Oncul Sule, Klymchenko Andrey S, Kucherak Oleksandr A, Demchenko Alexander P, Martin Sophie, Dontenwill Monique, Arntz Youri, Didier Pascal, Duportail Guy, Mély Yves
Laboratoire de Biophotonique et Pharmacologie, UMR 7213 CNRS, Université de Strasbourg, Faculté de Pharmacie, ILLKIRCH Cedex, France.
Biochim Biophys Acta. 2010 Jul;1798(7):1436-43. doi: 10.1016/j.bbamem.2010.01.013. Epub 2010 Jan 25.
Herein, using a recently developed hydration-sensitive ratiometric biomembrane probe based on 3-hydroxyflavone (F2N12S) that binds selectively to the outer leaflet of plasma membranes, we compared plasma membranes of living cells and lipid vesicles as model membranes. Through the spectroscopic analysis of the probe response, we characterized the membranes in terms of hydration and polarity (electrostatics). The hydration parameter value in cell membranes was in between the values obtained with liquid ordered (Lo) and liquid disordered (Ld) phases in model membranes, suggesting that cell plasma membranes exhibit a significant fraction of Lo phase in their outer leaflet. Moreover, two-photon fluorescence microscopy experiments show that cell membranes labeled with this probe exhibit a homogeneous lipid distribution, suggesting that the putative domains in Lo phase are distributed all over the membrane and are highly dynamic. Cholesterol depletion affected dramatically the dual emission of the probe suggesting the disappearance of the Lo phase in cell membranes. These conclusions were corroborated with the viscosity sensitive diphenylhexatriene derivative TMA-DPH, showing membrane fluidity in intact cells intermediate between those for Lo and Ld phases in model membranes, as well as a significant increase in fluidity after cholesterol depletion. Moreover, we observed that cell apoptosis results in a similar loss of Lo phase, which could be attributed to a flip of sphingomyelin from the outer to the inner leaflet of the plasma membrane due to apoptosis-driven lipid scrambling. Our data suggest a new methodology for evaluating the Lo phase in membranes of living cells.
在此，我们使用一种最近开发的基于3-羟基黄酮（F2N12S）的对水合作用敏感的比率生物膜探针，该探针能选择性地结合质膜的外小叶，我们将活细胞的质膜与作为模型膜的脂质囊泡进行了比较。通过对探针响应的光谱分析，我们从水合作用和极性（静电学）方面对膜进行了表征。细胞膜中的水合参数值介于模型膜中液晶有序（Lo）相和液晶无序（Ld）相所获得的值之间，这表明细胞质膜在外小叶中呈现出相当比例的Lo相。此外，双光子荧光显微镜实验表明，用该探针标记的细胞膜呈现出均匀的脂质分布，这表明Lo相中的假定结构域分布在整个膜上且具有高度动态性。胆固醇耗尽显著影响了探针的双发射，表明细胞膜中Lo相消失。这些结论通过对粘度敏感的二苯基己三烯衍生物TMA-DPH得到了证实，该衍生物显示完整细胞中的膜流动性介于模型膜中Lo相和Ld相的膜流动性之间，并且在胆固醇耗尽后流动性显著增加。此外，我们观察到细胞凋亡导致类似的Lo相丧失，这可能归因于凋亡驱动的脂质翻转导致鞘磷脂从质膜的外小叶翻转到内小叶。我们的数据提出了一种评估活细胞膜中Lo相的新方法。
