Ras diffusion is sensitive to plasma membrane viscosity
Ras diffusion is sensitive to plasma membrane viscosity
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DOI：
10.1529/biophysj.104.055640
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发表时间：
2005-08-01
影响因子：
3.4
通讯作者：
Kenworthy, AK
中科院分区：
文献类型：
--
作者：
Goodwin, JS;Drake, KR;Kenworthy, AK
细胞表面含有各种屏障和障碍，这些屏障和障碍减缓了糖基磷脂酰肌醇（GPI）锚定和跨膜蛋白的横向扩散，低于膜粘度所施加的理论极限。只存在于内层的蛋白质如何扩散。质膜的调控机制在很大程度上尚未被探索。我们在这里表明，小GTPase Ras的扩散对质膜的粘度很敏感。利用光漂白后的共聚焦荧光恢复，我们检测了绿色荧光蛋白（GFP）标记的HRas、NRas和KRas在装载或耗尽胆固醇（众所周知的膜双层粘度调节剂）的COS-7细胞中的扩散。在含有过量胆固醇的细胞中，GFP-HRas、GFP-NRas和GFP-KRas的扩散流动性显著降低，与粘度敏感脂质探针DiIC（16）和DiIC（18）的行为相似。然而，胆固醇消耗对细胞膜中蛋白质和脂质扩散的影响高度依赖于所使用的消耗方法。甲基- β -环糊精去除胆固醇通过一种与粘度无关的机制减缓Ras扩散，而隔夜去除胆固醇则略微增加蛋白质和脂质扩散。Ras感知膜粘度的能力可能代表了驻留在质膜细胞质表面的蛋白质的一般特征。
The cell surface contains a variety of barriers and obstacles that slow the lateral diffusion of glycosylphosphatidylinositol (GPI)-anchored and transmembrane proteins below the theoretical limit imposed by membrane viscosity. How the diffusion of proteins residing exclusively on the inner lea. et of the plasma membrane is regulated has been largely unexplored. We show here that the diffusion of the small GTPase Ras is sensitive to the viscosity of the plasma membrane. Using confocal fluorescence recovery after photobleaching, we examined the diffusion of green fluorescent protein (GFP)-tagged HRas, NRas, and KRas in COS-7 cells loaded with or depleted of cholesterol, a well-known modulator of membrane bilayer viscosity. In cells loaded with excess cholesterol, the diffusional mobilities of GFP-HRas, GFP-NRas, and GFP-KRas were significantly reduced, paralleling the behavior of the viscosity-sensitive lipid probes DiIC(16) and DiIC(18). However, the effects of cholesterol depletion on protein and lipid diffusion in cell membranes were highly dependent on the depletion method used. Cholesterol depletion with methyl-beta-cyclodextrin slowed Ras diffusion by a viscosity-independent mechanism, whereas overnight cholesterol depletion slightly increased both protein and lipid diffusion. The ability of Ras to sense membrane viscosity may represent a general feature of proteins residing on the cytoplasmic face of the plasma membrane.