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: 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
Note: Keep brain halves separate throughout the entire procedure. The following applies to the treatment of a single brain half.
2. Second Step: Crude Mitochondria Isolation
3. Isolation of Mitochondria-associated ER Membrane, MAMs
* Note: Make gradient buffer more concentrated (1.43 times) to achieve correct final concentration after diluting Percoll to 30%.
Note: At this step, you may also centrifuge at 100,000 x g for 1 hr, 4 °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
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 diffuse and broad band above the mitochondria. After careful recovery of the individual gradient bands, the purified fractions are resuspended in a lysis buffer, separated on SDS polyacrylamide gels and blotted onto PVDF membranes. Blots are then probed with a battery of antibodies for specific protein markers that will ascertain the purity of the fractions and their protein composition. Figure 2A shows the distribution of cytosolic, ER and mitochondrial markers in the isolated MAMs and mitochondrial fractions. Pure mitochondria preparations should be devoid of both ER and cytosolic markers. The close apposition between ER and mitochondrial membranes at the MAM contact sites explains the presence of calreticulin (ER marker) and Tom-20 (mitochondria marker) in these purified preparations. It is also possible to use MAMs specific markers FACL4 and PACS2, as well as other markers enriched within these domains, such as IP3R-1 and GRP754. The MAMs can be further extracted with Triton X-100 to obtain the GEMs. These microdomains, which contain components of lipid rafts and/or caveolae are caveolin-1 positive as shown in Figure 2B.
Figure 1. Shows a picture of the layering of the mitochondrial-associated ER membrane (MAMs) and the pure mitochondrial fractions (mito-p).
Figure 2. A) Shows western blots run to check the purity and the distribution of cytosolic, ER and mitochondrial markers in Cytosolic (cyto)-, ER-, pure mitochondrial fractions (mito-p)- and MAM-fractions. B) Western blots of the purified glycosphingolipid enriched microdomains (GEMs) and Triton X-100 extracted (Triton extr. MAMs) fractions isolated from MAMs.
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. This protocol has been successfully implemented for the identification of the molecular effectors that specify these microdomains, and underlie the apoptotic pathway downstream of Ca2+ imbalance leading to neuronal cell death in a neurodegenerative metabolic disease in children4.
The authors have nothing to disclose.
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).
Name of Reagent/Material | Company | Catalogue Number | Yorumlar |
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, 14×89 mm | Beckman-Coulter | 344059 | |
Parafilm | Cole-Parmer | PM996 | |
Disposable Borosilicate Glass Pasteur Pipets, 9″ | Fisher Scientific | 13-678-20C |