Sphingolipid-cholesterol rafts diffuse as small entities in the plasma membrane of mammalian cells.
Sphingolipid-cholesterol rafts diffuse as small entities in the plasma membrane of mammalian cells.
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DOI：
10.1083/jcb.148.5.997
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发表时间：
2000-03-06
影响因子：
7.8
通讯作者：
Horber, J K
中科院分区：
文献类型：
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作者：
Pralle, A;Keller, P;Florin, E L;Simons, K;Horber, J K
为了探测活细胞膜中脂筏的动态和大小，测量了单个膜蛋白的局部扩散。利用激光陷阱将与筏蛋白结合的珠子的运动限制在一个小区域内（直径≤ 100 nm），并通过高分辨率单粒子跟踪测量其局部扩散。使用相同的胞外域和不同的膜区域，反之亦然的蛋白质结构，我们证明了这种方法提供了在脂质双层的膜域的粘性阻尼。当糖基磷脂酰肌醇（GPI）锚定和跨膜蛋白筏相关，其扩散变得独立的膜锚的类型，并显着减少相比，nonraft跨膜蛋白。胆固醇耗竭加速跨膜筏蛋白的筏相关蛋白的扩散到跨膜非筏蛋白的水平，甚至进一步促进GPI锚定蛋白的扩散。筏相关的GPI锚定蛋白从未观察到解离的测量间隔内的筏长达10分钟。测量同意与脂筏胆固醇稳定的复合物的大小为26 ± 13 nm的扩散作为一个实体分钟。
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 ≤ 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.