Laboratoire de Bioimagerie et Pathologies, UMR 7021 CNRS, Université de Strasbourg, 74 route du Rhin, 67401 Illkirch, France.
MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, Headley Way, Oxford OX3 9DS, U.K.
Anal Chem. 2020 Nov 3;92(21):14798-14805. doi: 10.1021/acs.analchem.0c03559. Epub 2020 Oct 12.
Imaging of biological membranes by environmentally sensitive solvatochromic probes, such as Laurdan, provides information about the organization of lipids, their ordering, and their uneven distribution. To address a key drawback of Laurdan linked to its rapid internalization and subsequent labeling of internal membranes, we redesigned it by introducing a membrane anchor group based on negatively charged sulfonate and dodecyl chain. The obtained probe, Pro12A, stains exclusively the outer leaflet of lipid bilayers of liposomes, as evidenced by leaflet-specific fluorescence quenching with a viologen derivative, and shows higher fluorescence brightness than Laurdan. Pro12A also exhibits stronger spectral change between liquid-ordered and liquid-disordered phases in model membranes and distinguishes better lipid domains in giant plasma membrane vesicles (GPMVs) than Laurdan. In live cells, it stains exclusively the cell plasma membranes, in contrast to Laurdan and its carboxylate analogue C-Laurdan. Owing to its outer leaflet binding, Pro12A is much more sensitive to cholesterol extraction than Laurdan, which is redistributed within both plasma membrane leaflets and intracellular membranes. Finally, its operating range in the blue spectral region ensures the absence of crosstalk with a number of orange/red fluorescent proteins and dyes. Thus, Pro12A will enable accurate multicolor imaging of lipid organization of cell plasma membranes in the presence of fluorescently tagged proteins of interest, which will open new opportunities in biomembrane research.
环境敏感的溶致变色探针（如 Laurdan）对生物膜的成像提供了关于脂质组织、有序性及其不均匀分布的信息。为了解决与 Laurdan 快速内化及其随后对内膜标记相关的一个关键缺点，我们通过引入基于带负电荷的磺酸盐和十二烷基链的膜锚定基团对其进行了重新设计。所得探针 Pro12A 仅染色脂质体双层的外叶，这可以通过与紫精衍生物的叶层特异性荧光猝灭来证明，并且比 Laurdan 具有更高的荧光亮度。Pro12A 还在模型膜中表现出在液有序相与液无序相之间更强的光谱变化，并比 Laurdan 更好地区分巨质膜囊泡（GPMVs）中的脂质域。在活细胞中，它仅染色细胞膜，与 Laurdan 及其羧酸类似物 C-Laurdan 形成对比。由于其结合在外叶，Pro12A 比 Laurdan 对胆固醇提取更敏感，后者在质膜的两个叶层和细胞内膜内重新分布。最后，其在蓝色光谱区域的工作范围确保了与许多橙/红色荧光蛋白和染料的交叉干扰不存在。因此，Pro12A 将能够在存在荧光标记的感兴趣蛋白质的情况下准确地对细胞膜的脂质组织进行多色成像，这将为生物膜研究开辟新的机会。
