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Neuroscience

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

Published: March 4th, 2013

DOI:

10.3791/50215

1Department of Genetics, St Jude Children's Research Hospital
* These authors contributed equally

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 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–protein interactions4,15.

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:

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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 .......

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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. .......

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We acknowledge the contribution of Renata Sano in conceiving the initial protocol. A.d’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).

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

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