Fluorescence anisotropy measurements of lipid order in plasma membranes and lipid rafts from RBL-2H3 mast cells
Fluorescence anisotropy measurements of lipid order in plasma membranes and lipid rafts from RBL-2H3 mast cells
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DOI：
10.1021/bi010496c
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发表时间：
2001-10-16
期刊：
影响因子：
2.9
通讯作者：
Baird, B
中科院分区：
文献类型：
--
作者：
Gidwani, A;Holowka, D;Baird, B
被称为脂筏的专门质膜结构域参与信号转导和其他细胞过程，并且它们的液体有序（L-o）相似乎对其功能很重要。为了定量生物膜中的有序脂质，我们研究了两种脂质探针2-[3-（二苯基己三烯基）丙酰基]-1-十六烷酰基-sn-甘油基-3-磷酸胆碱（DPH-PC）和N-（7-硝基苯-2-氧杂-1，3-二氮唑-4-基）-1，2-二十六烷酰基-sn-甘油基-3-磷酸乙醇胺（NBD-PE）的稳态荧光各向异性。我们使用模型膜与不同量的胆固醇，稳态荧光各向异性是一个敏感的措施，胆固醇依赖的顺序。结果表明，DPH-PC是一种比NBD-PE更敏感的探针。在胆固醇的存在下，排序也取决于磷脂酰基链的饱和度。使用DPH-PC，我们发现RBL-2 H3肥大细胞的质膜基本上是有序的，大约40%，通过与完全处于液体有序（L-O）相和液体无序（L-alpha）相的模型膜的各向异性值进行比较来确定。该结果与约30%的质膜磷脂不溶于0.5%Triton X-100的发现一致。此外，耐洗涤剂的膜分离的Triton X-100细胞裂解物的蔗糖梯度分馏比质膜囊泡更有序，这表明，它们代表了一个更有序的子集的质膜。用甲基-β-环糊精处理质膜囊泡，导致75%胆固醇消耗，导致脂质顺序相应降低，如通过DPH-PC和NBD-PE的各向异性所测量的。这些结果表明，稳态荧光各向异性的DPH-PC是一个有用的方法来衡量的数量的脂质秩序的生物膜。
Specialized plasma membrane domains known as lipid rafts participate in signal transduction and other cellular processes, and their liquid ordered (L-o) phase appears to be important for their function. To quantify ordered lipids in biological membranes, we investigated steady-state fluorescence anisotropy of two lipid probes, 2-[3-(diphenylhexatrienyl)propanoyl]-1-hexadecanoyl-sn-glycero-3-phosphocholine (DPH-PC) and N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (NBD-PE). We show using model membranes with varying amounts of cholesterol that steady-state fluorescence anisotropy is a sensitive measure of cholesterol-dependent ordering. The results suggest that DPH-PC is a more sensitive probe than NBD-PE. In the presence of cholesterol, ordering also depends on the degree of saturation of the phospholipid acyl chains. Using DPH-PC, we find that the plasma membrane of RBL-2H3 mast cells is substantially ordered, roughly 40%, as determined by comparison with anisotropy values for model membranes entirely in a liquid ordered (L-o) phase and in a liquid disordered (L-alpha) phase. This result is consistent with the finding that similar to 30% of plasma membrane phospholipids are insoluble in 0.5% Triton X-100. Furthermore, detergent-resistant membranes isolated by sucrose gradient fractionation of Triton X-100 cell lysates are more ordered than plasma membrane vesicles, suggesting, that they represent a more ordered subset of the plasma membrane. Treatment of plasma membrane vesicles with methyl-beta -cyclodextrin resulting in 75% cholesterol depletion leads to commensurate decreases in lipid order as measured by anisotropy of DPH-PC and NBD-PE. These results demonstrate that steady-state fluorescence anisotropy of DPH-PC is a useful way to measure the amount of lipid order in biological membranes.