Nanoscale Science and Technology Laboratory, Institute for Advanced Study, Nanchang University, 999 Xuefu Ave., Honggutan District, Nanchang, 330031, Jiangxi, China.
J Membr Biol. 2014 Feb;247(2):189-200. doi: 10.1007/s00232-013-9624-x. Epub 2014 Jan 8.
The nonionic detergent extraction at 4 °C and the cholesterol-depletion-induced lipid raft disruption are the two widely used experimental strategies for lipid raft research. However, the effects of raft disruption and/or cold treatment on the ultrastructural and mechanical properties of cells are still unclear. Here, we evaluated the effects of raft disruption and/or cold (4 °C) treatment on these properties of living human umbilical vein endothelial cells (HUVECs). At first, the cholesterol-depletion-induced raft disruption was visualized by confocal microscopy and atomic force microscopy (AFM) in combination with fluorescent quantum dots. Next, the cold-induced cell contraction and the formation of end-branched filopodia were observed by confocal microscopy and AFM. Then, the cell-surface ultrastructures were imaged by AFM, and the data showed that raft disruption and cold treatment induced opposite effects on cell-surface roughness (a significant decrease and a significant increase, respectively). Moreover, the cell-surface mechanical properties (stiffness and adhesion force) of raft-disrupted- and/or cold-treated HUVECs were measured by the force measurement function of AFM. We found that raft disruption and cold treatment induced parallel effects on cell stiffness (increase) or adhesion force (decrease) and that the combination of the two treatments caused dramatically strengthened effects. Finally, raft disruption was found to significantly impair cell migration as previously reported, whereas temporary cold treatment only caused a slight but nonsignificant decrease in cell migration performed at physiological temperature. Although the mechanisms for causing these results might be complicated and more in-depth studies will be needed, our data may provide important information for better understanding the effects of raft disruption or cold treatment on cells and the two strategies for lipid raft research.
在 4°C 下使用非离子型去污剂提取和胆固醇耗竭诱导的脂筏破坏是两种广泛用于脂质筏研究的实验策略。然而，脂筏破坏和/或冷处理对细胞超微结构和力学性能的影响仍不清楚。在这里，我们评估了脂筏破坏和/或冷（4°C）处理对活的人脐静脉内皮细胞（HUVEC）这些性质的影响。首先，通过共聚焦显微镜和原子力显微镜（AFM）结合荧光量子点可视化胆固醇耗竭诱导的脂筏破坏。接下来，通过共聚焦显微镜和 AFM 观察冷诱导的细胞收缩和分支丝状伪足的形成。然后，通过 AFM 对细胞表面超微结构进行成像，数据显示脂筏破坏和冷处理对细胞表面粗糙度产生相反的影响（显著降低和显著增加）。此外，通过 AFM 的力测量功能测量了脂筏破坏和/或冷处理的 HUVEC 的细胞表面力学性质（刚性和粘附力）。我们发现，脂筏破坏和冷处理对细胞刚性（增加）或粘附力（降低）产生平行影响，两种处理的组合导致作用显著增强。最后，如先前报道的那样，发现脂筏破坏显著损害细胞迁移，而暂时的冷处理仅在生理温度下进行的细胞迁移中引起轻微但无统计学意义的降低。尽管引起这些结果的机制可能很复杂，需要进行更深入的研究，但我们的数据可为更好地理解脂筏破坏或冷处理对细胞的影响以及脂质筏研究的两种策略提供重要信息。
