's and Parkinson's disease), Barth Syndrome, Leigh syndrome, or cancer. The role of SC assembly alterations in disease progression still needs to be confirmed. Nevertheless, the availability of enough amounts of samples to determine the SC assembly status is often a challenge. This happens with biopsy or tissue samples that are small or have to be divided for multiple analyses, with cell cultures that have slow growth or come from microfluidic devices, with some primary cultures or rare cells, or when the effect of particular costly treatments has to be analyzed (with nanoparticles, very expensive compounds, etc.). In these cases, an efficient and easy-to-apply method is required. This paper presents a method adapted to obtain enriched mitochondrial fractions from small amounts of cells or tissues to analyze the structure and function of mitochondrial SCs by native electrophoresis followed by in-gel activity assays or western blot.

Author Spotlight: Unveiling Oxidative Phosphorylation System Dynamics and Mitochondrial Roles in Health and Disease

This protocol describes a technique for the analysis of respiratory supercomplexes when only small amounts of samples are available.

Over the last decades, the evidence accumulated about the existence of respiratory supercomplexes (SCs) has changed our understanding of the mitochondrial ...

Mitochondrial Isolation from Rodents for High-Quality Bioenergetic Studies

Robust Mitochondrial Isolation from Rodent Cardiac Tissue

Mitochondrial isolation has been practiced for decades, following procedures established by pioneers in the fields of molecular biology and biochemistry to study metabolic impairments and disease. Consistent mitochondrial quality is necessary to properly investigate mitochondrial physiology and bioenergetics; however, many different published isolation methods are available for researchers. Although different experimental strategies require different isolation methods, the basic principles and procedures are similar. This protocol details a method capable of extracting well-coupled mitochondria from a variety of tissue sources, including small animals and cells. The steps outlined include organ dissection, mitochondrial purification, protein quantification, and various quality control checks. The primary quality control metric used to identify high-quality mitochondria is the respiratory control ratio (RCR). The RCR is the ratio of the respiratory rate during oxidative phosphorylation to the rate in the absence of ADP. Alternative metrics are discussed. While high RCR values relative to their tissue source are obtained using this protocol, several steps can be optimized to suit the individual needs of researchers. This procedure is robust and has consistently resulted in isolated mitochondria with above-average RCR values across animal models and tissue sources.

Author Spotlight: Uncovering the Role of Mitochondrial Calcium Phosphate in Heart Failure and Bioenergetics

Bioenergetic and metabolomic studies on mitochondria have revealed their multifaceted role in many diseases, but the isolation methods for these organelles vary. The method detailed here is capable of purifying high-quality mitochondria from multiple tissue sources. Quality is determined by respiratory control ratios and other metrics assessed with high-resolution respirometry.

Mitochondrial isolation has been practiced for decades, following procedures established by pioneers in the fields of molecular biology and biochemistry ...

Rapid Isolation And Purification Of Mitochondria For Transplantation By Tissue Dissociation And Differential Filtration

Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 min to complete. We describe a method for the rapid isolation of mitochondria from mammalian biopsies using a commercial tissue dissociator and differential filtration. In this protocol, manual homogenization is replaced with the tissue dissociator&#8217;s standardized homogenization cycle. This allows for uniform and consistent homogenization of tissue that is not easily achieved with manual homogenization. Following tissue dissociation, the homogenate is filtered through nylon mesh filters, which eliminate repetitive centrifugation steps. As a result, mitochondrial isolation can be performed in less than 30 min. This isolation protocol yields approximately 2 x 1010 viable and respiration competent mitochondria from 0.18 &#177; 0.04 g (wet weight) tissue sample.

A method for rapid isolation of mitochondria from mammalian tissue biopsies is described. Rat liver or skeletal muscle preparations were homogenized with a commercial tissue dissociator and mitochondria were isolated by differential filtration through nylon mesh filters. Mitochondrial isolation time is &#60;30 min compared to 60 - 100 min using alternative methods.

Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 min to ...

Isolation and Functional Analysis of Mitochondria from Cultured Cells and Mouse Tissue

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of cell heath due to their role in both cell metabolism and energy production as well as control of apoptosis. Therefore, direct evaluation of isolated mitochondria and mitochondrial perturbation offers the ability to determine if organelle-specific (dys)function is occurring. The methods described in this protocol include isolation of intact, functional mitochondria from HEK cultured cells and mouse liver and spinal cord, but can be easily adapted for use with other cultured cells or animal tissues. Mitochondrial function assessed by TMRE and the use of common mitochondrial uncouplers and inhibitors in conjunction with a fluorescent plate reader allow this protocol not only to be versatile and accessible to most research laboratories, but also offers high throughput.

Comparison of mitochondrial membrane potential between samples yields valuable information about cellular status. Detailed steps for isolating mitochondria and assessing response to inhibitors and uncouplers using fluorescence are described. The method and utility of this protocol are illustrated by use of a cell culture and animal model of cellular stress.

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of ...

Isolation of Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High Throughput Microplate Respiratory Measurements

Dysfunctional skeletal muscle mitochondria play a role in altered metabolism observed with aging, obesity and Type II diabetes. Mitochondrial respirometric assays from isolated mitochondrial preparations allow for the assessment of mitochondrial function, as well as determination of the mechanism(s) of action of drugs and proteins that modulate metabolism. Current isolation procedures often require large quantities of tissue to yield high quality mitochondria necessary for respirometric assays. The methods presented herein describe how high quality purified mitochondria (~ 450 &#181;g) can be isolated from minimal quantities (~75-100 mg) of mouse skeletal muscle for use in high throughput respiratory measurements. We determined that our isolation method yields 92.5&#177; 2.0% intact mitochondria by measuring citrate synthase activity spectrophotometrically. In addition, Western blot analysis in isolated mitochondria resulted in the faint expression of the cytosolic protein, GAPDH, and the robust expression of the mitochondrial protein, COXIV. The absence of a prominent GAPDH band in the isolated mitochondria is indicative of little contamination from non-mitochondrial sources during the isolation procedure. Most importantly, the measurement of O2 consumption rate with micro-plate based technology and determining the respiratory control ratio (RCR) for coupled respirometric assays shows highly coupled (RCR; &#62;6 for all assays) and functional mitochondria. In conclusion, the addition of a separate mincing step and significantly reducing motor driven homogenization speed of a previously reported method has allowed the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle that results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

Here, we present a modification of a previously reported method that allows for the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle. This procedure results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

Dysfunctional skeletal muscle mitochondria play a role in altered metabolism observed with aging, obesity and Type II diabetes. Mitochondrial ...

Isolation of Mitochondria from Mouse Skeletal Muscle for Respirometric Assays

Most of the cell&#39;s energy is obtained through the degradation of glucose, fatty acids, and amino acids by different pathways that converge on the mitochondrial oxidative phosphorylation (OXPHOS) system, which is regulated in response to cellular demands. The lipid molecule Coenzyme Q (CoQ) is essential in this process by transferring electrons to complex III in the electron transport chain (ETC) through constant oxidation/reduction cycles. Mitochondria status and, ultimately, cellular health can be assessed by measuring ETC oxygen consumption using respirometric assays. These studies are typically performed in established or primary cell lines that have been cultured for several days. In both cases, the respiration parameters obtained may have deviated from normal physiological conditions in&#160;any given organ or tissue.
Additionally, the intrinsic characteristics of cultured single fibers isolated from skeletal muscle impede this type of analysis. This paper presents an updated and detailed protocol for the analysis of respiration in freshly isolated mitochondria from mouse skeletal muscle. We also provide solutions to potential problems that could arise at any step of the process. The method presented here could be applied to compare oxygen consumption rates in diverse transgenic mouse models and study the mitochondrial response to drug treatments or other factors such as aging or sex. This is a feasible method to respond to crucial questions about mitochondrial bioenergetics metabolism and regulation.

Here, we describe a detailed method for mitochondria isolation from mouse skeletal muscle and the subsequent analysis of respiration by Oxygen Consumption Rate (OCR) using microplate-based respirometric assays. This pipeline can be applied to study the effects of multiple environmental or genetic interventions on mitochondrial metabolism.

Most of the cell&#39;s energy is obtained through the degradation of glucose, fatty acids, and amino acids by different pathways that converge on the ...

Improved Protein Quantification Using Two-Step Mitochondrial Isolation

Two-Step Tag-Free Isolation of Mitochondria for Improved Protein Discovery and Quantification

Most physiological and disease processes, from central metabolism to immune response to neurodegeneration, involve mitochondria. The mitochondrial proteome is composed of more than 1,000 proteins, and the abundance of each can vary dynamically in response to external stimuli or during disease progression. Here, we describe a protocol for isolating high-quality mitochondria from primary cells and tissues. The two-step procedure comprises (1) mechanical homogenization and differential centrifugation to isolate crude mitochondria, and (2) tag-free immune capture of mitochondria to isolate pure organelles and eliminate contaminants. Mitochondrial proteins from each purification stage are analyzed by quantitative mass spectrometry, and enrichment yields are calculated, allowing the discovery of novel mitochondrial proteins by subtractive proteomics. Our protocol provides a sensitive and comprehensive approach to studying mitochondrial content in cell lines, primary cells, and tissues.

Author Spotlight: Two-Step Tag-Free Isolation of Mitochondria for Improved Protein Discovery and Quantification  

We present a two-step protocol for high-quality mitochondria isolation that is compatible with protein discovery and quantification at a proteome scale. Our protocol does not require genetic engineering and is thus suitable for studying mitochondria from any primary cells and tissues.

An Improved Method to Isolate Mitochondrial Contact Sites

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