Annunziata Ida, Patterson Annette, d'Azzo Alessandra
Department of Genetics, St Jude Children's Research Hospital, USA.
J Vis Exp. 2013 Mar 4(73):e50215. doi: 10.3791/50215.
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 components(1,2,3,4). The inter-organellar membrane microdomains that are formed between the endoplasmic reticulum (ER) and the mitochondria at the opening of the IP3-sensitive Ca(2+) channel are known as the mitochondria associated-ER membranes or MAMs(4,5,6). 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 Ca(2+) (4,5,6). The ER serves as the primary store of intracellular Ca(2+), and in this capacity regulates a myriad of cellular processes downstream of Ca(2+) signaling, including post-translational protein folding and protein maturation (7). Mitochondria, on the other hand, maintain Ca(2+) homeostasis, by buffering cytosolic Ca(2+) concentration thereby preventing the initiation of apoptotic pathways downstream of Ca(2+) unbalance(4,8). The dynamic nature of the MAMs makes them ideal sites to dissect basic cellular mechanisms, including Ca(2+) signaling and regulation of mitochondrial Ca(2+) 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 cells(13,14). 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 interactions(4,15).
细胞内细胞器是具有不同形状和组成的高度动态结构，受到细胞特异性的内在和外在信号的影响。它们的膜常常在特定的接触位点并列，这些位点成为信号分子和膜成分交换的中心（1,2,3,4）。在内质网（ER）和线粒体之间，在IP3敏感的Ca(2+)通道开口处形成的细胞器间膜微区被称为线粒体相关内质网膜或MAMs（4,5,6）。这些膜接触位点的蛋白质/脂质组成和生化特性已得到广泛研究，特别是关于它们在调节细胞内Ca(2+)方面的作用（4,5,6）。内质网作为细胞内Ca(2+)的主要储存库，并以此调节Ca(2+)信号下游的众多细胞过程，包括翻译后蛋白质折叠和蛋白质成熟（7）。另一方面，线粒体通过缓冲细胞质Ca(2+)浓度来维持Ca(2+)稳态，从而防止Ca(2+)失衡下游凋亡途径的启动（4,8）。MAMs的动态性质使其成为剖析基本细胞机制的理想位点，包括Ca(2+)信号传导和线粒体Ca(2+)浓度调节、脂质生物合成和运输、能量代谢以及细胞存活（4,9,10,11,12）。已经描述了几种从肝组织和培养细胞中纯化这些微区的方法（13,14）。考虑到先前发表的方法，我们改编了一种从成年小鼠脑中分离线粒体和MAMs的方案。在此过程中，我们增加了一个额外的纯化步骤，即Triton X100提取，这使得能够分离MAMs中富含糖鞘脂的微区（GEM）部分。这些GEM制剂与源自质膜或其他细胞内膜的小窝和脂筏共享几种蛋白质成分，并被认为作为受体蛋白聚集和蛋白质 - 蛋白质相互作用的聚集点（4,15）。
