Cholesterol segregates into submicrometric domains at the living erythrocyte membrane: evidence and regulation
Cholesterol segregates into submicrometric domains at the living erythrocyte membrane: evidence and regulation
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DOI：
10.1007/s00018-015-1951-x
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发表时间：
2015-12-01
影响因子：
8
通讯作者：
Tyteca, Donatienne
中科院分区：
文献类型：
--
作者：
Carquin, Melanie;Conrard, Louise;Tyteca, Donatienne
虽然胆固醇是必不可少的膜流动性和变形性，其水平的横向异质性在活细胞的质膜是知之甚少，由于缺乏适当的探针。我们在这里报告的有用性的产气荚膜梭菌毒素的D4片段融合到mCherry（θ *），作为具体的，无毒的，敏感的和定量的胆固醇标记工具，使用红细胞平膜。通过共聚焦显微镜，θ * 标签的胆固醇富集亚微米领域的盖玻片蔓延，但也凝胶悬浮（非拉伸）新鲜红细胞，这表明在体内的相关性。胆固醇结构域的传播红细胞是稳定的时间和空间，限制膜：血影蛋白锚定通过4.1R复合物，并依赖于温度和鞘磷脂，表明联合调节外在膜：细胞骨架相互作用和内在的脂质包装。胆固醇结构域与BODIPY-鞘磷脂富集结构域部分共定位。总之，我们表明，θ * 是一个有用的重要探针，研究胆固醇的组织和证明，胆固醇形式在活细胞中的亚微米域。
Although cholesterol is essential for membrane fluidity and deformability, the level of its lateral heterogeneity at the plasma membrane of living cells is poorly understood due to lack of appropriate probe. We here report on the usefulness of the D4 fragment of Clostridium perfringens toxin fused to mCherry (theta*), as specific, non-toxic, sensitive and quantitative cholesterol-labeling tool, using erythrocyte flat membrane. By confocal microscopy, theta* labels cholesterol-enriched submicrometric domains in coverslip-spread but also gel-suspended (non-stretched) fresh erythrocytes, suggesting in vivo relevance. Cholesterol domains on spread erythrocytes are stable in time and space, restricted by membrane:spectrin anchorage via 4.1R complexes, and depend on temperature and sphingomyelin, indicating combined regulation by extrinsic membrane:cytoskeleton interaction and by intrinsic lipid packing. Cholesterol domains partially co-localize with BODIPY-sphingomyelin-enriched domains. In conclusion, we show that theta* is a useful vital probe to study cholesterol organization and demonstrate that cholesterol forms submicrometric domains in living cells.