This study was financed by Instituto Serrapilheira, Funda&#231;&#227;o de Amparo &#224; Pesquisa do Estado do Rio de Janeiro (FAPERJ), Conselho Nacional de Desenvolvimento Cient&#237;fico e Tecnol&#243;gico (CNPq) and Coordena&#231;&#227;o de Aperfei&#231;oamento de Pessoal de N&#237;vel Superior-Brasil (CAPES). We are grateful to Dr. Antonio Galina Filho, Dr. Monica Montero Lomeli and Dr. Claudio Masuda for the support with laboratory facilities, and Dr. Martha Sorenson for kind and valuable comments in improving the article.

The present protocol is approved by the Ethics Committee on the Use of Animals in Research, CCS/UFRJ (CEUA-101/19), and National Institutes of Health guidelines for the care and use of experimental animals. The sciatic nerve is isolated from four-month-old male C57BL/6 mice, euthanized by cervical dislocation as per the institutional guidelines. The protocol steps are optimized to avoid mitochondrial deterioration. Therefore, in this protocol, calibration of polarographic oxygen sensors was performed prior to mouse sciatic nerve tissue dissection and permeabilization.
1. Preparation of reagents
<ol>
	<li>Prepare the Tissue Preservation Buffer (TP Buffer).
	<ol>
		<li>Prepare the following reagents in ultrapure water solution: 10 mM of Ca-EGTA buffer with 0.1 &#956;M of free calcium, 20 mM of imidazole, 20 mM of taurine, 50 mM of K-MES, 0.5 mM of DTT, 6.56 mM of MgCl2, 5.77 mM of ATP, 15 mM of phosphocreatine (see Table of Materials), pH 7.1. Store at -20&#176;C.</li>
	</ol>
	</li>
	<li>Prepare the Mitochondria Respiration Buffer (MR Buffer).
	<ol>
		<li>Prepare the following reagents in ultrapure water solution: 0.5 mM of K2EGTA, 3 mM of MgCl2, 60 mM of MES, 20 mM of taurine, 10 mM of KH2PO4, 20 mM of HEPES, 110 mM of D-sucrose, 1 mg/mL of BSA (fatty acid-free) (see Table of Materials), pH 7.4. Store at -20 &#176;C.</li>
	</ol>
	</li>
	<li>Prepare saponin stock solution by dissolving 5 mg of saponin (see Table of Materials) in 1 mL of TP Buffer (step 1.1) and keep it on ice. Saponin is prepared freshly.</li>
	<li>Prepare Amplex Red by resuspending the powder (see Table of Materials) with DMSO to obtain a stock concentration of 2 mM and store at -20 &#176;C in a vial protected from light. 
	&#8203;NOTE: To avoid wearing out the probe by freezing and thawing, make small aliquots for storage no longer than 6 months25.</li>
</ol>
2. Calibration of polarographic oxygen sensors for high-resolution respirometry (HRR)
<ol>
	<li>Clean the HRR chambers of the instrument and syringes (see Table of Materials).
	<ol>
		<li>Open HRR chambers, fill with distilled water up to the top and stir for 5 min 3x. Repeat with ethanol and then again with water. Wash stoppers and syringes with water/ethanol/water 3x each.</li>
	</ol>
	</li>
	<li>Apply the following calibration settings in the HRR software (see Table of Materials).
	<ol>
		<li>In HRR software control, add the experimental temperature (37 &#176;C), the parameters for oxygen sensor (gain, 2; polarization voltage, 800 mV), and for amperometric sensor (gain, 1000; polarization voltage, 100 mV).</li>
	</ol>
	</li>
	<li>Calibrate the oxygen sensors.
	<ol>
		<li>Pipette 2.1 mL of MR Buffer (step 1.2) into each chamber. Close with the stoppers and draw air into the chamber until a bubble is formed. Stir at 37 &#176;C for 1 h in calibration mode until the oxygen flux per mass is stable.</li>
		<li>Perform an air calibration of the polarographic oxygen sensors in the software according to the manufacturer&#39;s protocol24. 
		&#8203;NOTE: The calibration step is only performed once before an experiment. Additional experiments in the same respiration medium and temperature can be performed after merely washing chambers (step 2.1).</li>
	</ol>
	</li>
</ol>
3. Dissection and permeabilization of the sciatic nerve
<ol>
	<li>Remove the sciatic nerve following the steps below.
	<ol>
		<li>Euthanize the animal by cervical dislocation after removing from its cage and gently restrained to resting on the bench.</li>
		<li>In the euthanized animal, make an incision with scissors in the lower back, starting near the spine and proceeding down the thigh toward the foot. Remove skin and muscle attached to the nerve, and then cut and remove the entire sciatic nerve.</li>
		<li>Weigh the tissue immediately and place it in a vial filled with cold TB Buffer (4 &#176;C). Perform steps 3.2-3.3 on ice. 
		NOTE: The wet tissue weight is used to normalize oxygen consumption and ROS production flow in the following steps. If the tissue cannot be weighed immediately, conserve it in cold TB Buffer. The procedure is performed in fresh tissue and must not be frozen to avoid mitochondrial damage.</li>
	</ol>
	</li>
	<li>For the tissue preparation, place the sciatic nerve in a Petri dish with enough TP Buffer to cover it. Hold one end of the nerve with forceps, and with another pair of forceps, pull out the nerve bundles horizontally. 
	NOTE: This procedure needs to be done in less than 10 min to avoid tissue deterioration. The tissue will be ready when it can be visualized as transparent foggy layers, opposite the previous white opaque tissue (Figure 1).</li>
	<li>First, transfer the splayed tissue into a small dish containing 1 mL of TP Buffer for tissue permeabilization. To start permeabilization, transfer the tissue with forceps to a dish containing 1 mL of TP Buffer and 10 &#181;L of saponin (from stock solution, step 1.3).
	<ol>
		<li>Shake in a microplate shaker gently for 30 min, then transfer the tissue with forceps to a fresh dish containing MR Buffer (1 mL) and shake gently for 10 min. Transfer the tissue with forceps to a calibrated HRR chamber.</li>
	</ol>
	</li>
</ol>
4. Oxygen consumption and ROS production determination
<ol>
	<li>Fill the HRR chambers with 2.1 mL of MR Buffer, add Amplex Red (step 1.4) to a final concentration of 5 &#181;M and peroxidase to 2 U/mL, and add the permeabilized sciatic nerve (step 3).
	<ol>
		<li>Attach the instrument&#39;s fluorescence sensors, turn off the lights in the control section of the software, and press Connect to oxygraph. In &#34;edit protocols&#34; in the software, insert the tissue weight measured in step 3.1.3.</li>
	</ol>
	</li>
	<li>Go to &#34;layout&#34;, choose the &#34;specific flux per unit sample&#34; option, and select Plots to simultaneously access the oxygen consumption readout and, if desired, the H2O2 production. Wait ~10 min. 
	NOTE: This time is required to stabilize the basal flow of oxygen consumption with no added substrate (basal). Before further injections, ensure that the oxygen flow is stabilized.</li>
	<li>Inject two pulses of H2O2, each one to a final concentration of 260 &#181;M, for later calibration in the chamber.</li>
	<li>Inject 20 &#956;L of succinate (see Table of Materials), a mitochondrial complex II substrate, to activate the mitochondrial electron transport system. 
	NOTE: Different mitochondrial substrates can be added at this point to evaluate mitochondrial function by different complexes. Representative results with varying substrates for mitochondrial complexes I and II are shown in Figure 2 and Figure 3. At this point, an increase in O2 consumption and H2O2 production, simultaneously, is observed in Figure 3.</li>
	<li>Add 20 &#181;L of adenosine diphosphate (ADP) to activate adenosine triphosphate (ATP) synthesis. 
	NOTE: ADP stimulates ATP synthesis and reduces the membrane potential. An increase in O2 consumption and a decrease in the production of H2O2 are expected to be observed26,27.</li>
	<li>In sequence, add 5 &#181;L of cytochrome c (see Table of Materials) as an indicator of membrane integrity. 
	NOTE: If the tissue is well prepared and permeabilized, cytochrome c should not increase oxygen consumption by more than 15%. If it occurs, check the troubleshooting section for recommendations.</li>
	<li>Titrate with aliquots of 0.2 &#181;g/mL of oligomycin (see Table of Materials) until no further decrease in O2 consumption is observed. 
	NOTE: Oligomycin acts by inhibiting ATP synthesis leading to a decrease in O2 flow and an enhancement in H2O2 formation favored by the high membrane potential26,27.</li>
	<li>Titrate with aliquots of 0.5 &#181;mol/L of carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) (see Table of Materials), the mitochondrial uncoupler, until it is possible to observe no further increase in O2 consumption. To finish the experiment, inject 2 &#181;l of antimycin A to a final concentration of 5 &#956;M and wait for flow stabilization. 
	NOTE: A decrease in H2O2 production is observed due to the membrane potential dissipation after injecting FCCP. Antimycin A inhibits complex III, thereby preventing the flow of electrons. Therefore, mitochondria-dependent O2 consumption is impaired, decreasing oxygen flux per mass and stimulating electron leakage, increasing H2O2 production26,27.</li>
	<li>Go to the command bar, search for &#34;multisensory&#34; in the software, click on Control &#62; Save file&#160;and Disconnect. 
	NOTE: The addition of other substrates and inhibitors can be performed according to the question under study. An example is described in the representative results. H2O2 calibration is performed after finishing the experiment, according to the manufacturer&#39;s protocol28.</li>
	<li>Open the saved file and select the &#34;Oxygen Flux per Mass&#34; trace to obtain the experimental oxygen consumption results. Manually select the window between injections by pressing Shift + Left mouse button.
	<ol>
		<li>Go to Marks &#62; Statistics to visualize the results for each injection of substrate/inhibitors/uncoupled protocol. For H2O2 production, perform the same procedure with the &#34;Amp-Slope&#34; trace. 
		NOTE: When selecting the window, avoid artifacts of volume injections by choosing a window where oxygen (or H2O2) is more stable and constant. Examples of selected windows are represented by black braces in the representative results (Figure 2 and Figure 3).</li>
	</ol>
	</li>
</ol>

The authors have nothing to disclose.

Several diseases or conditions accompanying neuropathies have mitochondrial dysfunction as a risk factor. The evaluation of mitochondrial function in peripheral nerves is essential to elucidate how the mitochondria act in these neurodegenerative conditions. The assessment of mitochondrial function is laborious due to the difficulty of the isolation method and the scarcity of material. Thus, the development of tissue permeabilization techniques that do not require the isolation of mitochondria is essential.
<p class="...

Mitochondria are essential for maintaining cell viability and perform numerous cell functions such as energy metabolism (glucose, amino acid, lipid, and nucleotide metabolism pathways). As the primary site of reactive oxygen species (ROS) production, mitochondria are central in several cell signaling processes such as apoptosis and participate in the synthesis of iron-sulfur (Fe-S) clusters, mitochondrial protein import and maturation, and maintenance of their genome and ribosomes1,2,3. The mitochondrial membrane dynamics network is controlled by fusion and fission processes, and they also have machinery for quality control and mitophagy4,5,6.
Mitochondrial dysfunction is associated with the appearance of several pathological conditions such as cancer, diabetes, and obesity7. Disturbances in mitochondrial function are detected in neurodegenerative disorders that affect the central nervous system, as in Alzheimer&#39;s disease8,9, Parkinson&#39;s disease10,11, amyotrophic lateral sclerosis12,13, and Huntington&#39;s disease14,15. In the peripheral nervous system, loss of mitochondrial function in axons is observed in immune neuropathies, such as Guillain-Barr&#233; syndrome16,17, and in association with high mitochondrial ROS production in axons, these events lead to MAP Kinase activation in Schwann cells18. This demonstrates that mitochondrial physiology may be essential not only for a site-specific cell, but for an entire tissue. In HIV-associated distal sensory polyneuropathy (HIV-DSP), mitochondria have a role in the mechanism by which the trans-activator of transcription (HIV-TAT) protein allows HIV to replicate efficiently, as well as several other roles in HIV infection pathogenesis19,20.
Evaluation of sciatic nerve mitochondrial physiology has emerged as an essential target for investigating neuropathy7,21,22. In diabetic neuropathy, proteomic and metabolomic analyses suggest that most molecular alterations in diabetes affect sciatic nerve mitochondrial oxidative phosphorylation and lipid metabolism7. These alterations also seem to be early signs of obesity-induced diabetes21. In a mouse model of chemotherapy-induced painful neuropathy, mitochondrial impairment in the sciatic nerve is detected as a decrease in oxidative phosphorylation22, and a reduction of mitochondrial complexes activities, membrane potential, and ATP content23. However, although several groups have cited mitochondrial dysfunction in neuropathies, these studies are limited to the measurements of activity in mitochondrial complexes with no preservation of the mitochondrial membranes, lacking evaluation of mitochondrial integrity or measurements of ATP content as a parameter for mitochondrial ATP production. In general, a proper assessment of mitochondrial oxygen consumption and ROS production requires the isolation of mitochondria by differential centrifugation in a percoll/sucrose gradient. Isolation of mitochondria can also be a limiting factor for sciatic nerve tissue because of the large amount of tissue needed and mitochondria loss and disruption.
The present study aims to provide a protocol to measure mitochondrial physiology as mitochondrial oxygen consumption and ROS production in the sciatic nerve, preserving mitochondrial membranes and without the need for isolating mitochondria. This protocol is adapted from oxygen consumption measurements in permeabilized muscle fibers24 by high-resolution respirometry (HRR). The advantages of this procedure are the possibility of evaluating mitochondria in small amounts of tissue such as the sciatic nerve and evaluating mitochondrial parameters in situ, thereby preserving the mitochondrial environment, structure, and bioenergetic profile, to obtain a physiologically trustworthy result. The mitochondrial respiratory states were determined with substrates and inhibitors after sciatic nerve permeabilization to properly assess mitochondrial bioenergetics and cytochrome c coefficient for mitochondrial membrane integrity, providing a guide for steps of the mitochondrial electron transport system (ETS) evaluation and calculation of essential parameters. This study can provide tools for answering questions in pathophysiological mechanisms in which sciatic nerve metabolism is implicated, such as peripheral neuropathies.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Adenosine 5&#39; triphosphate dissodium salt hydrate</td>
			<td>Sigma-Aldrich</td>
			<td>A26209</td>
			<td></td>
		</tr>
		<tr>
			<td>Adenosine 5&#8242;-diphosphate sodium salt</td>
			<td>Sigma-Aldrich</td>
			<td>A2754</td>
			<td></td>
		</tr>
		<tr>
			<td>Amplex Red Reagent</td>
			<td>Thermo Fisher scientific</td>
			<td>A12222</td>
			<td>Amplex Red is prepared in DMSO accordindly with product datasheet</td>
		</tr>
		<tr>
			<td>Antimycin A (from Streptomyces sp.)</td>
			<td>Sigma-Aldrich</td>
			<td>A8674</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A7030</td>
			<td>heat shock fraction, protease free, fatty acid free, essentially globulin free, pH 7, &#8805;98%</td>
		</tr>
		<tr>
			<td>Calcium carbonate</td>
			<td>Sigma-Aldrich</td>
			<td>C6763</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP)</td>
			<td>Sigma-Aldrich</td>
			<td>C2920</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytochrome c</td>
			<td>Sigma-Aldrich</td>
			<td>C7752</td>
			<td>(from equine heart; small hemeprotein)</td>
		</tr>
		<tr>
			<td>DataLab version 5.1.1.91</td>
			<td>OROBOROS INSTRUMENTS, Austria</td>
			<td></td>
			<td>Copyright (c) 2002 - 13 by Dr. Erich Gnaiger</td>
		</tr>
		<tr>
			<td>Digital orbital microplate shaker 120V</td>
			<td>Thermo Fisher scientific</td>
			<td>88882005</td>
			<td></td>
		</tr>
		<tr>
			<td>DL-Dithiothreitol</td>
			<td>Sigma-Aldrich</td>
			<td>43819</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA sodium salt</td>
			<td>Sigma-Aldrich</td>
			<td>E8145</td>
			<td></td>
		</tr>
		<tr>
			<td>Hamilton syringe</td>
			<td>Sigma-Aldrich</td>
			<td>HAM80075</td>
			<td>10 uL, 25 uL and 50 uL</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrogen peroxide solution 30% W/W</td>
			<td>Merck</td>
			<td>H1009</td>
			<td></td>
		</tr>
		<tr>
			<td>Imidazole</td>
			<td>Sigma-Aldrich</td>
			<td>I2399</td>
			<td></td>
		</tr>
		<tr>
			<td>L-(&#8722;)-Malic acid</td>
			<td>Sigma-Aldrich</td>
			<td>M7397</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride hexahydrate</td>
			<td>Sigma-Aldrich</td>
			<td>M2393</td>
			<td></td>
		</tr>
		<tr>
			<td>MES sodium salt</td>
			<td>Sigma-Aldrich</td>
			<td>M3885</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-dissecting forceps, curved</td>
			<td>Sigma-Aldrich</td>
			<td>F4142</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-dissecting forceps, straight</td>
			<td>Sigma-Aldrich</td>
			<td>F4017</td>
			<td></td>
		</tr>
		<tr>
			<td>O2K - Filter set Amplex Red</td>
			<td>OROBOROS INSTRUMENTS, Austria</td>
			<td>44321-01</td>
			<td>Fasching M, Sumbalova Z, Gnaiger E (2013) O2k-Fluorometry: HRR and H2O2 production in mouse brain mitochondria. Mitochondr Physiol Network 17.17.</td>
		</tr>
		<tr>
			<td>O2K - Fluorescence LED2 - module component Fluorscence-Sensor Green</td>
			<td>OROBOROS INSTRUMENTS, Austria</td>
			<td>44210-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Oligomycin</td>
			<td>Sigma-Aldrich</td>
			<td>O4876</td>
			<td>(from Streptomyces diastatochromogenes; mixture of oligomycins A, B, and C</td>
		</tr>
		<tr>
			<td>OROBOROS Oxygraph-2k</td>
			<td>OROBOROS INSTRUMENTS, Austria</td>
			<td>http://www.oroboros.at</td>
			<td></td>
		</tr>
		<tr>
			<td>Palmitoylcarnitine (Palmitoyl-DL-carnitine-HCl)</td>
			<td>Sigma-Aldrich</td>
			<td>P4509</td>
			<td></td>
		</tr>
		<tr>
			<td>Peroxidase from horseradish</td>
			<td>Sigma-Aldrich</td>
			<td>P8375</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dishes, polystyrene</td>
			<td>MERCK</td>
			<td>P5606</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphocreatine disodium salt hydrate</td>
			<td>Sigma-Aldrich</td>
			<td>P7936</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium dihydrogen phosphate monobasic</td>
			<td>Sigma-Aldrich</td>
			<td>PHR1330</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td>221473</td>
			<td></td>
		</tr>
		<tr>
			<td>Rotenone</td>
			<td>Sigma-Aldrich</td>
			<td>R8875</td>
			<td></td>
		</tr>
		<tr>
			<td>Saponin</td>
			<td>Sigma-Aldrich</td>
			<td>SAE0073</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Sigma-Aldrich</td>
			<td>P5280</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium succinate dibasic hexahydrate</td>
			<td>Sigma-Aldrich</td>
			<td>S2378</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>S9378</td>
			<td></td>
		</tr>
		<tr>
			<td>Taurine</td>
			<td>Sigma-Aldrich</td>
			<td>T0625</td>
			<td></td>
		</tr>
	</tbody>
</table>

assessing mitochondrial function in sciatic nerve by high-resolution respirometry

Mitochondrial dysfunction in peripheral nerves accompanies several diseases associated with peripheral neuropathy, which can be triggered by multiple causes, including autoimmune diseases, diabetes, infections, inherited disorders, and tumors. Assessing mitochondrial function in mouse peripheral nerves can be challenging due to the small sample size, a limited number of mitochondria present in the tissue, and the presence of a myelin sheath. The technique described in this work minimizes these challenges by using a unique permeabilization protocol adapted from one used for muscle fibers, to assess sciatic nerve mitochondrial function instead of isolating the mitochondria from the tissue. By measuring fluorimetric reactive species production with Amplex Red/Peroxidase and comparing different mitochondrial substrates and inhibitors in saponin-permeabilized nerves, it was possible to detect mitochondrial respiratory states, reactive oxygen species (ROS), and the activity of mitochondrial complexes simultaneously. Therefore, the method presented here offers advantages compared to the assessment of mitochondrial function by other techniques.

The mitochondrial oxygen consumption by the permeabilized sciatic nerve is represented in Figure 2. The red trace represents the O2 flux per unit mass in pmol/s.mg. After recording a basal oxygen consumption with endogenous substrates (routine respiration), succinate (SUCC) is injected to record complex II (succinate dehydrogenase)-driven respiration, resulting in an increase in the oxygen consumption rate. In sequence, a saturating concentration of ADP is added, activating ATP sy...

Watch this Scientific Journal Video about Assessing Mitochondrial Function in Sciatic Nerve by High-Resolution Respirometry at JoVE.com

Assessing Mitochondrial Function in Sciatic Nerve by High-Resolution Respirometry

High-resolution respirometry coupled to fluorescence sensors determines mitochondrial oxygen consumption and reactive oxygen species (ROS) generation. The present protocol describes a technique to assess mitochondrial respiratory rates and ROS production in the permeabilized sciatic nerve.

Mitochondrial dysfunction in peripheral nerves accompanies several diseases associated with peripheral neuropathy, which can be triggered by multiple ...

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Biochemistry

2.3K Views.  Universidade Federal do Rio de Janeiro. High-resolution respirometry coupled to fluorescence sensors determines mitochondrial oxygen consumption and reactive oxygen species (ROS) generation. The present protocol describes a technique to assess mitochondrial respiratory rates and ROS production in the permeabilized sciatic nerve. 

Video: Assessing Mitochondrial Function in Sciatic Nerve by High-Resolution Respirometry

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 ...

High-resolution Single Particle Analysis from Electron Cryo-microscopy Images Using SPHIRE

SPHIRE (SPARX for High-Resolution Electron Microscopy) is a novel open-source, user-friendly software suite for the semi-automated processing of single particle electron cryo-microscopy (cryo-EM) data. The protocol presented here describes in detail how to obtain a near-atomic resolution structure starting from cryo-EM micrograph movies by guiding users through all steps of the single particle structure determination pipeline. These steps are controlled from the new SPHIRE graphical user interface and require minimum user intervention. Using this protocol, a 3.5 &#197; structure of TcdA1, a Tc toxin complex from Photorhabdus luminescens, was derived from only 9500 single particles. This streamlined approach will help novice users without extensive processing experience and a priori structural information, to obtain noise-free and unbiased atomic models of their purified macromolecular complexes in their native state.

This paper presents a protocol for processing cryo-EM images using the software suite SPHIRE. The present protocol can be applied for nearly all single particle EM projects that target near-atomic resolution.

SPHIRE (SPARX for High-Resolution Electron Microscopy) is a novel open-source, user-friendly software suite for the semi-automated processing of single ...

Simultaneous Measurement of Superoxide/Hydrogen Peroxide and NADH Production by Flavin-containing Mitochondrial Dehydrogenases

It has been reported that mitochondria can contain up to 12 enzymatic sources of reactive oxygen species (ROS). A majority of these sites include flavin-dependent respiratory complexes and dehydrogenases that produce a mixture of superoxide (O2&#9679;-) and hydrogen peroxide (H2O2). Accurate quantification of the ROS-producing potential of individual sites in isolated mitochondria can be challenging due to the presence of antioxidant defense systems&#160;and side reactions that also form O2&#9679;-/H2O2. Use&#160;of nonspecific inhibitors that can disrupt mitochondrial bioenergetics can also compromise measurements by altering&#160;ROS release from other sites of production. Here, we present an easy method for the simultaneous measurement of H2O2 release and nicotinamide adenine dinucleotide (NADH) production by purified flavin-linked dehydrogenases. For our purposes here, we have used purified pyruvate dehydrogenase complex (PDHC) and &#945;-ketoglutarate dehydrogenase complex (KGDHC) of porcine heart origin as examples. This method allows for an accurate measure of native H2O2 release rates by individual sites of production by eliminating other potential sources of ROS and antioxidant systems. In addition, this method allows for a direct comparison of the relationship between H2O2 release and enzyme activity and the screening of the effectiveness and selectivity of inhibitors for ROS production. Overall, this approach can allow for the in-depth assessment of native rates of ROS release for individual enzymes prior to conducting more sophisticated experiments with isolated mitochondria or permeabilized muscle fiber.

Mitochondria contain several flavin-dependent enzymes that can produce reactive oxygen species (ROS). Monitoring ROS release from individual sites in mitochondria is challenging due to unwanted side reactions. We present an easy, inexpensive method for direct assessment of native rates for ROS release using purified flavoenzymes and microplate fluorometry.

It has been reported that mitochondria can contain up to 12 enzymatic sources of reactive oxygen species (ROS). A majority of these sites include ...

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 ...

Human Peripheral Blood Neutrophil Isolation for Interrogating the Parkinson's Associated LRRK2 Kinase Pathway by Assessing Rab10 Phosphorylation

The leucine rich repeat kinase 2 (LRRK2) is the most frequently mutated gene in hereditary Parkinson&#8217; disease (PD) and all pathogenic LRRK2 mutations result in hyperactivation of its kinase function. Here, we describe an easy and robust assay to quantify LRRK2 kinase pathway activity in human peripheral blood neutrophils by measuring LRRK2-controlled phosphorylation of one of its physiological substrates, Rab10 at threonine 73. The immunoblotting analysis described requires a fully selective and phosphospecific antibody that recognizes the Rab10 Thr73 epitope phosphorylated by LRRK2, such as the MJFF-pRab10 rabbit monoclonal antibody. It uses human peripheral blood neutrophils, because peripheral blood is easily accessible and neutrophils are an abundant and homogenous constituent. Importantly, neutrophils express relatively high levels of both LRRK2 and Rab10. A potential drawback of neutrophils is their high intrinsic serine protease activity, which necessitates the use of very potent protease inhibitors such as the organophosphorus neurotoxin diisopropylfluorophosphate (DIFP) as part of the lysis buffer. Nevertheless, neutrophils are a valuable resource for research into LRRK2 kinase pathway activity in vivo and should be considered for inclusion into PD biorepository collections.

Mutations in the leucine rich repeat kinase 2 gene (LRRK2) cause hereditary Parkinson&#8217;s disease. We have developed an easy and robust method for assessing LRRK2-controlled phosphorylation of Rab10 in human peripheral blood neutrophils. This may help identify individuals with increased LRRK2 kinase pathway activity.

The leucine rich repeat kinase 2 (LRRK2) is the most frequently mutated gene in hereditary Parkinson&#8217; disease (PD) and all pathogenic LRRK2 ...

Interactions with and Membrane Permeabilization of Brain Mitochondria by Amyloid Fibrils

A growing body of evidence indicates that membrane permeabilization, including internal membranes such as mitochondria, is a common feature and primary mechanism of amyloid aggregate-induced toxicity in neurodegenerative diseases. However, most reports describing the mechanisms of membrane disruption are based on phospholipid model systems, and studies directly targeting events occurring at the level of biological membranes are rare. Described here is a model for studying the mechanisms of amyloid toxicity at the membrane level. For mitochondrial isolation, density gradient medium is used to obtain preparations with minimal myelin contamination. After mitochondrial membrane integrity confirmation, the interaction of amyloid fibrils arising from &#945;-synuclein, bovine insulin, and hen egg white lysozyme (HEWL) with rat brain mitochondria, as an in vitro biological model, is investigated. The results demonstrate that treatment of brain mitochondria with fibrillar assemblies can cause different degrees of membrane permeabilization and ROS content enhancement. This indicates structure-dependent interactions between amyloid fibrils and mitochondrial membrane. It is suggested that biophysical properties of amyloid fibrils and their specific binding to mitochondrial membranes may provide explanations for some of these observations.

Provided here is a protocol for investigating the interactions between native form, prefibrillar, and mature amyloid fibrils of different peptides and proteins with mitochondria isolated from different tissues and various areas of the brain.

A growing body of evidence indicates that membrane permeabilization, including internal membranes such as mitochondria, is a common feature and primary ...

High-Resolution Complexome Profiling by Cryoslicing BN-MS Analysis

Proteins generally exert biological functions through interactions with other proteins, either in dynamic protein assemblies or as a part of stably formed complexes. The latter can be elegantly resolved according to molecular size using native polyacrylamide gel electrophoresis (BN-PAGE). Coupling of such separations to sensitive mass spectrometry (BN-MS) has been well-established and theoretically allows for exhaustive assessment of the extractable complexome in biological samples. However, this approach is rather laborious and provides limited complex size resolution and sensitivity. Also, its application has remained restricted to abundant mitochondrial and plastid proteins. Thus, for a majority of proteins, information regarding integration into stable protein complexes is still lacking. Presented here is an optimized approach for complexome profiling comprising preparative-scale BN-PAGE separation, sub-millimeter sampling of broad gel lanes by cryomicrotome slicing, and mass spectrometric analysis with label-free protein quantification. The procedures and tools for critical steps are described in detail. As an application, the report describes complexome analysis of a solubilized endosome-enriched membrane fraction from mouse kidneys, with 2,545 proteins profiled in total. The results demonstrate identification of uniform, low-abundance membrane proteins such as intracellular ion channels as well as high resolution, complex protein assembly patterns, including glycosylation isoforms. The results are in agreement with independent biochemical analyses. In summary, this methodology allows for comprehensive and unbiased identification of protein (super)complexes and their subunit composition, providing a basis for investigating stoichiometry, assembly, and interaction dynamics of protein complexes in any biological system.

A versatile cryoslicing BN-MS protocol using a microtome is presented for high-resolution complexome profiling.

Proteins generally exert biological functions through interactions with other proteins, either in dynamic protein assemblies or as a part of stably formed ...

Routine Collection of High-Resolution cryo-EM Datasets Using 200 KV Transmission Electron Microscope

Cryo-electron microscopy (cryo-EM) has been established as a routine method for protein structure determination during the past decade, taking an ever-increasing share of published structural data. Recent advances in TEM technology and automation have boosted both the speed of data collection and quality of acquired images while simultaneously decreasing the required level of expertise for obtaining cryo-EM maps at sub-3 &#197; resolutions. While most of such high-resolution structures have been obtained using state-of-the-art 300 kV cryo-TEM systems, high-resolution structures can be also obtained with 200 kV cryo-TEM systems, especially when equipped with an energy filter. Additionally, automation of microscope alignments and data collection with real-time image quality assessment reduces system complexity and assures optimal microscope settings, resulting in increased yield of high-quality images and overall throughput of data collection. This protocol demonstrates the implementation of recent technological advances and automation features on a 200 kV cryo-transmission electron microscope and shows how to collect data for the reconstruction of 3D maps that are sufficient for de novo atomic model building. We focus on best practices, critical variables, and common issues that must be considered to enable the routine collection of such high-resolution cryo-EM datasets. Particularly the following essential topics are reviewed in detail: i) automation of microscope alignments, ii) selection of suitable areas for data acquisition, iii) optimal optical parameters for high-quality, high-throughput data collection, iv) energy filter tuning for zero-loss imaging, and v) data management and quality assessment. Application of the best practices and improvement of achievable resolution using an energy filter will be demonstrated on the example of apo-ferritin that was reconstructed to 1.6 &#197;, and Thermoplasma acidophilum 20S proteasome reconstructed to 2.1-&#197; resolution using a 200 kV TEM equipped with an energy filter and a direct electron detector.

High-resolution cryo-EM maps of macromolecules can be also achieved by using 200 kV TEM microscopes. This protocol shows the best practices for setting accurate optics alignments, data acquisition schemes, and selection of imaging areas that are all essential for the successful collection of high-resolution datasets using a 200 kV TEM.

Cryo-electron microscopy (cryo-EM) has been established as a routine method for protein structure determination during the past decade, taking an ...

High-Resolution Neutron Spectroscopy to Study Picosecond-Nanosecond Dynamics of Proteins and Hydration Water

Neutron scattering offers the possibility to probe the dynamics within samples for a wide range of energies in a nondestructive manner and without labeling other than deuterium. In particular, neutron backscattering spectroscopy records the scattering signals at multiple scattering angles simultaneously and is well suited to study the dynamics of biological systems on the ps-ns timescale. By employing D2O-and possibly deuterated buffer components-the method allows monitoring of both center-of-mass diffusion and backbone and side-chain motions (internal dynamics) of proteins in liquid state.
Additionally, hydration water dynamics can be studied by employing powders of perdeuterated proteins hydrated with H2O. This paper presents the workflow employed on the instrument IN16B at the Institut Laue-Langevin (ILL) to investigate protein and hydration water dynamics. The preparation of solution samples and hydrated protein powder samples using vapor exchange is explained. The data analysis procedure for both protein and hydration water dynamics is described for different types of datasets (quasielastic spectra or fixed-window scans) that can be obtained on a neutron backscattering spectrometer.
The method is illustrated with two studies involving amyloid proteins. The aggregation of lysozyme into &#181;m sized spherical aggregates-denoted particulates-is shown to occur in a one-step process on the space and time range probed on IN16B, while the internal dynamics remains unchanged. Further, the dynamics of hydration water of tau was studied on hydrated powders of perdeuterated protein. It is shown that translational motions of water are activated upon the formation of amyloid fibers. Finally, critical steps in the protocol are discussed as to how neutron scattering is positioned regarding the study of dynamics with respect to other experimental biophysical methods.

Neutron backscattering spectroscopy offers a nondestructive and label-free access to the ps-ns dynamics of proteins and their hydration water. The workflow is presented with two studies on amyloid proteins: on the time-resolved dynamics of lysozyme during aggregation and on the hydration water dynamics of tau upon fiber formation.

Neutron scattering offers the possibility to probe the dynamics within samples for a wide range of energies in a nondestructive manner and without ...

Advancing High-Resolution Imaging of Virus Assemblies in Liquid and Ice

Interest in liquid-electron microscopy (liquid-EM) has skyrocketed in recent years as scientists can now observe real-time processes at the nanoscale. It is extremely desirable to pair high-resolution cryo-EM information with dynamic observations as many events occur at rapid timescales - in the millisecond range or faster. Improved knowledge of flexible structures can also assist in the design of novel reagents to combat emerging pathogens, such as SARS-CoV-2. More importantly, viewing biological materials in a fluid environment provides a unique glimpse of their performance in the human body. Presented here are newly developed methods to investigate the nanoscale properties of virus assemblies in liquid and vitreous ice. To accomplish this goal, well-defined samples were used as model systems. Side-by-side comparisons of sample preparation methods and representative structural information are presented. Sub-nanometer features are shown for structures resolved in the range of ~3.5-&#197;-10 &#197;. Other recent results that support this complementary framework include dynamic insights of vaccine candidates and antibody-based therapies imaged in liquid. Overall, these correlative applications advance our ability to visualize molecular dynamics, providing a unique context for their use in human health and disease.

Here protocols are described to prepare virus assemblies suitable for liquid-EM and cryo-EM analysis at the nanoscale using transmission electron microscopy.

Interest in liquid-electron microscopy (liquid-EM) has skyrocketed in recent years as scientists can now observe real-time processes at the nanoscale. It ...

Assessing Mitochondrial Function in Sciatic Nerve by High-Resolution Respirometry

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Chapters in this video

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

High-resolution Single Particle Analysis from Electron Cryo-microscopy Images Using SPHIRE

Simultaneous Measurement of Superoxide/Hydrogen Peroxide and NADH Production by Flavin-containing Mitochondrial Dehydrogenases

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

Human Peripheral Blood Neutrophil Isolation for Interrogating the Parkinson's Associated LRRK2 Kinase Pathway by Assessing Rab10 Phosphorylation

Interactions with and Membrane Permeabilization of Brain Mitochondria by Amyloid Fibrils

High-Resolution Complexome Profiling by Cryoslicing BN-MS Analysis

Routine Collection of High-Resolution cryo-EM Datasets Using 200 KV Transmission Electron Microscope

High-Resolution Neutron Spectroscopy to Study Picosecond-Nanosecond Dynamics of Proteins and Hydration Water

Advancing High-Resolution Imaging of Virus Assemblies in Liquid and Ice