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 &#176;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 &#8722;20 &#176;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 &#176;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 &#176;C; however, it is recommended to store the sucrose-containing buffers at &#8722;20 &#176;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 &#176;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 &#176;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 &#176;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 &#181;L of the buffer to the refractometer, and detect the respective refractive indices. The calculation of sucrose concentration is as follows: 
		<img alt="Equation 1" src="/files/ftp_upload/65444/65444eq01v2.jpg" style="margin: auto;" /> 
		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 &#8722;20 &#176;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 &#8722;20 &#176;C. 
		NOTE: Remember to transfer the frozen gradients to 4 &#176;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 &#181;L) on top of 200 &#181;L 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 &#176;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 &#181;L fractions using a 1 mL pipette. This will result in 17 fractions, which allows for a sufficient resolution.</li>
</ol>
4.&#160;Analysis of submitochondrial vesicles
<ol>
	<li>Concentrate the proteins by subjecting each fraction to two sequentially performed TCA precipitations38&#160;to prepare the SDS-PAGE samples.
	<ol>
		<li>Add 200 &#181;L 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 &#176;C for 20 min. Discard the supernatant, add 500 &#181;L of 28 % TCA solution, mix well, and repeat the centrifugation step.</li>
		<li>Wash the pellets with 1 mL of acetone (&#8722;20&#176;C), and centrifuge for 10 min at 20,000 x g and 4 &#176;C. Discard the supernatant, and let the pellets air dry. Resuspend the pellets in 60 &#181;L of SDS sample buffer, and incubate the samples for 5 min at 95 &#176;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 &#181;L of each fraction by SDS-PAGE39&#160;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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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, 
			any pipet controller will suffice</td>
		</tr>
		<tr>
			<td>accu-jet pro Pipet Controller</td>
			<td>Brandtech, USA</td>
			<td>BR26320</td>
			<td>discontinued, 
			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&#160; 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>&#160;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, 
			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

1.2K Views.  Ludwig-Maximilians University Munich. 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.

Video: Author Spotlight: Unveiling Mitochondrial Contact Sites and Architectural Insights 

A Rapid Laser Probing Method Facilitates the Non-invasive and Contact-free Determination of Leaf Thermal Properties

Plants can produce valuable substances such as secondary metabolites and recombinant proteins. The purification of the latter from plant biomass can be streamlined by heat treatment (blanching). A blanching apparatus can be designed more precisely if the thermal properties of the leaves are known in detail, i.e., the specific heat capacity and thermal conductivity. The measurement of these properties is time consuming and labor intensive, and usually requires invasive methods that contact the sample directly. This can reduce the product yield and may be incompatible with containment requirements, e.g., in the context of good manufacturing practice. To address these issues, a non-invasive, contact-free method was developed that determines the specific heat capacity and thermal conductivity of an intact plant leaf in about one minute. The method involves the application of a short laser pulse of defined length and intensity to a small area of the leaf sample, causing a temperature increase that is measured using a near infrared sensor. The temperature increase is combined with known leaf properties (thickness and density) to determine the specific heat capacity. The thermal conductivity is then calculated based on the profile of the subsequent temperature decline, taking thermal radiation and convective heat transfer into account. The associated calculations and critical aspects of sample handling are discussed.

A method was developed to determine the specific heat capacity and thermal conductivity of leaf tissue by non-invasive, contact-free near infrared laser probing, which requires less than 1 min per sample.

Plants can produce valuable substances such as secondary metabolites and recombinant proteins. The purification of the latter from plant biomass can be ...

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating these studies, reincorporation of detergent-solubilized membrane proteins into liposomes is a stochastic process where protein topology is impossible to enforce. This paper offers an alternative method to these challenging techniques that utilizes a liposome-based scaffold. Protein solubility is enhanced by deletion of the transmembrane domain, and these amino acids are replaced with a tethering moiety, such as a His-tag. This tether interacts with an anchoring group (Ni2+ coordinated by nitrilotriacetic acid (NTA(Ni2+)) for His-tagged proteins), which enforces a uniform protein topology at the surface of the liposome. An example is presented wherein the interaction between Dynamin-related protein 1 (Drp1) with an integral membrane protein, Mitochondrial Fission Factor (Mff), was investigated using this scaffold liposome method. In this work, we have demonstrated the ability of Mff to efficiently recruit soluble Drp1 to the surface of liposomes, which stimulated its GTPase activity. Moreover, Drp1 was able to tubulate the Mff-decorated lipid template in the presence of specific lipids. This example demonstrates the effectiveness of scaffold liposomes using structural and functional assays and highlights the role of Mff in regulating Drp1 activity.

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

This paper describes a method for assessing the interactions and assemblies of integral membrane proteins in vitro with various partner factors in a lipid-proximal environment.

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

An Alternative Culture Method to Maintain Genomic Hypomethylation of Mouse Embryonic Stem Cells Using MEK Inhibitor PD0325901 and Vitamin C

Embryonic stem (ES) cells have the potential to differentiate into any of the three germ layers (endoderm, mesoderm, or ectoderm), and can generate many lineages for regenerative medicine. ES cell culture in vitro has long been the subject of widespread concerns. Classically, mouse ES cells are maintained in serum and leukemia inhibitory factor (LIF)-containing medium. However, under serum/LIF conditions, cells show heterogeneity in morphology and the expression profile of pluripotency-related genes, and are mostly in a metastable state. Moreover, cultured ES cells exhibit global hypermethylation, but na&#239;ve ES cells of the inner cell mass (ICM) and primordial germ cells (PGCs) are in a state of global hypomethylation. The hypomethylated state of ICM and PGCs is closely associated with their pluripotency. To improve mouse ES cell culture methods, we have recently developed a new method based on the selectively combined utilization of two small-molecule compounds to maintain the DNA hypomethylated and pluripotent state. Here, we present that the co-treatment of vitamin C (Vc) and PD0325901 can erase about 90% of 5-methylcytosine (5mC) at 5 days in mouse ES cells. The generated 5mC content is comparable to that in PGCs. The mechanistic investigation shows that PD0325901 up-regulates Prdm14 expression to suppress Dnmt3b (de novo DNA methyltransferase) and Dnmt3l (the cofactor of Dnmt3b), by reducing de novo 5mC synthesis. Vc facilitates the conversion of 5mC to 5-hydroxymethylcytosine (5hmC) catalyzed mainly by Tet1 and Tet2, indicating the involvement of both passive and active DNA demethylations. Moreover, under Vc/PD0325901 conditions, mouse ES cells show homogeneous morphology and pluripotent state. Collectively, we propose a novel and chemical-synergy culture method for achieving DNA hypomethylation and maintenance of pluripotency in mouse ES cells. The small-molecule chemical-dependent method overcomes the major shortcomings of serum culture, and holds promise to generate homogeneous ES cells for further clinical applications and researches.

We described in detail two chemical-based protocols for culturing mouse embryonic stem cells. This new method utilizes synergistic mechanisms of promoting Tet-mediated oxidation (by vitamin C) and repressing de novo synthesis of 5-methylcytosine (by PD0325901) to maintain DNA hypomethylation and pluripotency of mouse embryonic stem cells.

Embryonic stem (ES) cells have the potential to differentiate into any of the three germ layers (endoderm, mesoderm, or ectoderm), and can generate many ...

Improved Protocol for Chromatin Immunoprecipitation from Mouse Skeletal Muscle

We describe an efficient and reproducible protocol for the preparation of chromatin from adult mouse skeletal muscle, a physically resistant tissue with a high content of structural proteins. Dissected limb muscles from adult mice are physically disrupted by mechanical homogenisation, or a combination of mincing and douncing, in a hypotonic buffer before formaldehyde fixation of the cell lysate. The fixed nuclei are purified by further cycles of mechanical homogenisation or douncing and sequential filtrations to remove cell debris. The purified nuclei can be sonicated immediately or at a later stage after freezing. The chromatin can be efficiently sonicated and is suitable for chromatin immunoprecipitation experiments, as illustrated by the profiles obtained for transcription factors, RNA polymerase II, and covalent histone modifications. The binding events detected using chromatin prepared by this protocol are predominantly those taking place in the muscle fiber nuclei despite the presence of chromatin from other fiber-associated satellite and endothelial cells. This protocol is therefore adapted to study gene regulation in the adult mouse skeletal muscle.

A novel protocol for the preparation of chromatin from adult mouse skeletal muscle adapted to the study of gene regulation in muscle fibers by chromatin immunoprecipitation is presented.

We describe an efficient and reproducible protocol for the preparation of chromatin from adult mouse skeletal muscle, a physically resistant tissue with a ...

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

Cyclin-dependent kinase 1 (Cdk1) is a master controller for the cell cycle in all eukaryotes and phosphorylates an estimated 8 - 13% of the proteome; however, the number of identified targets for Cdk1, particularly in human cells is still low. The identification of Cdk1-specific phosphorylation sites is important, as they provide mechanistic insights into how Cdk1 controls the cell cycle. Cell cycle regulation is critical for faithful chromosome segregation, and defects in this complicated process lead to chromosomal aberrations and cancer.
Here, we describe an in vitro kinase assay that is used to identify Cdk1-specific phosphorylation sites. In this assay, a purified protein is phosphorylated in vitro by commercially available human Cdk1/cyclin B. Successful phosphorylation is confirmed by SDS-PAGE, and phosphorylation sites are subsequently identified by mass spectrometry. We also describe purification protocols that yield highly pure and homogeneous protein preparations suitable for the kinase assay, and a binding assay for the functional verification of the identified phosphorylation sites, which probes the interaction between a classical nuclear localization signal (cNLS) and its nuclear transport receptor karyopherin &#945;. To aid with experimental design, we review approaches for the prediction of Cdk1-specific phosphorylation sites from protein sequences. Together these protocols present a very powerful approach that yields Cdk1-specific phosphorylation sites and enables mechanistic studies into how Cdk1 controls the cell cycle. Since this method relies on purified proteins, it can be applied to any model organism and yields reliable results, especially when combined with cell functional studies.

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

Cyclin-dependent kinase 1 (Cdk1) is activated in the G2 phase of the cell cycle and regulates many cellular pathways. Here, we present a protocol for an in vitro kinase assay with Cdk1, which allows the identification of Cdk1-specific phosphorylation sites for establishing cellular targets of this important kinase.

Cyclin-dependent kinase 1 (Cdk1) is a master controller for the cell cycle in all eukaryotes and phosphorylates an estimated 8 - 13% of the proteome; ...

Measuring Liver Mitochondrial Oxygen Consumption and Proton Leak Kinetics to Estimate Mitochondrial Respiration in Holstein Dairy Cattle

Oxygen consumption, proton motive force (PMF) and proton leak are measurements of mitochondrial respiration, or how well mitochondria are able to convert NADH and FADH into ATP. Since mitochondria are also the primary site for oxygen use and nutrient oxidation to carbon dioxide and water, how efficiently they use oxygen and produce ATP directly relates to the efficiency of nutrient metabolism, nutrient requirements of the animal, and health of the animal. The purpose of this method is to examine mitochondrial respiration, which can be used to examine the effects of different drugs, diets and environmental effects on mitochondrial metabolism. Results include oxygen consumption measured as proton dependent respiration (State 3) and proton leak dependent respiration (State 4). The ratio of State 3 / State 4 respiration is defined as respiratory control ratio (RCR) and can represent mitochondrial energetic efficiency. Mitochondrial proton leak is a process that allows dissipation of mitochondrial membrane potential (MMP) by uncoupling oxidative phosphorylation from ADP decreasing the efficiency of ATP synthesis. Oxygen and TRMP+ sensitive electrodes with mitochondrial substrates and electron transport chain inhibitors are used to measure State 3 and State 4 respiration, mitochondrial membrane PMF (or the potential to produce ATP) and proton leak. Limitations to this method are that liver tissue must be as fresh as possible and all biopsies and assays must be performed in less than 10 h. This limits the number of samples that can be collected and processed by a single person in a day to approximately 5. However, only 1 g of liver tissue is needed, so in large animals, such as dairy cattle, the amount of sample needed is small relative to liver size and there is little recovery time needed.

Here, we share methods for measuring mitochondrial oxygen consumption, a defining concept of nutritional energetics, and proton leak, the primary cause of inefficiency in mitochondrial generation of ATP. These results can account for 30% of the energy lost in nutrient utilization to help evaluate mitochondrial function.

Oxygen consumption, proton motive force (PMF) and proton leak are measurements of mitochondrial respiration, or how well mitochondria are able to convert ...

Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ubiquitinated proteins, peptides with a diglycine remnant conjugated to the epsilon amino group of lysine (&#39;K-&#949;-diglycine&#39; or simply &#39;diGly&#39;) can be used to track back the original modification site. Efficient immunopurification of diGly peptides combined with sensitive detection by mass spectrometry has resulted in a huge increase in the number of ubiquitination sites identified up to date. We have made several improvements to this workflow, including offline high pH reverse-phase fractionation of peptides prior to the enrichment procedure, and the inclusion of more advanced peptide fragmentation settings in the ion routing multipole. Also, more efficient cleanup of the sample using a filter-based plug in order to retain the antibody beads results in a greater specificity for diGly peptides. These improvements result in the routine detection of more than 23,000 diGly peptides from human cervical cancer cells (HeLa) cell lysates upon proteasome inhibition in the cell. We show the efficacy of this strategy for in-depth analysis of the ubiquitinome profiles of several different cell types and of in vivo samples, such as brain tissue. This study presents an original addition to the toolbox for protein ubiquitination analysis to uncover the deep cellular ubiquitinome.

We present a method for the purification, detection, and identification of diGly peptides that originate from ubiquitinated proteins from complex biological samples. The presented method is reproducible, robust, and outperforms published methods with respect to the level of depth of the ubiquitinome analysis.

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ...

Improved UPLC-UV Method for the Quantification of Vitamin C in Lettuce Varieties (Lactuca sativa L.) and Crop Wild Relatives (Lactuca spp.)

Vitamins, especially vitamin C, are important micronutrients found in fruits and vegetables. Vitamin C is also a major contributor to their antioxidant capacity. Lettuce is one of the most popular vegetables among consumers worldwide. An accurate protocol to measure vitamin C content in lettuce and other related species is crucial. We describe here a method using the ultra-high-performance liquid chromatography-ultraviolet (UPLC-UV) technique, in which sample preparation, vitamin extraction and chromatography conditions were optimized.
Samples were collected to represent the entire plant, frozen at -80 &#176;C and lyophilized to prevent undesirable oxidation and make their manipulation easier. The extraction of vitamin C was carried out in acidic media, which also contributed to its stability. As vitamin C can be present in two different interconvertible forms, ascorbic acid (AA) and dehydroascorbic acid (DHAA), both compounds should be measured for accurate quantification. The DHAA was quantified indirectly after its reduction to AA because AA shows a higher absorptivity than DHAA in the UV range of the spectrum. From the same extract, two measurements were carried out, one before and one after that reduction reaction. In the first case, we were quantifying the AA content, and in the second one, we quantified the sum of AA and DHAA (TAA: total ascorbic acid) in the form of AA. Then, DHAA quantity was indirectly obtained by subtracting AA coming from the first measurement from TAA. They were determined by UPLC-UV, using a commercial AA standard to build a calibration curve and optimizing the chromatographic procedure, to obtain AA peaks that were completely resolved in a short time. This protocol could be easily extrapolated to any other plant material with slight or no changes. Its accuracy revealed statistically significant differences otherwise unperceived. Other strengths and limitations are discussed more in depth in the manuscript.

Improved UPLC-UV Method for the Quantification of Vitamin C in Lettuce Varieties Lactuca sativa L. and Crop Wild Relatives Lactuca spp.

We present a fast and reliable method to quantify vitamin C in Lactuca spp. using UPLC-UV, potentially transferable to other plants. The key steps are the sample preparation and vitamin C extraction under stable conditions, the reduction of dehydroascorbic acid to ascorbic acid and the optimization of the chromatographic procedure.

Vitamins, especially vitamin C, are important micronutrients found in fruits and vegetables. Vitamin C is also a major contributor to their antioxidant ...

Measuring Mitochondrial Electron Transfer Complexes in Previously Frozen Cardiac Tissue from the Offspring of Sow: A Model to Assess Exercise-Induced Mitochondrial Bioenergetics Changes

The mitochondrial electron transfer complex (ETC) profile is modified in the heart tissue of the offspring born to an exercised sow. The hypothesis proposed and tested was that a regular maternal exercise of a sow during pregnancy would increase the mitochondrial efficiency of offspring heart bioenergetics. This hypothesis was tested by isolating mitochondria using a mild-isolation procedure to assess mitochondrial ETC and supercomplex profiles. The procedure described here allowed for the processing of previously frozen archived heart tissues and eliminated the necessity of fresh mitochondria preparation for the assessment of mitochondrial ETC complexes, supercomplexes, and ETC complex activity profiles. This protocol describes the optimal ETC protein complex measurement in multiplexed antibody-based immunoblotting and super complex assessment using blue-native gel electrophoresis.

Preparation of mitochondria-enriched samples from previously frozen archived solid tissues allowed the investigators to perform both functional and analytical assessments of mitochondria in various experimental modalities. This study demonstrates how to prepare mitochondria-enriched preparations from frozen heart tissue and perform analytical assessments of mitochondria.

The mitochondrial electron transfer complex (ETC) profile is modified in the heart tissue of the offspring born to an exercised sow. The hypothesis ...

Deciphering Molecular Mechanism of Histone Assembly by DNA Curtain Technique

Chromatin is a higher-order structure that packages eukaryotic DNA. Chromatin undergoes dynamic alterations according to the cell cycle phase and in response to environmental stimuli. These changes are essential for genomic integrity, epigenetic regulation, and DNA metabolic reactions such as replication, transcription, and repair. Chromatin assembly is crucial for chromatin dynamics and is catalyzed by histone chaperones. Despite extensive studies, the mechanisms by which histone chaperones enable chromatin assembly remains elusive. Moreover, the global features of nucleosomes organized by histone chaperones are poorly understood. To address these problems, this work describes a unique single-molecule imaging technique named DNA curtain, which facilitates the investigation of the molecular details of nucleosome assembly by histone chaperones. DNA curtain is a hybrid technique that combines lipid fluidity, microfluidics, and total internal reflection fluorescence microscopy (TIRFM) to provide a universal platform for real-time imaging of diverse protein-DNA interactions.Using DNA curtain, the histone chaperone function of Abo1, the Schizosaccharomyces pombe bromodomain-containing AAA+ ATPase, is investigated, and the molecular mechanism underlying histone assembly of Abo1 is revealed. DNA curtain provides a unique approach for studying chromatin dynamics.

DNA curtain, a high-throughput single-molecule imaging technique, provides a platform for real-time visualization of diverse protein-DNA interactions. The present protocol utilizes the DNA curtain technique to investigate the biological role and molecular mechanism of Abo1, a Schizosaccharomyces pombe bromodomain-containing AAA+ ATPase.

Chromatin is a higher-order structure that packages eukaryotic DNA. Chromatin undergoes dynamic alterations according to the cell cycle phase and in ...