1. Cerveau mitochondrial Isolation (Adapté de Brown MR et al. 9) <ol><li> Sacrifier le rat en utilisant des méthodes approuvées par le Comité d&#39;utilisation Institutional Animal Care et (IACUC). </li><li> Couper la tête de l&#39;animal par décapitation, couper la peau et exposer le crâne. </li><li> Ouvrez le crâne doucement en coupant avec des ciseaux ou rongeur. Retirez le cerveau. </li><li> Hacher finement le cerveau sans cervelet dans le tampon d&#39;isolement (voir tableau 1) et de le transférer à un ml en verre / téflon homogénéisation 5 (voir la liste de l&#39;équipement). </li><li> Homogénéiser les tissus doucement 10 fois (sans bulles), environ 5 min. </li><li> Centrifuger à 1500 g pendant 10 min à 4 ° C dans une centrifugeuse de paillasse. </li><li> Enregistrer le surnageant (mitochondrial et synaptosomes) et jeter le culot (matières nucléaires et débris cellulaires). Centrifuger à 16000 g pendant 10 min à 4 ° C dans une centrifugeuse de paillasse. </li><li> Jetersurnageant et remettre en suspension le culot dans 500 ul de tampon d&#39;isolement. Perturber synaptosomes avec un navire de la perturbation de la cellule (voir la liste de l&#39;équipement). Appliquer une pression de 1200 psi pendant 10 minutes, suivie d&#39;une décompression rapide. </li><li> Couche du mélange sur Ficoll gradients (voir tableau 2), lieu SW-50.1 rotor et centrifuger à 126.500 g pendant 20 min à 4 ° C dans une ultracentrifugation (voir la liste de l&#39;équipement). Le culot est les mitochondries purifiées, la couche entre les différentes densités de Ficoll est synaptosomes sans perturbation. </li><li> Lavez granulés par centrifugation dans un tampon d&#39;isolement à 16.000 g pendant 10 min à 4 ° C dans une centrifugeuse de paillasse. </li></ol> 2. Submitochondriales vésicules (SMV) Isolation (Adapté de Chan et al. 10) <ol><li> Re-suspendre mitochondries dans 200 tampon d&#39;isolement ul combiné avec un volume égal de digitonine à 1% et laisser reposer sur la glace pendant 15 min. </li><li> Ajouter plus Buff Isolationer et centrifuger à 16000 g pendant 10 min à 4 ° C dans une centrifugeuse de paillasse. Pour ce faire, deux fois. </li><li> Re-suspendre le culot dans 200 ul de tampon d&#39;isolement et ajouter 2 pi de 10% Lubrol PX (C12E9). Mélanger et laisser reposer sur la glace pendant 15 min. </li><li> mélange de la couche tampon sur d&#39;isolation, lieu SW-50.1 rotor et centrifuger à 182 000 xg à 4 ° C pendant 1 heure. </li><li> Laver le culot final par centrifugation dans une centrifugeuse bureau haut dans le tampon d&#39;isolement à 16.000 g pendant 10 min à 4 ° C. </li></ol> 3. L&#39;enregistrement électrophysiologique <ol><li> Un dispositif typique électrophysiologique comprend un amplificateur, un ordinateur de type PC équipé d&#39;une interface de convertisseur analogique-numérique Digidata 1440A en conjonction avec le logiciel pClamp10.0, manipulateurs, un microscope, une table d&#39;isolation de vibration, cage de Faraday. </li><li> Tubes capillaires de verre borosilicate sont insérés dans un Flaming / Brown Micropipette Extracteur modèle P-87. Un programme pipette extracteur est optimisé pourgénérer des pipettes avec résistances entre 80 à 100 MQ. </li><li> SMVS sont placés dans une solution physiologique intracellulaire (ATP est ajoutée au moment approprié au cours de l&#39;enregistrement) (tableau 3). pipettes de patch-clamp sont remplis avec la même solution (pas ATP). Les enregistrements sont effectués en formant un joint giga-ohms sur SMVS à la température ambiante. Les vésicules sont visualisées par microscopie à contraste de phase avec un microscope inversé Nikon ou Zeiss. </li><li> Le potentiel de la membrane est maintenu à des tensions allant de -100 mV à +100 mV pour des périodes de 10 secondes. Les enregistrements sont filtrés à 5 kHz en utilisant le circuit de l&#39;amplificateur. Niveau de bruit électrique ou non-spécifique parasite de moins de 1 pA sont souhaitables pour l&#39;enregistrement réussi. </li><li> Les données sont analysées avec le logiciel Clampfit 10.0 par exemple pour déterminer la fréquence et l&#39;amplitude des événements à un seul canal. La dépendance de tension est déterminée en traçant une relation courant-tension. </li></ol>

<ol>
	<li>Cheng, W. C., Berman, S. B., Ivanovska, I., Jonas, E. A., Lee, S. J., Chen, Y., Kaczmarek, L. K., Pineda, F., Hardwick, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+factors+with+dual+roles+in+death+and+survival.">Mitochondrial factors with dual roles in death and survival.</a> Oncogene. 25, 4697-4705 (2006).</li><li>Duchen, M. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondria+and+calcium+in+health+and+disease.">Mitochondria and calcium in health and disease.</a> Cell Calcium. 44, 1-5 (2008).</li><li>Lemasters, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+mitochondrial+membrane+permeability+in+pathogenesis,+autophagy+and+control+of+metabolism.">Modulation of mitochondrial membrane permeability in pathogenesis, autophagy and control of metabolism.</a> J. Gastroenterol. Hepatol. 22, S31-S37 (2007).</li><li>Cox, G. B., Jans, D. A., Fimmel, A. L., Gibson, F., Hatch, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypothesis.+The+mechanism+of+ATP+synthase.+Conformational+change+by+rotation+of+the+beta-subunit.">Hypothesis. The mechanism of ATP synthase. Conformational change by rotation of the beta-subunit.</a> Biochim. Biophys. Acta. 768, 201-208 (1984).</li><li>Cox, G. B., Fimmel, A. L., Gibson, F., Hatch, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mechanism+of+ATP+synthase:+a+reassessment+of+the+functions+of+the+b+and+a+subunits.">The mechanism of ATP synthase: a reassessment of the functions of the b and a subunits.</a> Biochim. Biophys. Acta. 849, 62-69 (1986).</li><li>Cory, S., Huang, D. C., Adams, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Bcl-2+family:+roles+in+cell+survival+and+oncogenesis.">The Bcl-2 family: roles in cell survival and oncogenesis.</a> Oncogene. 22, 8590-8607 (2003).</li><li>Vander Heiden, M. G., Thompson, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bcl-2+proteins:+regulators+of+apoptosis+or+of+mitochondrial+homeostasis.">Bcl-2 proteins: regulators of apoptosis or of mitochondrial homeostasis.</a> Nat. Cell Biol. 1, 209-216 (1999).</li><li>Alavian, K. N., Li, H., Collis, L., Bonanni, L., Zeng, L., Sacchetti, S., Lazrove, E., Nabili, P., Flaherty, B., Graham, M., Chen, Y., Messerli, S. M., Mariggio, M. A., Rahner, C., McNay, E., Shore, G. C., Smith, P. J. S., Hardwick, J. M., Jonas, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bcl-xL+regulates+metabolic+efficiency+of+neurons+through+interaction+with+the+mitochondrial+F1FO+ATP+synthase.">Bcl-xL regulates metabolic efficiency of neurons through interaction with the mitochondrial F1FO ATP synthase.</a> Nat. Cell Biol. 13 (10), 1224-1233 (2011).</li><li>Brown, M. R., Sullivan, P. G., Dorenbos, K. A., Modafferi, E. A., Geddes, J. W., Steward, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nitrogen+disruption+of+synaptoneurosomes:+an+alternative+method+to+isolate+brain+mitochondria.">Nitrogen disruption of synaptoneurosomes: an alternative method to isolate brain mitochondria.</a> Journal of Neuroscience Methods. 137, 299-303 (2004).</li><li>Chan, T. L., Greenawalt, J. W., Pedersen, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+and+ultrastructural+properties+of+a+mitochondrial+inner+membrane+fraction+deficient+in+outer+membrane+and+matrix+activities.">Biochemical and ultrastructural properties of a mitochondrial inner membrane fraction deficient in outer membrane and matrix activities.</a> J. Cell Biol. 45 (2), 291-305 (1970).</li><li>Young, H. K. o., Delannoy, M., Hullihen, J., Chiu, W., Pedersen, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+ATP+Synthasomes.">Mitochondrial ATP Synthasomes.</a> J. Biol. Chem. 278 (14), 12305-12309 (2003).</li></ol>

Aucun conflit d&#39;intérêt déclaré.

Les procédés décrits ici permettent l&#39;isolement des mitochondries pur à la fin de l&#39;étape 1 et des vésicules submitochondriales (SMVS) après l&#39;étape 2 de cerveau entier, sans distinction de cellules phenotypes.SMVspurified par ce procédé sont essentiellement exempt de contamination par d&#39;autres organelles sous-cellulaires comme indiqué dans Figure 1 et notre travail précédent (Alavian KN et al. 8) et conserver leur intégrité structurale et fonctionnelle...

<table><tbody><tr><td>Potter-Elvehjem Tissue Grinder withPTFEPestle</td><td>Krackeler Scientific, Inc.</td><td>1-7725T-5</td><td></td></tr><tr><td>Eppendorf Centrifuge 5424 </td><td>Eppendorf</td><td>5424 000.410</td><td></td></tr><tr><td>4639 Cell Disruption Vessel</td><td>Parr Instrument Company</td><td>4639</td><td></td></tr><tr><td>Ficoll</td><td>Sigma-Aldrich</td><td>F5415</td><td></td></tr><tr><td>Polycarbonate centrifuge tubes</td><td>Beckman Coulter</td><td>P20314</td><td></td></tr><tr><td>SW-50.1 rotor </td><td>Beckman Coulter</td><td></td><td></td></tr><tr><td>L8-70M Ultracentrifuge</td><td>Beckman Coulter</td><td></td><td></td></tr><tr><td>Digitonin</td><td>Sigma-Aldrich</td><td>D5628</td><td></td></tr><tr><td>Lubrol PX (C12E9) </td><td>Calbiochem</td><td>205534</td><td></td></tr><tr><td>Axopatch 200B </td><td>Axon Instruments</td><td></td><td></td></tr><tr><td>Digidata 1440A </td><td>Molecular Device</td><td></td><td></td></tr><tr><td>pClamp10.0</td><td>Molecular Device</td><td></td><td></td></tr><tr><td>Manipulator</td><td>Sutter Instrument</td><td></td><td></td></tr><tr><td>Borosilicate glass capillary</td><td>World Precision Instruments</td><td>1308325</td><td></td></tr><tr><td>Flaming/Brown Micropipette Puller Model P-87</td><td>Sutter Instrument</td><td></td><td></td></tr></tbody></table>

f1fo atpase vesicle preparation and technique for performing patch clamp recordings of submitochondrial vesicle membranes

Les mitochondries sont impliquées dans de nombreuses fonctions cellulaires importantes, y compris le métabolisme, la survie à 1, le développement et la signalisation calcium 2. Deux des fonctions mitochondriales les plus importants sont liés à la production efficace de l&#39;ATP, la devise de l&#39;énergie de la cellule, par phosphorylation oxydative et la médiation des signaux de mort cellulaire programmée 3. L&#39;enzyme principalement responsable de la production de l&#39;ATP synthase est le F1FO-ATP, aussi appelé ATP synthase 4-5. Au cours des dernières années, le rôle des mitochondries dans la mort cellulaire par apoptose et nécrose a reçu une attention considérable. Dans la mort cellulaire par apoptose, BCL-2 protéines de la famille tels que Bax entrent dans la membrane mitochondriale externe, s&#39;oligomériser et perméabilisent la membrane externe, libérant des facteurs pro-apoptotiques dans le cytosol 6. Dans la mort cellulaire nécrotique classique, telle que celle produite par une ischémie ou excitotoxicité des neurones, un large, augmentation mal régulé en calcium de la matrice contribue à l&#39;ouverture d&#39;un pore de la membrane intérieure, le pore de transition de perméabilité mitochondriale ou mPTP. Cette dépolarise la membrane interne et provoque des changements osmotiques, en contribuant à la rupture de la membrane externe, libération de facteurs pro-apoptotiques, et un dysfonctionnement métabolique. De nombreuses protéines dont la protéine Bcl-xL 7 interagir avec F1FO ATP synthase, de moduler sa fonction. Bcl-xL interagit directement avec la sous-unité bêta de l&#39;ATP-synthase F1FO, et cette interaction diminue une conductance de fuite à l&#39;intérieur de la F1FOATPasecomplex, augmentant le transport net de H + par F1FO pendant F1FO activité ATPase 8 et augmentant ainsi l&#39;efficacité mitochondriale. Pour étudier l&#39;activité et de la modulation de l&#39;ATP synthase, nous avons isolé à partir de rongeurs cerveau submitochondriales vésicules (SMVS) contenant F1FO ATPase. Les SMVS conserver l&#39;intégrité structurelle et fonctionnelle de la F1FO ATPase comme indiqué dans Alavian et al. Ici, nous décrivons une méthodeque nous avons utilisé avec succès pour l&#39;isolement du SMVS de cerveau de rat et nous délimiter la technique de patch-clamp pour analyser l&#39;activité du canal (ion fuite conductance) de la SMVS.

La première étape de notre protocole permet l&#39;isolement des mitochondries purifiées comme indiqué par Western blot à la figure 1. Dans la figure 2 est représenté un exemple de submitochondriales enregistrement de patch des vésicules dérivé du cerveau. Utilisation de la configuration de patch inside-out, nous démontrons l&#39;activité du canal modulé par l&#39;ATP. Le contrôle (CTL) enregistrement (à gauche) montre l&#39;activité du canal multi-conductance avec une co...

Watch this Scientific Journal Video about F1FO ATPase Vesicle Preparation and Technique for Performing Patch Clamp Recordings of Submitochondrial Vesicle Membranes at JoVE.com

F1FO ATPase Vesicle Preparation and Technique for Performing Patch Clamp Recordings of Submitochondrial Vesicle Membranes

Une méthode pour isoler les vésicules submitochondriales enrichis en F1FO ATP synthase complexes du cerveau de rat est décrite. Ces vésicules permettent l&#39;étude de l&#39;activité de F1FO complexe ATPase et sa modulation en utilisant la technique de l&#39;enregistrement de patch-clamp.

<sub&gt; 1</sub&gt; F<sub&gt; O</sub&gt; ATPase Vesicle Préparation et Technique for the Performing Patch enregistrements de serrage des membranes des vésicules submitochondriales

Mitochondria are involved in many important cellular  functions including metabolism, survival1, development and, calcium signaling2. Two of the most ...

f1fo-atpase-vesicle-preparation-technique-for-performing-patch-clamp

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

F1FO ATPase Vesicle Preparation and Technique for Performing Patch Clamp Recordings of Submitochondrial Vesicle Membranes

10.2K vues.  Yale University. Une méthode pour isoler les vésicules submitochondriales enrichis en F1FO ATP synthase complexes du cerveau de rat est décrite. Ces vésicules permettent l'étude de l'activité de F1FO complexe ATPase et sa modulation en utilisant la technique de l'enregistrement de patch-clamp.

Vidéo: F1FO ATPase Vesicle Preparation and Technique for Performing Patch Clamp Recordings of Submitochondrial Vesicle Membranes

Detergent-free Ultrafast Reconstitution of Membrane Proteins into Lipid Bilayers Using Fusogenic Complementary-charged Proteoliposomes.

Detergents are indispensable for delivery of membrane proteins into 30-100 nm small unilamellar vesicles, while more complex, larger model lipid bilayers are less compatible with detergents.
Here we describe a strategy for bypassing this fundamental limitation using fusogenic oppositely charged liposomes bearing a membrane protein of interest. Fusion between such vesicles occurs within 5 min in a low ionic strength buffer. Positively charged fusogenic liposomes can be used as simple shuttle vectors for detergent-free delivery of membrane proteins into biomimetic target lipid bilayers, which are negatively charged. We also show how to reconstitute membrane proteins into fusogenic proteoliposomes with a fast 30-min protocol.
Combining these two approaches, we demonstrate a fast assembly of an electron transport chain consisting of two membrane proteins from E. coli, a primary proton pump bo3-oxidase and F1Fo ATP synthase, in membranes of vesicles of various sizes, ranging from 0.1 to &#62;10 microns, as well as ATP production by this chain.

Here we present two ultrafast protocols for reconstitution of membrane proteins into fusogenic proteoliposomes, and fusion of such proteoliposomes with target lipid bilayers for detergent-free delivery of these membrane proteins into the postfusion bilayer. The combination of these approaches enables fast and easily controlled assembly of complex multi-component membrane systems.

Sans tensioactifs ultrarapide Reconstitution des protéines membranaires dans les bicouches lipidiques à l’aide de fusogène complémentaire chargée protéoliposomes.

Detergents are indispensable for delivery of membrane proteins into 30-100 nm small unilamellar vesicles, while more complex, larger model lipid bilayers ...

Recherche

Biochimie

Isolation of F1-ATPase from the Parasitic Protist Trypanosoma brucei

F1-ATPase is a membrane-extrinsic catalytic subcomplex of F-type ATP synthase, an enzyme that uses the proton motive force across biological membranes to produce adenosine triphosphate (ATP). The isolation of the intact F1-ATPase from its native source is an essential prerequisite to characterize the enzyme&#39;s protein composition, kinetic parameters, and sensitivity to inhibitors. A highly pure and homogeneous F1-ATPase can be used for structural studies, which provide insight into molecular mechanisms of ATP synthesis and hydrolysis. This article describes a procedure for the purification of the F1-ATPase from Trypanosoma brucei, the causative agent of African trypanosomiases. The F1-ATPase is isolated from mitochondrial vesicles, which are obtained by hypotonic lysis from in vitro cultured trypanosomes. The vesicles are mechanically fragmented by sonication and the F1-ATPase is released from the inner mitochondrial membrane by the chloroform extraction. The enzymatic complex is further purified by consecutive anion exchange and size-exclusion chromatography. Sensitive mass spectrometry techniques showed that the purified complex is devoid of virtually any protein contaminants and, therefore, represents suitable material for structure determination by X-ray crystallography or cryo-electron microscopy. The isolated F1-ATPase exhibits ATP hydrolytic activity, which can be inhibited fully by sodium azide, a potent inhibitor of F-type ATP synthases. The purified complex remains stable and active for at least three days at room temperature. Precipitation by ammonium sulfate is used for long-term storage. Similar procedures have been used for the purification of F1-ATPases from mammalian and plant tissues, yeasts, or bacteria. Thus, the presented protocol can serve as a guideline for the F1-ATPase isolation from other organisms.

Isolation of F1-ATPase from the Parasitic Protist Trypanosoma brucei

This protocol describes the purification of F1-ATPase from the cultured insect stage of Trypanosoma brucei. The procedure yields a highly pure, homogeneous, and active complex suitable for structural and enzymatic studies.

Isolation de F1-ATPase de la protiste parasite Trypanosoma brucei

F1-ATPase is a membrane-extrinsic catalytic subcomplex of F-type ATP synthase, an enzyme that uses the proton motive force across biological membranes to ...

Membrane Remodeling of Giant Vesicles in Response to Localized Calcium Ion Gradients

In a wide variety of fundamental cell processes, such as membrane trafficking and apoptosis, cell membrane shape transitions occur concurrently with local variations in calcium ion concentration. The main molecular components involved in these processes have been identified; however, the specific interplay between calcium ion gradients and the lipids within the cell membrane is far less known, mainly due to the complex nature of biological cells and the difficultly of observation schemes. To bridge this gap, a synthetic approach is successfully implemented to reveal the localized effect of calcium ions on cell membrane mimics. Establishing a mimic to resemble the conditions within a cell is a severalfold problem. First, an adequate biomimetic model with appropriate dimensions and membrane composition is required to capture the physical properties of cells. Second, a micromanipulation setup is needed to deliver a small amount of calcium ions to a particular membrane location. Finally, an observation scheme is required to detect and record the response of the lipid membrane to the external stimulation. This article offers a detailed biomimetic approach for studying the calcium ion-membrane interaction, where a lipid vesicle system, consisting of a giant unilamellar vesicle (GUV) connected to a multilamellar vesicle (MLV), is exposed to a localized calcium gradient formed using a microinjection system. The dynamics of the ionic influence on the membrane were observed using fluorescence microscopy and recorded at video frame rates. As a result of the membrane stimulation, highly curved membrane tubular protrusions (MTPs) formed inside the GUV, oriented away from the membrane. The described approach induces the remodeling of the lipid membrane and MTP production in an entirely contactless and controlled manner. This approach introduces a means to address the details of calcium ion-membrane interactions, providing new avenues to study the mechanisms of cell membrane reshaping.

We present a technique for contactless micromanipulation of vesicles, using localized calcium ion gradients. Microinjection of a calcium ion solution, in the vicinity of a giant lipid vesicle, is utilized to remodel the lipid membrane, resulting in the production of membrane tubular protrusions.

Remodelage de membrane de vésicules géants en réponse aux Gradients d’Ion Calcium localisée

In a wide variety of fundamental cell processes, such as membrane trafficking and apoptosis, cell membrane shape transitions occur concurrently with local ...

Chimie

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.

Une plate-forme membranaire modèle pour reconstituer la dynamique memitochondriale de membrane

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

Assessment of Submitochondrial Protein Localization in Budding Yeast Saccharomyces cerevisiae

Despite recent advances in the characterization of yeast mitochondrial proteome, the submitochondrial localization of a significant number of proteins remains elusive. Here, we describe a robust and effective method for determining the suborganellar localization of yeast mitochondrial proteins, which is considered a fundamental step during mitochondrial protein function elucidation. This method involves an initial step that consists of obtaining highly pure intact mitochondria. These mitochondrial preparations are then subjected to a subfractionation protocol consisting of hypotonic shock (swelling) and incubation with proteinase K (protease). During swelling, the outer mitochondrial membrane is selectively disrupted, allowing the proteinase K to digest proteins of the intermembrane space compartment. In parallel, to obtain information about the topology of membrane proteins, the mitochondrial preparations are initially sonicated, and then subjected to alkaline extraction with sodium carbonate. Finally, after centrifugation, the pellet and supernatant fractions from these different treatments are analyzed by SDS-PAGE and western blot. The submitochondrial localization as well as the membrane topology of the protein of interest is obtained by comparing its western blot profile with known standards.

Assessment of Submitochondrial Protein Localization in Budding Yeast Saccharomyces cerevisiae

Despite recent advances, many yeast mitochondrial proteins still remain with their functions completely unknown. This protocol provides a simple and reliable method to determine the submitochondrial localization of proteins, which has been fundamental for the elucidation of their molecular functions.

Évaluation de la localisation des protéines submitochondriales dans la levure bourgeonnante Saccharomyces cerevisiae

Despite recent advances in the characterization of yeast mitochondrial proteome, the submitochondrial localization of a significant number of proteins ...

Isolement des sites de contact mitochondriaux

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.

Pleins feux sur l’auteur : Dévoilement des sites de contact mitochondriaux et des aperçus architecturaux 

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.

Une méthode améliorée pour isoler les sites de contact mitochondrial

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

Preparation, Purification, and Use of Fatty Acid-containing Liposomes

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due to the unique chemical properties of these amphiphiles. In particular, fatty acid liposomes enhance dynamic character, due to the relatively high solubility of single-chain amphiphiles. Similarly, liposomes containing free fatty acids are more sensitive to salt and divalent cations, due to the strong interactions between the carboxylic acid head groups and metal ions. Here we illustrate techniques for preparation, purification, and use of liposomes comprised in part or whole of single chain amphiphiles (e.g., oleic acids).

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due to the unique chemical properties of single chain amphiphiles. Here we describe techniques for the preparation, purification, and use of liposomes comprised in part or whole of these amphiphiles.

Préparation, Purification et l’utilisation de Liposomes contenant de l’acide gras

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due ...

Bio-ingénierie

Reconstitution of a Kv Channel into Lipid Membranes for Structural and Functional Studies

To study the lipid-protein interaction in a reductionistic fashion, it is necessary to incorporate the membrane proteins into membranes of well-defined lipid composition. We are studying the lipid-dependent gating effects in a prototype voltage-gated potassium (Kv) channel, and have worked out detailed procedures to reconstitute the channels into different membrane systems. Our reconstitution procedures take consideration of both detergent-induced fusion of vesicles and the fusion of protein/detergent micelles with the lipid/detergent mixed micelles as well as the importance of reaching an equilibrium distribution of lipids among the protein/detergent/lipid and the detergent/lipid mixed micelles. Our data suggested that the insertion of the channels in the lipid vesicles is relatively random in orientations, and the reconstitution efficiency is so high that no detectable protein aggregates were seen in fractionation experiments. We have utilized the reconstituted channels to determine the conformational states of the channels in different lipids, record electrical activities of a small number of channels incorporated in planar lipid bilayers, screen for conformation-specific ligands from a phage-displayed peptide library, and support the growth of 2D crystals of the channels in membranes. The reconstitution procedures described here may be adapted for studying other membrane proteins in lipid bilayers, especially for the investigation of the lipid effects on the eukaryotic voltage-gated ion channels.

Procedures for complete reconstitution of a prototype voltage-gated potassium channel into lipid membranes are described. The reconstituted channels are suitable for biochemical assays, electrical recordings, ligand screening and electron crystallographic studies. These methods may have general applications to the structural and functional studies of other membrane proteins.

Reconstitution d&#39;un canal Kv dans les membranes lipidiques des études structurales et fonctionnelles

To study the lipid-protein interaction in a reductionistic fashion, it is necessary to incorporate the membrane proteins into membranes of well-defined ...

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Reconstitution d'un canal Kv dans les membranes lipidiques des études structurales et fonctionnelles