Asano Atsushi, Selvaraj Vimal, Buttke Danielle E, Nelson Jacquelyn L, Green Karin M, Evans James E, Travis Alexander J
The Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853, USA.
J Cell Physiol. 2009 Mar;218(3):537-48. doi: 10.1002/jcp.21623.
Despite enormous interest in membrane raft micro-domains, no studies in any cell type have defined the relative compositions of the raft fractions on the basis of their major components--sterols, phospholipids, and proteins--or additional raft-associating lipids such as the ganglioside, G(M1). Our previous localization data in live sperm showed that the plasma membrane overlying the acrosome represents a stabilized platform enriched in G(M1) and sterols. These findings, along with the physiological requirement for sterol efflux for sperm to function, prompted us to characterize sperm membrane fractions biochemically. After confirming limitations of commonly used detergent-based approaches, we utilized a non-detergent-based method, separating membrane fractions that were reproducibly distinct based on sterol, G(M1), phospholipid, and protein compositions (both mass amounts and molar ratios). Based on fraction buoyancy and biochemical composition, we identified at least three highly reproducible sub-types of membrane raft. Electron microscopy revealed that raft fractions were free of visible contaminants and were separated by buoyancy rather than morphology. Quantitative proteomic comparisons and fluorescence localization of lipids suggested that different organelles contributed differentially to individual raft sub-types, but that multiple membrane micro-domain sub-types could exist within individual domains. This has important implications for scaffolding functions broadly associated with rafts. Most importantly, we show that the common practice of characterizing membrane domains as either "raft" or "non-raft" oversimplifies the actual biochemical complexity of cellular membranes.
尽管人们对膜筏微结构域有着浓厚兴趣，但在任何细胞类型中，都没有研究基于其主要成分——固醇、磷脂和蛋白质——或其他与筏相关的脂质（如神经节苷脂G(M1)）来定义筏组分的相对组成。我们之前在活精子中的定位数据表明，顶体上方的质膜代表了一个富含G(M1)和固醇的稳定平台。这些发现，连同精子功能所需的固醇流出的生理需求，促使我们对精子膜组分进行生化表征。在确认了常用的基于去污剂的方法的局限性后，我们采用了一种非基于去污剂的方法，根据固醇、G(M1)、磷脂和蛋白质组成（质量含量和摩尔比）分离出可重复区分的膜组分。基于组分浮力和生化组成，我们鉴定出至少三种高度可重复的膜筏亚型。电子显微镜显示，筏组分没有可见污染物，并且是通过浮力而非形态分离的。脂质的定量蛋白质组学比较和荧光定位表明，不同的细胞器对各个筏亚型的贡献不同，但在单个结构域内可能存在多种膜微结构域亚型。这对于与筏广泛相关的支架功能具有重要意义。最重要的是，我们表明将膜结构域简单地分为“筏”或“非筏”的常见做法过于简化了细胞膜实际的生化复杂性。
