Name: Mitochondria-associated ER Membranes (MAMs) and Glycosphingolipid Enriched Microdomains (GEMs): Isolation from Mouse Brain (Video) | JoVE Journal
Uploaded: 2013-03-04T12:15:00.000+00:00
Duration: 10 min 32 s
Description: Learn to isolate mitochondria-associated ER membranes (MAMs) and glycosphingolipid-enriched microdomains (GEMs) from adult mouse brain using homogenization and sucrose gradient centrifugation. This method enables neurobiology research.

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5 minutes

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: 0.32 M Sucrose, 1 mM NaHCO3 + Protease Inhibitors (add fresh, as needed).
MAMs Isolation:
Isolation Medium: 250 mM Mannitol, 5 mM HEPES pH 7.4, 0.5 mM EGTA, 0.1% BSA.
Gradient Buffer: 225 mM Mannitol, 25 mM HEPES pH 7.5, 1 mM EGTA, 0.1% BSA (final concentration).
GEMs Isolation:
GEM Extraction Buffer: 25 mM HEPES pH 7.5, 0.15 M NaCl, 1% Triton X-100 + Protease Inhibitors (add fresh, as needed).
GEM Solubilising Buffer: 50 mM Tris-HCl, pH 8.8, 5 mM EDTA , 1% SDS + Protease Inhibitors (add fresh, as needed).
1. First Step Subcellular Fractionation: Removal of Nuclei, Unlysed Cells and Cellular Debris
<ol>
 <li>Euthanize mouse in CO2 chamber.
 </li>
 <li>Immediately remove brain, halve, place in 2 ml tubes on ice and weigh. </li>
</ol>
Note: Keep brain halves separate throughout the entire procedure. The following applies to the treatment of a single brain half. 
<ol start="3">
 <li>Place half brain in a pre-chilled 2 ml glass Dounce tissue grinder containing 1 ml cold Solution A. Homogenize with 15 total strokes of a large clearance pestle. </li>
 <li>Transfer to a 15 ml falcon tube, on ice, and dilute homogenates up to 10 volumes w/v, e.g. 0.225 g to 2.25 ml. </li>
 <li>Centrifuge sample at 1,400 x g for 10 min at 4 &deg;C.
 </li>
 <li>Carefully remove the supernatant and transfer to a 30 ml round-bottom glass centrifuge tube, on ice. Make sure not to disturb the pellet. </li>
 <li>Resuspend the pellet in the same 10 volumes of Solution A. Homogenize in the same grinder, with 3-6 strokes of a small clearance pestle, 1 ml at a time. </li>
 <li>Transfer to a fresh 15 ml falcon tube and centrifuge at 710 x g for 10 min at 4 &deg;C. This results in a pellet of nuclei and cell debris. </li>
 <li>Carefully remove the supernatant and pool with the supernatant saved in step 1.6. </li>
</ol>
2. Second Step: Crude Mitochondria Isolation
<ol>
 <li>Centrifuge supernatants at 13,800 x g for 10 min at 4 &deg;C. </li>
 <li>Transfer supernatant to an Ultra-Clear Beckman Centrifuge Tube, on ice </li>
 <li>Resuspend pellet in 10 volumes Solution A. Homogenize in the same grinder, with 3-6 strokes of a small clearance pestle, 1 ml at a time. </li>
 <li>Centrifuge as step 2.1. </li>
 <li>Repeat steps 2.2-2.4. </li>
 <li>The resulting pellet is an enriched mitochondrial fraction. Pool the supernatants (cytosol and ER), cover with parafilm and keep on ice.</li>
 <li>Resuspend the pellet with 6 strokes of a small clearance pestle, in 4.8 ml/g (g is referred to the original brain weight) of Solution B, in a fresh homogeniser. </li>
 <li>Using glass Pasteur pipettes, prepare a Discontinuous Sucrose Gradient in an Ultra-Clear Beckman Centrifuge Tube, adding subsequently to the bottom of the tube as follows, carefully, ensuring no bubbles are formed: 
 Resupended Pellet (0.32 M Sucrose) 
 3 ml 850 mM Sucrose (in 1 mM NaHCO3) 
 3 ml 1 M Sucrose (in 1 mM NaHCO3) 
 3 ml 1.2 M Sucrose (in 1 mM NaHCO3) </li>
 <li>Centrifuge at 82,500 x g for 2 hr at 4 &deg;C. The resulting separation at the end of the gradient will produce three bands and a pellet: 1) myelin and other membrane contaminants (0.32 M - 0.85 M interface); 2) ER, Golgi, plasma membranes (0.85 M - 1 M interface); 3) synaptosomes (1 M - 1.2 M interface); 4) crude mitochondria (pellet) that is used for the subsequent purification steps
 
 </li>
</ol>
3. Isolation of Mitochondria-associated ER Membrane, MAMs
<ol>
 <li>Resuspend the freshly isolated crude mitochondria from one brain half, in 2 ml Isolation Medium containing freshly added protease inhibitors. 
 </li>
 <li>Prepare a 30% Percoll gradient with Gradient Buffer 8 ml per half brain, and place in an Ultra-Clear Beckman Centrifuge Tube. </li>
</ol>
* Note: Make gradient buffer more concentrated (1.43 times) to achieve correct final concentration after diluting Percoll to 30%.
<ol start="3">
 <li>Layer mitochondrial suspension on top of prepared gradient SLOWLY to avoid any bubbles.</li>
 <li>Centrifuge at 95,000 x g for 30 min, 4 &deg;C.
 </li>
 <li><ol class="lroman">
 <li> Remove the Heavy Fraction (lower band) FIRST with a glass Pasteur pipette and transfer to a fresh round-bottom glass tube, on ice.</li>
 <li> Remove the Light Fraction (upper band) SECOND with another glass Pasteur pipette and transfer to a separate fresh round-bottom glass tube, on ice. </li>
 </ol>
 </li>
 <li>Dilute both fractions collected in step 3.5 with 10 ml Isolation Medium and centrifuge at 6,300 x g for 10 min at 4 &deg;C.
 </li>
 <li>Discard the supernatant from the Heavy Fraction obtained in step 3.6, resuspend the pellet with another 10 ml Isolation Medium and centrifuge again, as step 3.6. The resulting pellet will be the pure mitochondrial fraction. </li>
 <li>Transfer the supernatant from the Light Fraction obtained in step 3.6 to an Ultra-Clear Beckman Centrifuge Tube, on ice. Discard the pellet. </li>
 <li>Add sufficient Isolation Medium to the supernatant obtained in step 3.8 to fill the tube and centrifuge at 100,000 x g for 1 hr, 4 &deg;C. The resulting pellet will be the MAMs fraction. Carefully remove and discard the supernatant. </li>
</ol>
Note: At this step, you may also centrifuge at 100,000 x g for 1 hr, 4 &deg;C the supernatant saved at step 2.6. The pellet will be the ER fraction and supernatant the Cytosolic fraction.
4. Extraction of Glycosphingolipid-enriched Microdomains, GEMs 
<ol>
 <li>Lyse pure mitochondria and/or MAM fractions in 500 &mu;l-1 ml of Extraction Buffer for 20 min on ice.</li>
 <li>Centrifuge lysates at 15,300 x g for 2 min at 4 &deg;C. </li>
 <li>Collect supernatants (Triton X-100 soluble material) and re-centrifuge pellets for 2 min to remove remaining soluble material. (Triton X-100 extracted Mitochondria and/or Triton X-100 extracted MAMs).</li>
 <li>Solubilize pellets in Solubilising Buffer. This solubilized material represents the GEM fractions and can be used for further analysis.</li>
</ol>

<ol><li>Poburko, D., Kuo, K. H., Dai, J., Lee, C. H., van Breemen, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Organellar+junctions+promote+targeted+Ca2%2B+signaling+in+smooth+muscle%3A+why+two+membranes+are+better+than+one%5BTitle%5D)+AND+%22Trends+Pharmacol.+Sci%22%5BJournal%5D)">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/pubmed?term=((Lipid+rafts+determine+clustering+of+STIM1+in+endoplasmic+reticulum-plasma+membrane+junctions+and+regulation+of+store-operated+Ca2%2B+entry+%28SOCE%5BTitle%5D)+AND+%22J.+Biol.+Chem%22%5BJournal%5D)">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/pubmed?term=((Endoplasmic+reticulum%3A+one+continuous+network+compartmentalized+by+extrinsic+cues%5BTitle%5D)+AND+%22Curr.+Opin.+Cell+Biol%22%5BJournal%5D)">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/pubmed?term=((GM1-ganglioside+accumulation+at+the+mitochondria-associated+ER+membranes+links+ER+stress+to+Ca%282%2B%29-dependent+mitochondrial+apoptosis%5BTitle%5D)+AND+%22Mol.+Cell%22%5BJournal%5D)">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/pubmed?term=((Where+the+endoplasmic+reticulum+and+the+mitochondrion+tie+the+knot%3A+The+mitochondria-associated+membrane+%28MAM%29%5BTitle%5D)+AND+%22Biochim.+Biophys.+Acta%22%5BJournal%5D)">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/pubmed?term=((Structural+and+functional+link+between+the+mitochondrial+network+and+the+endoplasmic+reticulum%5BTitle%5D)+AND+%22Int.+J.+Biochem.+Cell+Biol%22%5BJournal%5D)">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/pubmed?term=((Sano+Gangliosides+as+apoptotic+signals+in+ER+stress+response%5BTitle%5D)+AND+%22Cell+Death+Differ%22%5BJournal%5D)">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/pubmed?term=((Mitochondrial+membrane+permeabilization+in+cell+death%5BTitle%5D)+AND+%22Physiol.+Rev%22%5BJournal%5D)">Mitochondrial membrane permeabilization in cell death.</a> Physiol. Rev. 87, 99-163 (2007).</li>
<li>Rizzuto, R., Brini, M., Murgia, M., Pozzan, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microdomains+with+high+Ca2%2B+close+to+IP3-sensitive+channels+that+are+sensed+by+neighboring+mitochondria%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Microdomains with high Ca2+ close to IP3-sensitive channels that are sensed by neighboring mitochondria.</a> Science. 262, 744-747 (1993).</li>
<li>Lynes, E. M., Bui, M., Yap, M. C., Benson, M. D., Schneider, B., Ellgaard, L., Berthiaume, L. G., Simmen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Palmitoylated+TMX+and+calnexin+target+to+the+mitochondria-associated+membrane%5BTitle%5D)+AND+%22EMBO+J%22%5BJournal%5D)">Palmitoylated TMX and calnexin target to the mitochondria-associated membrane.</a> EMBO J. 31 (2), 457-470 (2011).</li>
<li>Fujimoto, M., Hayashi, T., Su, T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+role+of+cholesterol+in+the+association+of+endoplasmic+reticulum+membranes+with+mitochondria%5BTitle%5D)+AND+%22Biochem.+Biophys.+Res.+Commun%22%5BJournal%5D)">The role of cholesterol in the association of endoplasmic reticulum membranes with mitochondria.</a> Biochem. Biophys. Res. Commun. 417 (1), 635-639 (2012).</li>
<li>Grimm, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+ER-mitochondria+interface%3A+the+social+network+of+cell+death%5BTitle%5D)+AND+%22Biochim.+Biophys.+Acta%22%5BJournal%5D)">The ER-mitochondria interface: the social network of cell death.</a> Biochim. Biophys. Acta. 1823 (2), 327-334 (2012).</li>
<li>Vance, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Phospholipid+synthesis+in+a+membrane+fraction+associated+with+mitochondria%5BTitle%5D)+AND+%22J.+Biol.+Chem%22%5BJournal%5D)">Phospholipid synthesis in a membrane fraction associated with mitochondria.</a> J. Biol. Chem. 265, 7248-7256 (1990).</li>
<li>Wieckowski, M. R., Giorgi, C., Lebiedzinska, M., Duszynski, J., Pinton, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+of+mitochondria-associated+membranes+and+mitochondria+from+animal+tissues+and+cells%5BTitle%5D)+AND+%22Nat.+Protoc%22%5BJournal%5D)">Isolation of mitochondria-associated membranes and mitochondria from animal tissues and cells.</a> Nat. Protoc. 4 (11), 1582-1590 (2009).</li>
<li>Pizzo, P., Viola, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lymphocyte+lipid+rafts%3A+structure+and+function%5BTitle%5D)+AND+%22Curr.+Opin.+Immunol%22%5BJournal%5D)">Lymphocyte lipid rafts: structure and function.</a> Curr. Opin. Immunol. 15 (3), 255-260 (2003).</li>
</ol>

None of the authors have any conflicts of interest to declare.

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><tbody><tr><td colspan="4">&lt;strong&gt;REAGENTS&lt;/strong&gt;</td></tr><tr><td colspan="4">&lt;strong&gt;Fractionation&lt;/strong&gt;</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 colspan="4">&lt;strong&gt;MAM&lt;/strong&gt; </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 colspan="4">&lt;strong&gt;GEM&lt;/strong&gt; </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 colspan="4">&lt;strong&gt;Common&lt;/strong&gt; </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 colspan="4">&lt;strong&gt;EQUIPMENT&lt;/strong&gt; </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&quot;</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

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 ...

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.

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

The overall goal of this procedure is to isolate the mitochondrion, endoplasmic reticular membrane, micro domains, or MAMs from mouse brain purified MAMs are further extracted to obtain the glyco finger lipid enriched micro domain fractions or gems. This is accomplished by first dissecting the mouse brain, which is then homogenized in a specific buffer using a dance homogenizer. The resulting homogenate is centrifuged at low speed to remove nuclei, unli cells, and day debris.The second step is to prepare a crude mitochondrial fraction. Through further centrifugation of the supinate, the resulting pellet is resuspended and further separated using a discontinuous sucrose gradient. Next, the crude mitochondrial fraction is applied to a peral gradient.After centrifugation and several washing steps, the pure mitochondrial fraction and the MAMs are obtained. The final step is to treat the MAMs with a solution containing TRITTON X 100 to extract the glyco finger lipid enriched micro domain fraction gem. Ultimately, the purity of the fractions is validated by western blos analyses by using antibodies against protein markers that a characteristic of the various organelle fractions.The main advantage of this procedure of an existing methods is that it has been adapted to the isolation of mitochondria mam and gem fractions from adult mouse brain using relatively small amount of study material. This optimized procedure is very reproducible and enables the isolation of sufficient quantities of the different organal fractions to be used by researchers, especially those in the neurobiology field. For the analysis of their proteins of interest, demonstrating the procedure will be a a, a technician from my laboratory.After removing the brain from a carbon dioxide euthanized mouse, begin this procedure by cutting the brain in half. Place each half into a separate two milliliter tube on ice. Weigh the tubes using an empty pre chilled two milliliter tube to zero the balance and store them on ice.Transfer half of the brain into a pre chilled two milliliter glass down tissue grinder containing one milliliter of cold fractionation solution. A homogenized with 15 total strokes of a large clearance pestle. Place the homogenate in a sterile 15 milliliter Falcon tube on ice and dilute up to 10 volumes of cold fractionation solution A based on the weight of the sample.Then centrifuge the sample at 1, 400 times G for 10 minutes of four degrees. SIUs carefully remove the supinate taking care not to disturb the pellet and transfer to a 30 milliliter round bottom glass centrifuge tube on ice. Next, resus, suspend the pellet in the same 10 volumes of fractionation solution.A homogenize the resuspended pellet in the same grinder, one milliliter at a time. This time use three to six strokes of a small clearance pestle. Combine the homogenous in a 15 milliliter Falcon tube and centrifuge at 710 times G for 10 minutes at four degrees Celsius.This results in appellative nuclei and cell debris. Carefully remove the sup natant and pull it with the previous supine natant saved from the earlier centrifugation to begin mitochondrial isolation centrifuge the S supernatants at 13, 800 times G.The 10 minutes of four degrees Celsius. Then transfer the snat to an UltraClear Beckman centrifuge tube on ice.Re suspend the pellets containing the mitochondria fraction in 10 volumes of fractionation solution A and homogenize one milliliter at a time. This time use three to six strokes of a small clearance Pele, combining the homogenates in a new 30 milliliter round bottom glass centrifuge tube on ice. Further purify the sample by repeating these steps one more time.Centrifuge the homogenate. Pull the S supernatants and re homogenize the cell pellet one milliliter at a time after suspending it in 10 volumes of solution A.The next step is to resuspend the pellet containing the enriched mitochondrial fraction in 4.8 milliliters of fractionation solution B per gram of original brain weight homogenize using six strokes of a small clearance pestle in a fresh homogenizer store this homogenate on ice. Then prepare a discontinuous sucrose gradient in an UltraClear Beckman centrifuge tube.First, add the resuspended palette followed by three milliliters of 850 millimolar sucrose, three milliliters of one molar sucrose, and finally three milliliters of 1.2 molar sucrose. Be sure to add the solution slowly from the bottom to ensure that no bubbles are formed. Finally, centrifuge at 82, 500 times G for two hours at four degrees Celsius.The resulting separation will produce three bands and appellate. The appellate contains the crude mitochondria that will be used in the subsequent purification steps. Reus bend the freshly isolated crude mitochondria from one brain half with two milliliters of isolation medium.Next, add eight milliliters of a 30%per core gradient to a fresh UltraClear Beckman centrifuge tube. Layer the mitochondrial suspension on top of the prepared gradient very slowly to avoid any bubble formation. Then centrifuge at 95, 000 times G for 30 minutes at four degrees Celsius following gradient separation.First, remove the heavy fraction, which is the lower band with a glass pasta pipette, and transfer it to a fresh round bottom glass tube. Store the tube on ice. Next, remove the light fraction, which is the upper band with a new glass pasta pipette and transfer to a separate fresh round bottom glass tube.Place this tube on ice as well. Dilute both fractions with 10 milliliters of isolation, medium and centrifuge at 6, 300 times G for 10 minutes at four degrees Celsius. Discard the supinate from the heavy fraction and re suspend the pellet with another 10 milliliters of isolation.Medium.Centrifuge the sample again with the same conditions. The resulting pellet will be the pure mitochondrial fraction. Next, transfer the supinate from the light fraction to an UltraClear Beckman centrifuge tube on ice.The pellet from this fraction is discarded as it contains contaminating mitochondria. Add sufficient isolation medium to the supinate from the light fraction to fill the tube and centrifuge at 100, 000 times G for one hour at four degrees Celsius. The resulting pellet will contain the mitochondria associated ER membranes or MAMs.Carefully remove and discard the supinate. Begin extraction of the glyco finger lipid enriched micro domains or gems by izing the MAM's fraction in 500 microliters to one milliliter of extraction buffer on ice for 20 minutes. Next, centrifuge the lysate 15, 300 times G for two minutes of four degrees Celsius.Collect the sup natant and centrifuge the pellet for two minutes to remove all remaining tritons 100 soluble material, and finally resuspend the pellet with the solubilizing buffer. This solubilized material represents the gem fractions and can be used for further analysis. Western blots were run to check the purity and distribution of markers for endoplasmic reticulum, cytosolic proteins, and mitochondrial markers in the cytosolic fraction.The endoplasmic reticulum fraction, the pure mitochondrial fraction and the MAMs fraction. Western blots also showed that the gems were positive for cavi in one, a marker of cli and negative for endoplasmic reticulum and mitochondrial proteins. On the other hand, the Triton X 100 extracted MAMs were positive for endoplasmic reticulum and mitochondrial markers and showed little cavi in one While attempting this procedure.It is important to remember to use freshly dissected brain tissue and to subsequently keep all solutions samples and tubes on ice. Also, it is recommended to save a small QUT of sample from each step of the fractionation to be able to determine via immuno blotting the overall success of the purification procedure.

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

Research

JoVE Journal

Neuroscience

Mitochondrial visualization and analysis from mammalian brain tissue is a challenging task. Here, we describe how three dimensional (3D) reconstruction analysis from the serial block-face scanning electron microscopy (SBFSEM) can be used to gain insights on the morphological and volumetric analysis of this critical energy generating organelle.



Analysis of Brain Mitochondria Using Serial Block-Face Scanning Electron Microscopy

Here, we describe a procedure to visualize and quantify with high sensitivity the endogenous interactions between the endoplasmic reticulum and mitochondria in fixed cells. The protocol features an optimized in situ proximity ligation assay targeting the inositol 1,4,5-triphosphate receptor/glucose-regulated protein 75/voltage-dependent anion channel/cyclophilin D complex at the mitochondria-associated membrane interface.

Study of Endoplasmic Reticulum and Mitochondria Interactions by In Situ Proximity Ligation Assay in Fixed Cells

We present a protocol to study the distribution of mitochondria and endoplasmic reticulum in whole cells after genetic modification using correlative light and volume electron microscopy including ascorbate peroxidase 2 and horseradish peroxidase staining, serial sectioning of cells with and without the target gene in the same section, and serial imaging via electron microscopy.

Mitochondria and Endoplasmic Reticulum Imaging by Correlative Light and Volume Electron Microscopy

We provide a protocol for isolation of microglia from different dissected regions of an adult mouse brain hemisphere, followed by semi-automated library preparation for deep single-cell RNA sequencing of full-length transcriptomes. This method will help to elucidate functional heterogeneity of microglia in health and disease.

Isolation of Region-specific Microglia from One Adult Mouse Brain Hemisphere for Deep Single-cell RNA Sequencing

We describe a detailed protocol for the generation of human induced pluripotent stem cell-derived brain organoids and their use in modeling mitochondrial diseases.

Generation of Human Brain Organoids for Mitochondrial Disease Modeling

Intensive preparation of intact mouse cerebral endothelial "tubes" from cerebral parenchymal arterioles is illustrated for studying cerebral blood flow regulation. Further, we demonstrate the experimental strengths of this endothelial study model for fluorescence imaging and electrophysiology measurement of key cellular signaling pathways, including changes in intracellular [Ca2+] and membrane potential.

Isolation and Functional Analysis of Arteriolar Endothelium of Mouse Brain Parenchyma

We present a two-step protocol for high-quality mitochondria isolation that is compatible with protein discovery and quantification at a proteome scale. Our protocol does not require genetic engineering and is thus suitable for studying mitochondria from any primary cells and tissues.

Two-Step Tag-Free Isolation of Mitochondria for Improved Protein Discovery and Quantification

Here, we describe measuring the axonal transport rate of constitutive stabilizers of mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs) by increasing or maintaining neurotoxic β-amyloid (Aβ) generation from Alzheimer's disease (AD) neurons in real-time to serve as a direct and quantitative metric to measure MAM stabilization and aid the development of AD therapeutics.

Quantitative Analysis of Mitochondria-Associated Endoplasmic Reticulum Membrane (MAM) Stabilization in a Neural Model of Alzheimer's Disease (AD)

This protocol describes a technique for the analysis of respiratory supercomplexes when only small amounts of samples are available.

Isolation of Mitochondria for Mitochondrial Supercomplex Analysis from Small Tissue and Cell Culture Samples

A protocol was developed for the preparation of purified mitochondria from microglial cells, isolation of mitochondrial proteins for N-glycan release, and rapid detection of subcellular, mitochondrial glycans using infrared matrix-assisted laser desorption electrospray ionization coupled to high-resolution accurate mass analyzer mass spectrometry.

Mitochondrial Preparation from Microglia for Glycan Analysis

Learn to isolate mitochondria-associated ER membranes (MAMs) and glycosphingolipid-enriched microdomains (GEMs) from adult mouse brain using homogenization and sucrose gradient centrifugation. This method enables neurobiology research.

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

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