Kabbani Abir M, Kelly Christopher V
Department of Physics and Astronomy, Wayne State University, Detroit, Michigan.
Department of Physics and Astronomy, Wayne State University, Detroit, Michigan.
Biophys J. 2017 Oct 17;113(8):1795-1806. doi: 10.1016/j.bpj.2017.08.031.
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 <50 nm radius, grew to >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)× more concentrated on the positive curvature top and (26 ± 11)× 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.
对于内吞作用和外排作用，细胞膜通过脂质、蛋白质和碳水化合物的纳米级重组在平面、出芽和囊泡拓扑结构之间转变。然而，此前理解纳米级弯曲初始阶段的尝试受到实验分辨率的限制。通过实施偏振定位显微镜技术，本文报道了霍乱毒素B亚基（CTxB）在准单组分支撑脂质双层膜中固有的膜弯曲能力。首次检测到半径小于50纳米的膜芽，其生长至半径大于200纳米，并延伸成更长的小管，这取决于膜张力和CTxB浓度。与平面支撑脂质双层膜的浓度相比，CTxB在纳米级膜芽的正曲率顶部浓度高（12±4）倍，在负高斯曲率颈部浓度高（26±11）倍。CTxB经常被用作有序脂质相的标记物；然而，CTxB与膜弯曲之间的耦合为CTxB诱导的膜重组提供了另一种理解方式。这些发现通过将CTxB聚集和扩散与CTxB诱导的膜弯曲相关联，使得能够重新解释先前的观察结果。对单个脂质和CTxB进行了单粒子追踪，以揭示单分子扩散、CTxB积累和膜拓扑结构之间的相关性。在纳米级芽位置观察到脂质和CTxB扩散减慢，这表明膜弯曲时有效膜粘度或分子拥挤程度局部增加。这些结果表明，CTxB诱导的固有膜弯曲是细胞中启动CTxB内化的一种机制，可能独立于网格蛋白、小窝蛋白、肌动蛋白和脂质相分离。
