Nanoscale Membrane Budding Induced by CTxB and Detected via Polarized Localization Microscopy
Nanoscale Membrane Budding Induced by CTxB and Detected via Polarized Localization Microscopy
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DOI：
10.1016/j.bpj.2017.08.031
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发表时间：
2017-10-17
影响因子：
3.4
通讯作者：
Kelly, Christopher V.
中科院分区：
文献类型：
--
作者：
Kabbani, Abir M.;Kelly, Christopher V.
对于内吞作用和胞吐作用，膜通过脂质、蛋白质和碳水化合物的纳米级重组在平面、出芽和囊泡形态之间转换。然而，先前试图了解纳米尺度弯曲的初始阶段受到实验分辨率的限制。本文通过极化定位显微镜的应用，报道了霍乱毒素亚单位B （CTxB）在准单组分支持的脂质双分子层中固有的膜弯曲能力。膜芽首先在200 nm半径处被检测到，随着膜张力和CTxB浓度的增加，膜芽扩展成更长的小管。与平面支撑脂质双分子层相比，CTxB在纳米膜芽正曲率顶部的浓度高（12 +/- 4）倍，在负高斯曲率颈部的浓度高（26 +/- 11）倍。CTxB经常被用作液体有序脂质相的标记物；然而，CTxB和膜弯曲之间的耦合为CTxB诱导的膜重组提供了另一种理解。这些发现允许通过将CTxB聚集和扩散与CTxB诱导的膜弯曲相关来重新解释先前的观察结果。对单个脂质和CTxB进行单粒子跟踪，以揭示单分子扩散、CTxB积累和膜形貌之间的相关性。在纳米级芽位置观察到脂质和CTxB扩散减慢，表明膜弯曲时有效膜粘度或分子拥挤的局部增加。这些结果表明，固有的CTxB诱导的膜弯曲是细胞内启动CTxB内化的机制，可以独立于网格蛋白、小窝蛋白、肌动蛋白和脂质相分离。
For endocytosis and exocytosis, membranes transition among planar, budding, and vesicular topographies through nanoscale reorganization of lipids, proteins, and carbohydrates. However, prior attempts to understand the initial stages of nanoscale bending have been limited by experimental resolution. Through the implementation of polarized localization microscopy, this article reports the inherent membrane bending capability of cholera toxin subunit B (CTxB) in quasi-one-component-supported lipid bilayers. Membrane buds were first detected with 200 nm radius, and extended into longer tubules with dependence on the membrane tension and CTxB concentration. Compared to the concentration of the planar-supported lipid bilayers, CTxB was (12 +/- 4)x more concentrated on the positive curvature top and (26 +/- 11)x more concentrated on the negative Gaussian curvature neck of the nanoscale membrane buds. CTxB is frequently used as a marker for liquid-ordered lipid phases; however, the coupling between CTxB and membrane bending provides an alternate understanding of CTxB-induced membrane reorganization. These findings allow for the reinterpretation of prior observations by correlating CTxB clustering and diffusion to CTxB-induced membrane bending. Single-particle tracking was performed on single lipids and CTxB to reveal the correlations among single-molecule diffusion, CTxB accumulation, and membrane topography. Slowed lipid and CTxB diffusion was observed at the nanoscale bud locations, suggesting a local increase in the effective membrane viscosity or molecular crowding upon membrane bending. These results suggest inherent CTxB-induced membrane bending as a mechanism for initiating CTxB internalization in cells that could be independent of clathrin, caveolin, actin, and lipid phase separation.