Pralle A, Keller P, Florin E L, Simons K, Hörber J K
Cell Biology and Biophysics, European Molecular Biology Laboratory, D-69117 Heidelberg, Germany.
J Cell Biol. 2000 Mar 6;148(5):997-1008. doi: 10.1083/jcb.148.5.997.
To probe the dynamics and size of lipid rafts in the membrane of living cells, the local diffusion of single membrane proteins was measured. A laser trap was used to confine the motion of a bead bound to a raft protein to a small area (diam < or = 100 nm) and to measure its local diffusion by high resolution single particle tracking. Using protein constructs with identical ectodomains and different membrane regions and vice versa, we demonstrate that this method provides the viscous damping of the membrane domain in the lipid bilayer. When glycosylphosphatidylinositol (GPI) -anchored and transmembrane proteins are raft-associated, their diffusion becomes independent of the type of membrane anchor and is significantly reduced compared with that of nonraft transmembrane proteins. Cholesterol depletion accelerates the diffusion of raft-associated proteins for transmembrane raft proteins to the level of transmembrane nonraft proteins and for GPI-anchored proteins even further. Raft-associated GPI-anchored proteins were never observed to dissociate from the raft within the measurement intervals of up to 10 min. The measurements agree with lipid rafts being cholesterol-stabilized complexes of 26 +/- 13 nm in size diffusing as one entity for minutes.
为了探究活细胞膜中脂筏的动力学和大小，我们测量了单个膜蛋白的局部扩散。使用激光阱将与筏蛋白结合的珠子的运动限制在一个小区域（直径≤100 nm），并通过高分辨率单粒子追踪测量其局部扩散。通过使用具有相同胞外结构域和不同膜区域的蛋白构建体，反之亦然，我们证明了该方法可提供脂质双层中膜结构域的粘性阻尼。当糖基磷脂酰肌醇（GPI）锚定蛋白和跨膜蛋白与筏相关时，它们的扩散变得与膜锚类型无关，并且与非筏跨膜蛋白相比显著降低。胆固醇耗竭会加速与筏相关的跨膜蛋白的扩散，使其达到跨膜非筏蛋白的水平，对于GPI锚定蛋白甚至更高。在长达10分钟的测量间隔内，从未观察到与筏相关的GPI锚定蛋白从筏上解离。这些测量结果与脂筏是大小为26±13 nm的胆固醇稳定复合物且作为一个整体扩散数分钟的观点一致。
