PHOSPHOLIPID TRANSMEMBRANE DOMAINS AND LATERAL DIFFUSION IN FIBROBLASTS
PHOSPHOLIPID TRANSMEMBRANE DOMAINS AND LATERAL DIFFUSION IN FIBROBLASTS
复制标题
DOI：
10.1073/pnas.90.2.447
复制
发表时间：
1993-01-15
影响因子：
11.1
通讯作者：
DEVAUX, PF
中科院分区：
文献类型：
--
作者：
CHAHINE, JMEH;CRIBIER, S;DEVAUX, PF
用调制条纹图光漂白法研究了荧光磷脂在培养的中国仓鼠肺成纤维细胞中的侧向扩散。当细胞被标记并保持在7 ℃时，荧光仍然定位在质膜上。N-[6-（7-硝基苯-2-氧杂-1，3-二唑-4-基-氨基）己酰基]鞘氨醇磷酸胆碱（C6-NBD-SphPCho）和1-酰基-2-[6-（7-硝基苯-2-氧杂-1，3-二唑-4-基-氨基）己酰基]磷脂酰胆碱（C6-NBD-PtdCho）均以相同的表观侧向扩散系数（D1几乎等于0.3 x 10（-9）cm 2/s）扩散。相比之下，磷脂酰丝氨酸衍生物{1-酰基-2-[6-（7-硝基苯-2-氧杂-1，3-二唑-4-基-氨基）己酰基]磷脂酰丝氨酸（C6-NBD-PtdSer）}产生两个扩散分量：一个慢分量D1，类似于含胆碱脂质的测量值，和一个快分量（D2几乎等于2 × 10（-9）cm 2/s）。快速组分仅存在于含ATP的细胞中。研究表明，它与C6-NBD-PtdSer移位到内瓣叶有关。这表明两个小叶形成非常不同的膜结构域。在较高的温度下，在含胆碱脂质和氨基脂质之间观察到相同的迁移率差异。然而，使用C6-NBD-SphPCho，随着时间的推移逐渐出现一部分非常缓慢扩散或准固定化的探针。这可能是由于鞘磷脂在内化后位于小细胞器中。从这个组件的幅度在不同的时间间隔注册，我们计算出，几乎等于50%的质膜鞘磷脂回收在不到30分钟的中国仓鼠成纤维细胞的ATP和微管依赖性的过程。
The lateral diffusion of fluorescent phospholipids in cultured Chinese hamster lung fibroblasts was examined by modulated fringe pattern photobleaching. When cells were labeled and maintained at 7-degrees-C, the fluorescence remained localized at the plasma membrane. N-[6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl-amino)caproyl]sphingosylphosphocholine (C6-NBD-SphPCho) and 1-acyl-2-[6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl-amino)caproyl] phosphatidylcholine (C6-NBD-PtdCho) both diffused with the same apparent lateral diffusion coefficient (D1 almost-equal-to 0.3 x 10(-9) cm2/s). By contrast, the phosphatidylserine derivative {1-acyl-2-[6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl-amino)caproyl] phosphatidylserine (C6-NBD-PtdSer)} gave rise to two diffusional components: a slow component, D1, analogous to that measured with the choline-containing lipids, and a fast component (D2 almost-equal-to 2 x 10(-9) cm2/s). The fast component only exists in ATP-containing cells. It was shown to be associated with C6-NBD-PtdSer translocated to the inner leaflet. This indicates that the two leaflets form very different membranous domains. At higher temperature, the same difference in mobility was, observed between the choline-containing lipids and the aminolipid. However, with C6-NBD-SphPCho, a fraction of very slowly diffusing or quasi-immobilized probes gradually appeared with time. This could be attributed to sphingomyelin located in small organelles after internalization. From the amplitude of this component registered at different intervals, we calculated that almost-equal-to 50% of the plasma membrane sphingomyelin is recycled in less than 30 min in Chinese hamster fibroblasts by an ATP- and microtubule-dependent process.