Switchable Nile Red-Based Probe for Cholesterol and Lipid Order at the Outer Leaflet of Biomembranes
Switchable Nile Red-Based Probe for Cholesterol and Lipid Order at the Outer Leaflet of Biomembranes
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DOI：
10.1021/ja100351w
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发表时间：
2010-04-07
影响因子：
15
通讯作者：
Klymchenko, Andrey S.
中科院分区：
文献类型：
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作者：
Kucherak, Oleksandr A.;Oncul, Sule;Klymchenko, Andrey S.
胆固醇和鞘磷脂一起形成高度有序的膜相，其被认为在哺乳动物细胞的质膜中发挥重要的生物学功能。由于鞘磷脂主要存在于细胞膜的外部小叶，监测其脂质顺序需要能够在该小叶特异性结合并表现出可忽略的缓慢翻转的分子探针。在目前的工作中，这样的探针开发通过修改的溶剂化显色荧光染料尼罗红与两亲性锚基团。为了评估所获得的探针（NR 12 S）的触发器，我们开发了使用连二亚硫酸钠在一个小叶处可逆氧化还原切换其荧光的方法。该方法显示，与母体尼罗红相反，NR 12 S仅结合模型脂质囊泡和活细胞的外膜小叶，在小时的时间尺度内具有可忽略的触发。此外，NR 12 S在模型囊泡中的发射最大值在液体有序相（鞘磷脂-胆固醇）中与液体无序相（不饱和磷脂）相比表现出显著的蓝移。因此，这两个阶段可以清楚地区分在NR 12 S染色的巨大囊泡的荧光显微镜成像的探针发射光谱的蓝色和红色部分之间的强度比。被添加到活细胞中，NR 12 S主要（如果不是排他的话）结合它们的质膜，并且显示出介于模型膜的液体有序相和无序相之间的发射光谱。重要的是，NR 12 S的发射颜色与细胞膜中的胆固醇含量密切相关，这允许通过荧光光谱和显微镜监测甲基-β-环糊精的胆固醇消耗过程。NR 12 S具有吸引人的生物物理和开关特性，以及其选择性的外小叶染色和对胆固醇和脂质顺序的敏感性，使其成为研究模型和细胞膜的新的强大工具。
Cholesterol and sphingomyelin form together a highly ordered membrane phase, which is believed to play important biological functions in plasma membranes of mammalian cells. Since sphingomyelin is present mainly at the outer leaflet of cell membranes, monitoring its lipid order requires molecular probes capable to bind specifically at this leaflet and exhibit negligibly slow flip-flop. In the present work, such a probe was developed by modifying the solvatochromic fluorescent dye Nile Red with an amphiphilic anchor group. To evaluate the flip-flop of the obtained probe (NR12S), we developed a methodology of reversible redox switching of its fluorescence at one leaflet using sodium dithionite. This method shows that NR12S, in contrast to parent Nile Red, binds exclusively the outer membrane leaflet of model lipid vesicles and living cells with negligible flip-flop in the time scale of hours. Moreover, the emission maximum of NR12S in model vesicles exhibits a significant blue shift in liquid ordered phase (sphingomyelin-cholesterol) as compared to liquid disordered phase (unsaturated phospholipids). As a consequence, these two phases could be clearly distinguished in NR12S-stained giant vesicles by fluorescence microscopy imaging of intensity ratio between the blue and red parts of the probe emission spectrum. Being added to living cells, NR12S binds predominantly, if not exclusively, their plasma membranes and shows an emission spectrum intermediate between those in liquid ordered and disordered phases of model membranes. Importantly, the emission color of NR12S correlates well with the cholesterol content in cell membranes, which allows monitoring the cholesterol depletion process with methyl-beta-cyclodextrin by fluorescence spectroscopy and microscopy. The attractive photophysical and switching properties of NR12S, together with its selective outer leaflet staining and sensitivity to cholesterol and lipid order, make it a new powerful tool for studying model and cell membranes.