Structure and cholesterol dynamics of caveolae/raft and nonraft plasma membrane domains
Structure and cholesterol dynamics of caveolae/raft and nonraft plasma membrane domains
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DOI：
10.1021/bi0602720
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发表时间：
2006-10-03
期刊：
影响因子：
2.9
通讯作者：
Ball, Judith M.
中科院分区：
文献类型：
--
作者：
Gallegos, Adalberto M.;Storey, Stephen M.;Ball, Judith M.
尽管认识到质膜由脂筏结构域组成，这些结构域是多种信号通路和其他细胞功能的关键组织部位，但关于这些微域的结构和功能的信息有限。为了开始解决这些问题，通过三种不同的技术对MDCK膜进行了细分，以产生(I)耐洗涤剂膜(DRM)和洗涤剂可溶膜(DSM)，(Ii)非洗涤剂小窝/筏(NDCR)，和(Iii)非洗涤剂亲和纯化小窝/筏(ACR)和非小窝/非筏(NR)。ACR与其他PM结构域或细胞内膜的交叉污染最少，与包含最高水平交叉污染的DRM形成鲜明对比。脱氢麦角甾醇(DHE)是一种天然存在的荧光甾醇，其光谱性质表明ACR、NDCR和NR不含结晶甾醇，这与完整细胞的质膜中缺少结晶甾醇是一致的。相反，DRM含有大量的结晶甾醇。膜探针的荧光偏振分析表明，ACR的流动性最小，跨双层流动性梯度最大，液体有序度最高，对甾醇载体蛋白-2(SCP-2)反应最灵敏。相反，DRM具有与NR相似的结构特性、异常(非常快)的自发甾醇动力学以及对SCP-2无反应的甾醇动力学。DRM与非洗涤剂制剂(ACR和NDCR)的结构和功能特性之间的差异不是由于洗涤剂的存在。首次从MDCK细胞中分离到一个非去污剂、亲和纯化(ACR)的脂类结构域组分，与非脂筏(NR)相比，它具有独特的结构(非晶态甾醇、液体有序、高跨双层流动性梯度)和功能(胆固醇动力学)特性。综上所述，这项研究表明，用三种不同方法分离的膜微域具有独特的结构、功能和组织特征。
Despite recognition that the plasma membrane (PM) is comprised of lipid raft domains that are key organizing sites of multiple signaling pathways and other cell functions, limited information is available regarding the structure and function in sterol dynamics of these microdomains. To begin to resolve these issues, MDCK membranes were subfractionated by three different techniques to produce (i) detergent-resistant membranes (DRM) and detergent-soluble membranes (DSM), (ii) nondetergent caveolae/rafts (NDCR), and (iii) nondetergent, affinity-purified caveolae/rafts (ACR) and noncaveolae/nonrafts (NR). ACR exhibited the least cross contamination with other PM domains or intracellular membranes, in marked contrast to DRM that contained the highest level of cross contaminants. Spectral properties of dehydroergosterol (DHE), a naturally occurring fluorescent sterol, showed that ACR, NDCR, and NR did not contain crystalline sterol, consistent with the lack of crystalline sterol in PM of intact cells. In contrast, DRM contained significant levels of crystalline sterol. Fluorescence polarization of membrane probes showed that ACR were the least fluid and had the highest transbilayer fluidity gradient, the most liquid ordered phase, and the sterol dynamics most responsive to sterol carrier protein-2 (SCP-2). In contrast, DRM had structural properties similar to those of NR, anomalous (very fast) spontaneous sterol dynamics, and sterol dynamics that were unresponsive to SCP-2. Differences between the structural and functional properties of DRM and those of the nondetergent preparations (ACR and NDCR) were not due to the presence of detergent. A nondetergent, affinity-purified (ACR) lipid domain fraction isolated from MDCK cells for the first time revealed unique structural (noncrystalline sterol, liquid-ordered, high transbilayer fluidity gradient) and functional (cholesterol dynamics) properties of lipid rafts as compared to nonrafts (NR). In summary, this study showed membrane microdomains (rafts/caveolae) isolated by three different methodologies have unique structural, functional, and organizational characteristics.