We acknowledge the contribution of Renata Sano in conceiving the initial protocol. A.d&#x2019;A. holds the Jewelers For Children (JFC) Endowed Chair in Genetics and Gene Therapy. This work was funded in part by NIH grants GM60905, DK52025 and CA021764, and the American Lebanese Syrian Associated Charities (ALSAC).

The following protocol is intended for the isolation and purification of MAMs and GEMs from mouse brain.
Solutions required for Isolation of mitochondria, MAMs and GEMs.
Fractionation to obtain crude mitochondrial preparation:
Solution A: 0.32 M Sucrose, 1 mM NaHCO3, 1 mM MgCl2, 0.5 mM CaCl2 + Protease Inhibitors (add fresh, as needed).
Solution B: </stron...

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	<li>Poburko, D., Kuo, K. H., Dai, J., Lee, C. H., van Breemen, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organellar+junctions+promote+targeted+Ca2++signaling+in+smooth+muscle:+why+two+membranes+are+better+than+one.">Organellar junctions promote targeted Ca2+ signaling in smooth muscle: why two membranes are better than one.</a> Trends Pharmacol. Sci. 25 (1), 8-15 (2004).</li><li>Pani, B., Ong, H. L., Liu, X., Rauser, K., Ambudkar, I. S., Singh, B. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+rafts+determine+clustering+of+STIM1+in+endoplasmic+reticulum-plasma+membrane+junctions+and+regulation+of+store-operated+Ca2++entry+(SOCE.">Lipid rafts determine clustering of STIM1 in endoplasmic reticulum-plasma membrane junctions and regulation of store-operated Ca2+ entry (SOCE.</a> J. Biol. Chem. 283 (25), 17333-17340 (2008).</li><li>Levine, T., Rabouille, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endoplasmic+reticulum:+one+continuous+network+compartmentalized+by+extrinsic+cues.">Endoplasmic reticulum: one continuous network compartmentalized by extrinsic cues.</a> Curr. Opin. Cell Biol. 17 (4), 362-368 (2005).</li><li>Sano, R., Annunziata, I., Patterson, A., Moshiach, S., Gomero, E., Opferman, J., Forte, M., d'Azzo, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GM1-ganglioside+accumulation+at+the+mitochondria-associated+ER+membranes+links+ER+stress+to+Ca(2+)-dependent+mitochondrial+apoptosis.">GM1-ganglioside accumulation at the mitochondria-associated ER membranes links ER stress to Ca(2+)-dependent mitochondrial apoptosis.</a> Mol. Cell. 36 (3), 500-511 (2009).</li><li>Raturi, A., Simmen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Where+the+endoplasmic+reticulum+and+the+mitochondrion+tie+the+knot:+The+mitochondria-associated+membrane+(MAM).">Where the endoplasmic reticulum and the mitochondrion tie the knot: The mitochondria-associated membrane (MAM).</a> Biochim. Biophys. Acta. , (2012).</li><li>Giorgi, C., De Stefani, D., Bononi, A., Rizzuto, R., Pinton, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+and+functional+link+between+the+mitochondrial+network+and+the+endoplasmic+reticulum.">Structural and functional link between the mitochondrial network and the endoplasmic reticulum.</a> Int. J. Biochem. Cell Biol. 41 (10), 1817-1827 (2009).</li><li>d'Azzo, A., Tessitore, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sano+Gangliosides+as+apoptotic+signals+in+ER+stress+response.">Sano Gangliosides as apoptotic signals in ER stress response.</a> Cell Death Differ. 13, 404-414 (2006).</li><li>Kroemer, G., Galluzzi, L., Brenner, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+membrane+permeabilization+in+cell+death.">Mitochondrial membrane permeabilization in cell death.</a> Physiol. 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Protoc. 4 (11), 1582-1590 (2009).</li><li>Pizzo, P., Viola, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphocyte+lipid+rafts:+structure+and+function.">Lymphocyte lipid rafts: structure and function.</a> Curr. Opin. Immunol. 15 (3), 255-260 (2003).</li></ol>

The sites of contact between intracellular membranes or between organelles and the plasma membrane of cells represent dynamic signaling platforms for basic cellular processes. The accurate characterization of their function and composition under both physiological and pathological conditions requires reliable and reproducible purification protocols. The methods detailed here have been specifically optimized by our lab for the isolation and purification of the MAMs and their corresponding GEMs from the adult mouse brain. ...

<table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalogue Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>REAGENTS</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Fractionation</td>
 </tr>
 <tr>
 <td>Sucrose</td>
 <td>Fisher Scientific</td>
 <td>S5-500</td>
 <td></td>
 </tr>
 <tr>
 <td>Sodium Bicarbonate</td>
 <td>Sigma-Aldrich</td>
 <td>S-5761</td>
 <td></td>
 </tr>
 <tr>
 <td>Magnesium Chloride, Hexahydrate</td>
 <td>Fisher Scientific</td>
 <td>BP214-500</td>
 <td></td>
 </tr>
 <tr>
 <td>Calcium Chloride, Dihydrate</td>
 <td>Sigma-Aldrich</td>
 <td>C-5080</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>MAM </td>
 </tr>
 <tr>
 <td>D-Mannitol</td>
 <td>Sigma-Aldrich</td>
 <td>M9546-250G</td>
 <td></td>
 </tr>
 <tr>
 <td>Hepes</td>
 <td>Fisher Scientific</td>
 <td>BP310-500</td>
 <td></td>
 </tr>
 <tr>
 <td>EGTA</td>
 <td>Sigma-Aldrich</td>
 <td>E4378-250G</td>
 <td></td>
 </tr>
 <tr>
 <td>BSA, Fraction V, Heat Shock, Lyophilizate</td>
 <td>Roche</td>
 <td>03-116-964-001</td>
 <td></td>
 </tr>
 <tr>
 <td>Percoll</td>
 <td>GE</td>
 <td>17-0891-02</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>GEM </td>
 </tr>
 <tr>
 <td>Triton X-100</td>
 <td>Sigma-Aldrich</td>
 <td>T9284-500 ml</td>
 <td></td>
 </tr>
 <tr>
 <td>Sodium Chloride</td>
 <td>Fisher Scientific</td>
 <td>S271-3</td>
 <td></td>
 </tr>
 <tr>
 <td>Tris Base</td>
 <td>Roche</td>
 <td>03-118-142-001</td>
 <td></td>
 </tr>
 <tr>
 <td>HCl</td>
 <td>Fisher Scientific</td>
 <td>A144S-500</td>
 <td></td>
 </tr>
 <tr>
 <td>EDTA</td>
 <td>Fisher Scientific</td>
 <td>BP120-500</td>
 <td></td>
 </tr>
 <tr>
 <td>Sodium Dodecyl Sulfate (SDS)</td>
 <td>Fisher Scientific</td>
 <td>BP166-500</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Common </td>
 </tr>
 <tr>
 <td>Protease Inhibitors Tablets, Complete EDTA-free</td>
 <td>Roche</td>
 <td>11-873-580-001</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>EQUIPMENT </td>
 </tr>
 <tr>
 <td>2 ml Douce All-Glass Tissue Grinders</td>
 <td>Kimble Chase</td>
 <td>885300-0002</td>
 <td></td>
 </tr>
 <tr>
 <td>15 ml Polypropylene Conical Centrifuge Tubes, BD Falcon</td>
 <td>BD</td>
 <td>352097</td>
 <td></td>
 </tr>
 <tr>
 <td>30 ml Round-Bottom Glass Centrifuge Tubes</td>
 <td>Kimble Chase</td>
 <td>45500-30</td>
 <td></td>
 </tr>
 <tr>
 <td>15 ml Round-Bottom Glass Centrifuge Tubes</td>
 <td>Kimble Chase</td>
 <td>45500-15</td>
 <td></td>
 </tr>
 <tr>
 <td>Ultracentrifuge tubes, Ultra-Clear, Thinwall, 14x89 mm</td>
 <td>Beckman-Coulter</td>
 <td>344059</td>
 <td></td>
 </tr>
 <tr>
 <td>Parafilm</td>
 <td>Cole-Parmer</td>
 <td>PM996</td>
 <td></td>
 </tr>
 <tr>
 <td>Disposable Borosilicate Glass Pasteur Pipets, 9"</td>
 <td>Fisher Scientific</td>
 <td>13-678-20C</td>
 <td></td>
 </tr>
 </tbody>
</table>

mitochondria-associated er membranes (mams) and glycosphingolipid enriched microdomains (gems): isolation from mouse brain

Intracellular organelles are highly dynamic structures with varying shape and composition, which are subjected to cell-specific intrinsic and extrinsic cues. Their membranes are often juxtaposed at defined contact sites, which become hubs for the exchange of signaling molecules and membrane components1,2,3,4. The inter-organellar membrane microdomains that are formed between the endoplasmic reticulum (ER) and the mitochondria at the opening of the IP3-sensitive Ca2+ channel are known as the mitochondria associated-ER membranes or MAMs4,5,6. The protein/lipid composition and biochemical properties of these membrane contact sites have been extensively studied particularly in relation to their role in regulating intracellular Ca2+ 4,5,6. The ER serves as the primary store of intracellular Ca2+, and in this capacity regulates a myriad of cellular processes downstream of Ca2+ signaling, including post-translational protein folding and protein maturation7. Mitochondria, on the other hand, maintain Ca2+ homeostasis, by buffering cytosolic Ca2+ concentration thereby preventing the initiation of apoptotic pathways downstream of Ca2+ unbalance4,8. The dynamic nature of the MAMs makes them ideal sites to dissect basic cellular mechanisms, including Ca2+ signaling and regulation of mitochondrial Ca2+ concentration, lipid biosynthesis and transport, energy metabolism and cell survival 4,9,10,11,12. Several protocols have been described for the purification of these microdomains from liver tissue and cultured cells13,14. 
	Taking previously published methods into account, we have adapted a protocol for the isolation of mitochondria and MAMs from the adult mouse brain. To this procedure we have added an extra purification step, namely a Triton X100 extraction, which enables the isolation of the glycosphingolipid enriched microdomain (GEM) fraction of the MAMs. These GEM preparations share several protein components with caveolae and lipid rafts, derived from the plasma membrane or other intracellular membranes, and are proposed to function as gathering points for the clustering of receptor proteins and for protein&#x2013;protein interactions4,15.

Based on our experience with using this protocol we can safely recommend it for the isolation and purification of MAMs, GEMs and mitochondrial fractions from mouse brain. The procedure as outlined is highly reproducible and consistent. In Figure 1 we show a representative image of the way pure mitochondria and MAMs layer on a Percoll gradient (step 3.4). A defined, milky band containing purified mitochondria (Mito-P) segregates at the bottom of the ultracentrifuge tube, while the MAM fraction makes up a ...

Watch this Scientific Journal Video about Mitochondria-associated ER Membranes MAMs and Glycosphingolipid Enriched Microdomains GEMs: Isolation from Mouse Brain at JoVE.com

Mitochondria-associated ER Membranes MAMs and Glycosphingolipid Enriched Microdomains GEMs: Isolation from Mouse Brain

This procedure illustrates how to isolate from the adult mouse brain the mitochondria-associated ER membranes or MAMs and the glycosphingolipid-enriched microdomain fractions from MAMs and mitochondrial preparations.

Mitochondria-associated ER Membranes (MAMs) and Glycosphingolipid Enriched Microdomains (GEMs): Isolation from Mouse Brain

Intracellular organelles are highly dynamic structures with varying shape and composition, which are subjected to cell-specific intrinsic and extrinsic ...

mitochondria-associated-er-membranes-mams-glycosphingolipid-enriched

Research

JoVE Journal

Neuroscience

24.2K ビュー.  St Jude Children's Research Hospital. この手順では、成体マウスの脳ミトコンドリア関連のER膜またはMAMSとMAMSとミトコンドリア調製物からスフィンゴ糖脂質に富むマイクロドメイン画分から単離する方法を示しています。