We thank Dr. R. Mart&#237;nez-de-Mena, M. M. Mu&#241;oz-Hernandez, A., Dr C. Jimenez and E. R. Mart&#237;nez-Jimenez for technical assistance. This study was supported by MICIN: RTI2018-099357-B-I00 and HFSP (RGP0016/2018). The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia, Innovaci&#243;n y Universidades (MCNU) and the Pro CNIC Foundation and is a Severo Ochoa Center of Excellence (SEV-2015-0505). Figure 2 created with BioRender.com.

All animal experiments were performed following the Guide for the Care and Use of Laboratory Animals and were approved by the institutional ethics committee of the Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC), Spain, in accordance with the European Union Directive of 22 September 2010 (2010/63/UE) and with the Spanish Royal Decree of 1 February 2013 (53/2013). All efforts were made to minimize the number of animals used and their suffering.
NOTE: This comparative assay to study the segmentation of mitochondrial CoQ pools is described as follows:
1. Protein quantification
<ol>
	<li>Freeze and thaw the isolated mitochondria26 from a wild-type mouse liver three times (i.e., mitochondrial membranes) before experimentation to make the organelles permeable to the reaction substrates.</li>
	<li>Quantify the protein amount of the isolated mitochondria sample by Bradford or Bicinchoninic acid (BCA) methods. In the case of Bradford, add 2 &#181;L of sample into 1 mL of 1x Bradford reagent.</li>
	<li>Split the sample into four subsamples of 20 &#181;g each (namely: A, B, C, D; Figure 2A).</li>
</ol>
2. Measuring CI+CIII activity
NOTE: This part of the protocol uses samples A and B to measure CI+CIII activity (Figure 2B).
<ol>
	<li>Split samples A and B into two subsamples of 10 &#181;g each (namely A1, A2, B1, and B2). Mix each of the subsamples in a 1 mL cuvette with 30 &#181;L of cyt c (10 mg/mL), 10 &#181;L of 100 mM malonate, and add preheated C1/C2 buffer (Table 1) at 37 &#176;C up to 980 &#181;L (979 &#181;L for cuvettes A2 and B2). 
	CAUTION: This step involves the use of the toxic reagents malonate and potassium cyanide. 
	NOTE: cyt c (10 mg/mL) must be prepared fresh by mixing 10 mg of cyt c in 1 mL of 10 mM K2HPO4 solution, pH adjusted to 7.2, and it must be maintained in ice throughout the experiment.</li>
	<li>Add 10 &#181;L of 1 M KCl in cuvettes A1 and A2, and add 10 &#181;L of 1 M NaCl in cuvettes B1 and B2.</li>
	<li>Add 1 &#181;L of 1 mM rotenone into the cuvette containing subsamples A2 and B2. 
	CAUTION: This step involves the use of the toxic reagent rotenone.</li>
	<li>Right before the measurement, add 10 &#181;L of NADH (10 mM) into all cuvettes. 
	NOTE: The 10 &#181;L is preferably added on the step of the cuvette, so the reaction starts upon mixing.</li>
	<li>Mix the cuvette by carefully flipping it three times. Place it in the absorbance cuvette reader (UV/VISJASCO spectrophotometer).</li>
	<li>Click on Measure &#62; Parameters &#62; General and set the measurement parameters at Wavelength: 550 nm, and Time: 4 min of reading; press Accept and Start buttons to begin the experiment.</li>
	<li>At the end of the measurement, save the slope comprising the linear increase of absorbance by clicking on File and Save As. The slope can also be collected manually.</li>
</ol>
3.&#160;Measuring CII+CIII activity
NOTE: This part of the protocol uses samples C and D to measure CII+CIII activity (Figure 2C).
<ol>
	<li>Split samples C and D into two subsamples of 10 &#181;g each (namely C1, C2, D1, and D2). Mix each of the subsamples in a 1 mL cuvette with 30 &#181;L of cyt c (10 mg/mL), 1 &#181;L of 1 mM rotenone, and add preheated C1/C2 buffer at 37 &#176;C up to 980 &#181;L (970 &#181;L for cuvettes C2 and D2). 
	CAUTION: This step involves the use of the toxic reagents potassium cyanide and rotenone. 
	NOTE: cyt c (10 mg/mL) must be prepared fresh by mixing 10 mg of cyt c in 1 mL of 10 mM K2HPO4 solution, pH adjusted to 7.2, and it must be maintained in ice throughout the experiment.</li>
	<li>Add 10 &#181;L of 1 M KCl in cuvettes C1 and C2, and add 10 &#181;L of 1 M NaCl in cuvettes D1 and D2.</li>
	<li>Add 1 &#181;L of 1 mM antimycin A into the cuvette containing subsamples C2 and D2. 
	CAUTION: This step involves the use of the toxic reagent antimycin A.</li>
	<li>Right before the measurement, add 10 &#181;L of succinate (1 M) into all cuvettes. 
	NOTE: The 10 &#181;L is preferably added on the step of the cuvette, so the reaction starts upon mixing.</li>
	<li>Mix the cuvette carefully, flipping it three times. Place it in the absorbance cuvette reader (UV/VIS spectrophotometer).</li>
	<li>Click on Measure &#62; Parameters &#62; General and set the measurement parameters at Wavelength: 550 nm, and Time: 4 min of reading; press Accept and Start buttons to begin the experiment.</li>
	<li>At the end of the measurement, save the slope comprising the linear increase of absorbance by clicking on File and Save As. The slope can also be collected manually.</li>
</ol>

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The authors declare no conflicts of interest.

Though this protocol represents a very straightforward procedure to identify the existence of the partially segmented CoQ pools, there are a few critical steps to take into account. Substrates (i.e., NADH or succinate) are preferably added last since autooxidation of these compounds may occur. Cuvette&#39;s flipping must be careful in order to avoid the formation of bubbles which may interfere with the reading.
In addition, the present technique presents a few limitations which are worth menti...

Mitochondrial oxidative phosphorylation system (OXPHOS) is the main pathway driving adenosine triphosphate (ATP) synthesis, reactive oxygen species (ROS) production, and consumption of reducing equivalents, such as nicotinamide adenine dinucleotide (NADH) or succinate, by mitochondria. OXPHOS system is composed of five protein complexes: Complex I (CI) oxidizes NADH and reduces CoQ into ubiquinol (CoQH2). Complex II (CII) oxidizes succinate into fumarate and reduces CoQ into CoQH2. Complex III (CIII) oxidizes CoQH2 back into CoQ, reducing cytochrome c (cyt c). Finally, complex IV (CIV) oxidizes cyt c and reduces oxygen to water. This oxidoreduction chain, the so-called electron transport chain (mETC), is coupled to pumping of H+ across the IMM, which creates an electrochemical gradient used by complex V (CV) to phosphorylate adenosine diphosphate (ADP) into ATP.
mETC complexes can either be alone in the IMM or assemble into quaternary structures called supercomplexes. CIV can assemble with CIII, forming the III2+IV or Q-respirasome (as it is able to respire in the presence of CoQH2)1,2,3 or forming homodimers or homooligomers4. CIII can interact with CI, forming the supercomplex I+III25. Finally, CI is also able to interact with the Q-respirasome, building the I+III2+IV or N-respirasome (as it can respire consuming NADH)1,6,7,8,9,10.
CoQ and cyt c are mobile electron carriers in charge of transferring electrons from CI/CII to CIII, and from CIII to CIV, respectively. Whether or not supercomplexes impose a functional local restriction for these carriers has been a matter of intense debate through the last two decades2,7,11,12,13,14,15,16,17. However, several independent groups have demonstrated that CoQ and cyt c can be functionally segmented into pools in the IMM. In respect of the CoQ, it can be functionally segmented into a specific CoQ pool for CI (CoQNAD) and another pool dedicated to FAD-dependent enzymes (CoQFAD)1,7,12,18,19. However, in order to differentiate the existence of partially segmented functional CoQ pools, the overexpression of the alternative oxidase (AOX) and the generation of specific mtDNA mutants, which can assemble CI in the absence of CIII, were required1,19,20.
The mechanism of reactive oxygen species (ROS) production during hypoxia was unknown until recently. Upon acute hypoxia, CI undergoes the active/deactive (A/D) transition, which involves the decrease in its H+ pumping NADH-CoQ oxidoreductase activity. Such a decrease in H+ pumping acidifies the mitochondrial matrix and partially dissolves the calcium-phosphate precipitates in the mitochondrial matrix, releasing soluble Ca2+. This increase in soluble Ca2+ activates the Na+/Ca2+ exchanger (NCLX), which extrudes Ca2+ in exchange for Na+. Mitochondrial Na+ increase interacts with phospholipids in the inner side of the IMM, decreasing its fluidity and CoQ transfer between CII and CIII, finally producing superoxide anion, a redox signal21. Interestingly, CoQ transfer was only diminished between CII and CIII, but not between CI and CIII, highlighting that 1) Na+ was able to modulate only one of the existing CoQ pools in the mitochondria; 2) there exists functionally differentiated CoQ pools in the IMM. Thus, a widely used protocol for the study of mitochondrial enzyme activities can be used to assess the existence of the mentioned CoQ pools.
The current protocol is based on the measurement of the reduction of oxidized cyt c, the substrate of CIII, by absorbance in the presence of succinate (i.e., CII substrate) or NADH (i.e., CI substrate). The same sample is divided into two, one of which will be treated with KCl, and the other one with the same concentration of NaCl. In this way, given that Na+ decreases IMM fluidity, if CoQ existed in a unique pool in the IMM, both CI+CIII and CII+CIII would decrease in the presence of Na+. However, if CoQ existed in partially segmented functional CoQ pools, the effect of Na+ would mostly (or only) be evident on the CII+CIII activity, but not on the CI+CIII. As recently published21, Na+ only affects the CoQ transfer between CII and CIII (Figure 1C,D), but not between CI and CIII (Figure 1A,B).
This protocol, together with a panoply of techniques, has been used to confirm the existence of partially segmented functional CoQ pools in the IMM, one dedicated to CI (i.e., CoQNAD), and another dedicated to FAD-linked enzymes (i.e., CoQFAD)1,3,7; an observation that, though it continues to be debated22, has been corroborated independently by several groups7,19. Thus, the superassembly of CI into supercomplexes impacts on the local mobility of CoQ, facilitating its usage by the CIII within the supercomplex1,7,13,14,23,24,25.

<table><tbody><tr><td>Antimycin A</td><td>Sigma-Aldrich</td><td>A8674</td><td></td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Sigma-Aldrich</td><td>10775835001</td><td></td></tr><tr><td>Bradford protein assay</td><td>Bio-Rad</td><td>5000001</td><td></td></tr><tr><td>Cytochrome c from equine heart</td><td>Sigma-Aldrich</td><td>C7752</td><td></td></tr><tr><td>K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>P3786</td><td></td></tr><tr><td>KCl</td><td>Sigma-Aldrich</td><td>P3911</td><td></td></tr><tr><td>Malonic acid</td><td>Sigma-Aldrich</td><td>M1296</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>M8266</td><td></td></tr><tr><td>NaCl</td><td>Sigma-Aldrich</td><td>S9888</td><td></td></tr><tr><td>NADH</td><td>Roche</td><td>10107735001</td><td></td></tr><tr><td>Potassium cyanide</td><td>Sigma-Aldrich</td><td>207810</td><td></td></tr><tr><td>Rotenone</td><td>Sigma-Aldrich</td><td>R8875</td><td></td></tr><tr><td>Spectra Manager software</td><td>JASCO</td><td>version 2</td><td></td></tr><tr><td>Spectrophotometer</td><td>UV/VISJASCO</td><td></td><td></td></tr><tr><td>Succinate</td><td>Sigma-Aldrich</td><td>398055</td><td></td></tr></tbody></table>

inner mitochondrial membrane sensitivity to na+ reveals partially segmented functional coq pools

Ubiquinone (CoQ) pools in the inner mitochondrial membrane (IMM) are partially segmented to either complex I or FAD-dependent enzymes. Such subdivision can be easily assessed by a comparative assay using NADH or succinate as electron donors in frozen-thawed mitochondria, in which cytochrome c (cyt c) reduction is measured. The assay relies on the effect of Na+ on the IMM, decreasing its fluidity. Here, we present a protocol to measure NADH-cyt c oxidoreductase activity and succinate-cyt c oxidoreductase activities in the presence of NaCl or KCl. The reactions, which rely on the mixture of reagents in a cuvette in a stepwise manner, are measured spectrophotometrically during 4 min in the presence of Na+ or K+. The same mixture is performed in parallel in the presence of the specific enzyme inhibitors in order to subtract the unspecific change in absorbance. NADH-cyt c oxidoreductase activity does not decrease in the presence of any of these cations. However, succinate-cyt c oxidoreductase activity decreases in the presence of NaCl. This simple experiment highlights: 1) the effect of Na+ in decreasing IMM fluidity and CoQ transfer; 2) that supercomplex I+III2 protects ubiquinone (CoQ) transfer from being affected by lowering IMM fluidity; 3) that CoQ transfer between CI and CIII is functionally different from CoQ transfer between CII and CIII. These facts support the existence of functionally differentiated CoQ pools in the IMM and show that they can be regulated by the changing Na+ environment of mitochondria.

Typical results from this protocol are represented below (Figure 3). As reduced cyt c absorbance locates at 550 nm, all uninhibited subsamples must show an increase in the absorbance at 550 nm. Inhibited subsamples ideally show a flat-line or slightly increasing slope (Figure 3). Slopes from inhibited subsamples are to be subtracted from uninhibited subsamples.
Samples A and B, both corrected by their correspondent inhibition and whic...

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Inner Mitochondrial Membrane Sensitivity to Na+ Reveals Partially Segmented Functional CoQ Pools

This protocol describes a comparative assay, using mitochondrial complex activities CI+CIII and CII+CIII in the presence or absence of Na+, to study the existence of partially segmented functional CoQ pools.

Inner Mitochondrial Membrane Sensitivity to Na+ Reveals Partially Segmented Functional CoQ Pools

Ubiquinone (CoQ) pools in the inner mitochondrial membrane (IMM) are partially segmented to either complex I or FAD-dependent enzymes. Such subdivision ...

inner-mitochondrial-membrane-sensitivity-to-na-reveals-partially

Research

JoVE Journal

Biochemistry

1.8K Views.  Fundación Centro Nacional de Investigaciones Cardiovasculares Carlos III CNIC. Dit protocol benadrukt de rol van natrium als tweede boodschapper en toont ook het bestaan van gedeeltelijk gedifferentieerde ubiquinolpools. Deze techniek demonstreert een uiterst eenvoudige benadering voor de studie van ubiquinolpools die anders zeer specifieke celmodellen vereist. Deze benadering kan worden gebruikt voor de studie van elektronenoverdracht in andere membranen, met name van enzymen in lipidedruppels.Splits de monsters in vier submonsters van elk 20 microgram en label ...

Video: Inner Mitochondrial Membrane Sensitivity to Na+ Reveals Partially Segmented Functional CoQ Pools

Analyzing Supercomplexes of the Mitochondrial Electron Transport Chain with Native Electrophoresis, In-gel Assays, and Electroelution

The mitochondrial electron transport chain (ETC) transduces the energy derived from the breakdown of various fuels into the bioenergetic currency of the cell, ATP. The ETC is composed of 5 massive protein complexes, which also assemble into supercomplexes called respirasomes (C-I, C-III, and C-IV) and synthasomes (C-V) that increase the efficiency of electron transport and ATP production. Various methods have been used for over 50 years to measure ETC function, but these protocols do not provide information on the assembly of individual complexes and supercomplexes. This protocol describes the technique of native gel polyacrylamide gel electrophoresis (PAGE), a method that was modified more than 20 years ago to study ETC complex structure. Native electrophoresis permits the separation of ETC complexes into their active forms, and these complexes can then be studied using immunoblotting, in-gel assays (IGA), and purification by electroelution. By combining the results of native gel PAGE with those of other mitochondrial assays, it is possible to obtain a completer picture of ETC activity, its dynamic assembly and disassembly, and how this regulates mitochondrial structure and function. This work will also discuss limitations of these techniques. In summary, the technique of native PAGE, followed by immunoblotting, IGA, and electroelution, presented below, is a powerful way to investigate the functionality and composition of mitochondrial ETC supercomplexes.

This protocol describes the separation of functional mitochondrial electron transport chain complexes (Cx) I-V and supercomplexes thereof using native electrophoresis to reveal information about their assembly and structure. The native gel can be subjected to immunoblotting, in-gel assays, and purification by electroelution to further characterize individual complexes.

The mitochondrial electron transport chain (ETC) transduces the energy derived from the breakdown of various fuels into the bioenergetic currency of the ...

A Model Membrane Platform for Reconstituting Mitochondrial Membrane Dynamics

Mitochondrial dynamics is essential for the organelle&#8217;s diverse functions and cellular responses. The crowded, spatially complex, mitochondrial membrane is a challenging environment to distinguish regulatory factors. Experimental control of protein and lipid components can help answer specific questions of regulation. Yet, quantitative manipulation of these factors is challenging in cellular assays. To investigate the molecular mechanism of mitochondria inner-membrane fusion, we introduced an in vitro reconstitution platform that mimics the lipid environment of the mitochondrial inner-membrane. Here we describe detailed steps for preparing lipid bilayers and reconstituting mitochondrial membrane proteins. The platform allowed analysis of intermediates in mitochondrial inner-membrane fusion, and the kinetics for individual transitions, in a quantitative manner. This protocol describes the fabrication of bilayers with asymmetric lipid composition and describes general considerations for reconstituting transmembrane proteins into a cushioned bilayer. The method may be applied to study other membrane systems.

Mitochondrial fusion is an important homeostatic reaction underlying mitochondrial dynamics. Described here is an in vitro reconstitution system to study mitochondrial inner-membrane fusion that can resolve membrane tethering, docking, hemifusion, and pore opening. The versatility of this approach in exploring cell membrane systems is discussed.

Mitochondrial dynamics is essential for the organelle&#8217;s diverse functions and cellular responses. The crowded, spatially complex, mitochondrial ...

Single-Molecule Imaging of Lateral Mobility and Ion Channel Activity in Lipid Bilayers using Total Internal Reflection Fluorescence (TIRF) Microscopy

High-resolution imaging techniques have shown that many ion channels are not static, but subject to highly dynamic processes, including the transient association of pore-forming and auxiliary subunits, lateral diffusion, and clustering with other proteins. However, the relationship between lateral diffusion and function is poorly understood. To approach this problem, we describe how lateral mobility and activity of individual channels in supported lipid membranes can be monitored and correlated using total internal reflection fluorescence (TIRF) microscopy. Membranes are fabricated on an ultrathin hydrogel substrate using the droplet interface bilayer (DIB) technique. Compared to other types of model membranes, these membranes have the advantage of being mechanically robust and suitable for highly sensitive analytical techniques. This protocol measures Ca2+ ion flux through single channels by observing the fluorescence emission of a Ca2+-sensitive dye in close proximity to the membrane. In contrast to classical single-molecule tracking approaches, no fluorescent fusion proteins or labels, which can interfere with lateral movement and function in the membrane, are required. Possible changes in ion flux associated with conformational changes of the protein are only due to protein lateral motion in the membrane. Representative results are shown using the mitochondrial protein translocation channel TOM-CC and the bacterial channel OmpF. In contrast to OmpF, the gating of TOM-CC is very sensitive to molecular confinement and the nature of lateral diffusion. Hence, supported droplet-interface bilayers are a powerful tool to characterize the link between lateral diffusion and the function of ion channels.

Single-Molecule Imaging of Lateral Mobility and Ion Channel Activity in Lipid Bilayers using Total Internal Reflection Fluorescence TIRF Microscopy

This protocol describes how to use TIRF microscopy to track individual ion channels and determine their activity in supported lipid membranes, thereby defining the interplay between lateral membrane movement and channel function. It describes how to prepare the membranes, record the data, and analyze the results.

High-resolution imaging techniques have shown that many ion channels are not static, but subject to highly dynamic processes, including the transient ...

Isolation of Mitochondrial Contact Sites

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.

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.

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

Multi-parameter Measurement of the Permeability Transition Pore Opening in Isolated Mouse Heart Mitochondria

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with a molecular mass smaller than 1.5 kDa. Although the definitive molecular identity of the pore is still under debate, proteins such as cyclophilin D, VDAC and ANT contribute to mtPTP formation. While the involvement of mtPTP opening in cell death is well established1, accumulating evidence indicates that the mtPTP serves a physiologic role during mitochondrial Ca2+ homeostasis2, bioenergetics and redox signaling 3.
mtPTP opening is triggered by matrix Ca2+ but its activity can be modulated by several other factors such as oxidative stress, adenine nucleotide depletion, high concentrations of Pi, mitochondrial membrane depolarization or uncoupling, and long chain fatty acids4. In vitro, mtPTP opening can be achieved by increasing Ca2+ concentration inside the mitochondrial matrix through exogenous additions of Ca2+ (calcium retention capacity). When Ca2+ levels inside mitochondria reach a certain threshold, the mtPTP opens and facilitates Ca2+ release, dissipation of the proton motive force, membrane potential collapse and an increase in mitochondrial matrix volume (swelling) that ultimately leads to the rupture of the outer mitochondrial membrane and irreversible loss of organelle function.
Here we describe a fluorometric assay that allows for a comprehensive characterization of mtPTP opening in isolated mouse heart mitochondria. The assay involves the simultaneous measurement of 3 mitochondrial parameters that are altered when mtPTP opening occurs: mitochondrial Ca2+ handling (uptake and release, as measured by Ca2+ concentration in the assay medium), mitochondrial membrane potential, and mitochondrial volume. The dyes employed for Ca2+ measurement in the assay medium and mitochondrial membrane potential are Fura FF, a membrane impermeant, ratiometric indicator which undergoes a shift in the excitation wavelength in the presence of Ca2+, and JC-1, a cationic, ratiometric indicator which forms green monomers or red aggregates at low and high membrane potential, respectively. Changes in mitochondrial volume are measured by recording light scattering by the mitochondrial suspension. Since high-quality, functional mitochondria are required for the mtPTP opening assay, we also describe the steps necessary to obtain intact, highly coupled and functional isolated heart mitochondria.

A spectrofluorometric protocol for the measurement of the mitochondrial permeability transition pore opening in isolated mouse heart mitochondria is presented here. The assay involves the simultaneous measurement of mitochondria Ca2+ handling, mitochondrial membrane potential and mitochondrial volume. The procedure for obtaining high-quality and functional heart mitochondria is also described.

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with ...

Biology

Functional Characterization of Na+/H+ Exchangers of Intracellular Compartments Using Proton-killing Selection to Express Them at the Plasma Membrane

Endosomal acidification is critical for a wide range of processes, such as protein recycling and degradation, receptor desensitization, and neurotransmitter loading in synaptic vesicles. This acidification is described to be mediated by proton ATPases, coupled to ClC chloride transporters. Highly-conserved electroneutral protons transporters, the Na+/H+ exchangers (NHE) 6, 7 and 9 are also expressed in these compartments. Mutations in their genes have been linked with human cognitive and neurodegenerative diseases. Paradoxically, their roles remain elusive, as their intracellular localization has prevented detailed functional characterization. This manuscript shows a method to solve this problem. This consists of the selection of mutant cell lines, capable of surviving acute cytosolic acidification by retaining intracellular NHEs at the plasma membrane. It then depicts two complementary protocols to measure the ion selectivity and activity of these exchangers: (i) one based on intracellular pH measurements using fluorescence video microscopy, and (ii) one based on the fast kinetics of lithium uptake. Such protocols can be extrapolated to measure other non-electrogenic transporters. Furthermore, the selection procedure presented here generates cells with an intracellular retention defective phenotype. Therefore these cells will also express other vesicular membrane proteins at the plasma membrane. The experimental strategy depicted here may therefore constitute a potentially powerful tool to study other intracellular proteins that will be then expressed at the plasma membrane together with the vesicular Na+/H+ exchangers used for the selection.

Functional Characterization of Na+/H+ Exchangers of Intracellular Compartments Using Proton-killing Selection to Express Them at the Plasma Membrane

The first part of this article shows how to select mutant cell lines expressing vesicular Na+/H+ exchangers at their plasma membrane. The second part provides protocols based on intracellular pH measurements and fast ion uptake, which are used to determine the ion selectivity and the kinetic parameters of these exchangers.

Endosomal acidification is critical for a wide range of processes, such as protein recycling and degradation, receptor desensitization, and ...

Unveiling Mitochondrial Ultrastructure in Neurons Using STED Microscopy

Using Live Cell STED Imaging to Visualize Mitochondrial Inner Membrane Ultrastructure in Neuronal Cell Models

Mitochondria play many essential roles in the cell, including energy production, regulation of Ca2+ homeostasis, lipid biosynthesis, and production of reactive oxygen species (ROS). These mitochondria-mediated processes take on specialized roles in neurons, coordinating aerobic metabolism to meet the high energy demands of these cells, modulating Ca2+ signaling, providing lipids for axon growth and regeneration, and tuning ROS production for neuronal development and function. Mitochondrial dysfunction is therefore a central driver in neurodegenerative diseases. Mitochondrial structure and function are inextricably linked. The morphologically complex inner membrane with structural infolds called cristae harbors many molecular systems that perform the signature processes of the mitochondrion. The architectural features of the inner membrane are ultrastructural and therefore, too small to be visualized by traditional diffraction-limited resolved microscopy. Thus, most insights on mitochondrial ultrastructure have come from electron microscopy on fixed samples. However, emerging technologies in super-resolution fluorescence microscopy now provide resolution down to tens of nanometers, allowing visualization of ultrastructural features in live cells. Super-resolution imaging therefore offers an unprecedented ability to directly image fine details of mitochondrial structure, nanoscale protein distributions, and cristae dynamics, providing fundamental new insights that link mitochondria to human health and disease. This protocol presents the use of stimulated emission depletion (STED) super-resolution microscopy to visualize the mitochondrial ultrastructure of live human neuroblastoma cells and primary rat neurons. This procedure is organized into five sections: (1) growth and differentiation of the SH-SY5Y cell line, (2) isolation, plating, and growth of primary rat hippocampal neurons, (3) procedures for staining cells for live STED imaging, (4) procedures for live cell STED experiments using a STED microscope&#160;for reference, and (5) guidance for segmentation and image processing using examples to measure and quantify morphological features of the inner membrane.

Author Spotlight: Decoding Mitochondrial Aging 

This protocol presents a workflow for the propagation, differentiation, and staining of cultured SH-SY5Y cells and primary rat hippocampal neurons for mitochondrial ultrastructure visualization and analysis using stimulated emission depletion (STED) microscopy.

Mitochondria play many essential roles in the cell, including energy production, regulation of Ca2+ homeostasis, lipid biosynthesis, and production of ...

Neuroscience

High-resolution Respirometry to Assess Mitochondrial Function in Permeabilized and Intact Cells

A high-resolution oxygraph is a device for measuring cellular oxygen consumption in a closed-chamber system with very high resolution and sensitivity in biological samples (intact and permeabilized cells, tissues or isolated mitochondria). The high-resolution oxygraph device is equipped with two chambers and uses polarographic oxygen sensors to measure oxygen concentration and calculate oxygen consumption within each chamber. Oxygen consumption rates are calculated using software and expressed as picomoles per second per number of cells. Each high-resolution oxygraph chamber contains a stopper with injection ports, which makes it ideal for substrate-uncoupler-inhibitor titrations or detergent titration protocols for determining effective and optimum concentrations for plasma membrane permeabilization. The technique can be applied to measure respiration in a wide range of cell types and also provides information on mitochondrial quality and integrity, and maximal mitochondrial respiratory electron transport system capacity.

High-resolution respirometry is used to determine mitochondrial oxygen consumption. This is a straightforward technique to determine mitochondrial respiratory chain complexes&#39; (I-IV) respiratory rates, maximal mitochondrial electron transport system capacity, and mitochondrial outer membrane integrity.

A high-resolution oxygraph is a device for measuring cellular oxygen consumption in a closed-chamber system with very high resolution and sensitivity in ...

Assessment of Open Probability of the Mitochondrial Permeability Transition Pore in the Setting of Coenzyme Q Excess

The mitochondrial permeability transition pore (mPTP) is a voltage-gated, nonselective, inner mitochondrial membrane (IMM) mega-channel important in health and disease. The mPTP mediates leakage of protons across the IMM during low-conductance opening and is specifically inhibited by cyclosporine A (CsA). Coenzyme Q (CoQ) is a regulator of the mPTP, and tissue-specific differences have been found in CoQ content and open probability of the mPTP in forebrain and heart mitochondria in a newborn mouse model of fragile X syndrome (FXS, Fmr1 knockout). We developed a technique to determine the voltage threshold for mPTP opening in this mutant strain, exploiting the role of the mPTP as a proton leak channel.
To do so, oxygen consumption and membrane potential (&#916;&#936;) were simultaneously measured in isolated mitochondria using polarography and a tetraphenylphosphonium (TPP+) ion-selective electrode during leak respiration. The threshold for mPTP opening was determined by the onset of CsA-mediated inhibition of proton leak at specific membrane potentials. Using this approach, differences in voltage gating of the mPTP were precisely defined in the context of CoQ excess. This novel technique will permit future investigation for enhancing the understanding of physiological and pathological regulation of low-conductance opening of the mPTP.

This method exploits the contribution of the mitochondrial permeability transition pore to low-conductance proton leak to determine the voltage threshold for pore opening in neonatal fragile X syndrome mice with increased cardiomyocyte mitochondrial coenzyme Q content compared to wildtype control.

The mitochondrial permeability transition pore (mPTP) is a voltage-gated, nonselective, inner mitochondrial membrane (IMM) mega-channel important in ...

The Inner Mitochondrial Membrane

The inner mitochondrial membrane is the primary site of ATP synthesis. The inner membrane domain that forms a smooth layer adjacent to the outer membrane is called the inner boundary membrane. This domain contains membrane transporters that drive metabolites in and out of the mitochondria.&#160; In contrast, the inner membrane network that invaginates into the matrix space is called the cristae membrane. This domain accounts for principle mitochondrial function as it accommodates the protein complexes of the respiratory chain. The cristae usually form tubular invaginations; however, triangular and vesicular cristae are observed in specific cell types.
The composition of the inner boundary membrane and cristae membrane differs significantly. Heterogeneity between the inner membrane domains and their specialized curved structure is important for respiratory functions. This compositional asymmetry is maintained by the tubular neck-like structure known as crista junctions that connect the inner boundary membrane and the cristae membrane. The cristae membrane has a small diameter of 20-40 nm and consequently has an extreme curvature. This distinct membrane structure is molded by several protein components, including the mitochondrial contact site and cristae organizing system (MICOS) and ATP synthase.
The MICOS is a conserved multi-protein complex that works with mitochondrial lipid cardiolipin, respiratory complexes, and additional factors. In addition to stabilizing the inner-membrane structure, MICOS plays multiple roles, including contact site formation between the inner and the outer membrane and promoting biosynthesis of specific proteins and polypeptides. Mutations in the MICOS subunits are linked to many human diseases such as Parkinson&#8217;s disease, hepatic-encephalopathy, and acute coronary conditions. In addition to MICOS, ATP synthase dimers that localize at the rims of cristae are crucial in the inner membrane morphology. Defective ATP synthase dimerization has been associated with Leigh&#8217;s syndrome, a neurometabolic disorder that progresses into acute respiratory failure.

The inner mitochondrial membrane is the primary site of ATP synthesis. The inner membrane domain that forms a smooth layer adjacent to the outer membrane ...

Inner Mitochondrial Membrane Sensitivity to Na⁺ Reveals Partially Segmented Functional CoQ Pools

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