Hiramoto-Yamaki Nao, Tanaka Kenji A K, Suzuki Kenichi G N, Hirosawa Koichiro M, Miyahara Manami S H, Kalay Ziya, Tanaka Koichiro, Kasai Rinshi S, Kusumi Akihiro, Fujiwara Takahiro K
Institute for Integrated Cell-Material Sciences (WPI-iCeMS) and Institute for Frontier Medical Sciences, Kyoto University, Kyoto, 606-8507, Japan.
Traffic. 2014 Jun;15(6):583-612. doi: 10.1111/tra.12163. Epub 2014 Mar 11.
Cholesterol distribution and dynamics in the plasma membrane (PM) are poorly understood. The recent development of Bodipy488-conjugated cholesterol molecule (Bdp-Chol) allowed us to study cholesterol behavior in the PM, using single fluorescent-molecule imaging. Surprisingly, in the intact PM, Bdp-Chol diffused at the fastest rate ever found for any molecules in the PM, with a median diffusion coefficient (D) of 3.4 µm²/second, which was ∼10 times greater than that of non-raft phospholipid molecules (0.33 µm²/second), despite Bdp-Chol's probable association with raft domains. Furthermore, Bdp-Chol exhibited no sign of entrapment in time scales longer than 0.5 milliseconds. In the blebbed PM, where actin filaments were largely depleted, Bdp-Chol and Cy3-conjugated dioleoylphosphatidylethanolamine (Cy3-DOPE) diffused at comparable Ds (medians = 5.8 and 6.2 µm²/second, respectively), indicating that the actin-based membrane skeleton reduces the D of Bdp-Chol only by a factor of ∼2 from that in the blebbed PM, whereas it reduces the D of Cy3-DOPE by a factor of ∼20. These results are consistent with the previously proposed model, in which the PM is compartmentalized by the actin-based membrane-skeleton fence and its associated transmembrane picket proteins for the macroscopic diffusion of all of the membrane molecules, and suggest that the probability of Bdp-Chol passing through the compartment boundaries, once it enters the boundary, is ∼10× greater than that of Cy3-DOPE. Since the compartment sizes are greater than those of the putative raft domains, we conclude that raft domains coexist with membrane-skeleton-induced compartments and are contained within them.
人们对胆固醇在质膜（PM）中的分布和动态了解甚少。最近，硼二吡咯488缀合胆固醇分子（Bdp-Chol）的开发使我们能够通过单荧光分子成像研究胆固醇在质膜中的行为。令人惊讶的是，在完整的质膜中，Bdp-Chol以质膜中任何分子所发现的最快速率扩散，中位扩散系数（D）为3.4μm²/秒，尽管Bdp-Chol可能与筏结构域相关联，但该系数比非筏磷脂分子的扩散系数（0.33μm²/秒）大10倍。此外，在时间尺度超过0.5毫秒时，Bdp-Chol没有表现出被困的迹象。在肌动蛋白丝大量缺失的泡状质膜中，Bdp-Chol和Cy3缀合的二油酰磷脂酰乙醇胺（Cy3-DOPE）以相当的扩散系数扩散（中位数分别为5.8和6.2μm²/秒），这表明基于肌动蛋白的膜骨架仅使Bdp-Chol的扩散系数比泡状质膜中的扩散系数降低约2倍，而它使Cy3-DOPE的扩散系数降低约20倍。这些结果与先前提出的模型一致，在该模型中，质膜由基于肌动蛋白的膜骨架围栏及其相关的跨膜桩蛋白分隔，以实现所有膜分子的宏观扩散，并表明Bdp-Chol一旦进入边界，穿过隔室边界的概率比Cy3-DOPE大10倍。由于隔室大小大于假定的筏结构域的大小，我们得出结论，筏结构域与膜骨架诱导的隔室共存并包含在其中。
