Effect of receptor dimerization on membrane lipid raft structure continuously quantified on single cells by camera based fluorescence correlation spectroscopy.
Effect of receptor dimerization on membrane lipid raft structure continuously quantified on single cells by camera based fluorescence correlation spectroscopy.
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DOI：
10.1371/journal.pone.0121777
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Pralle A
中科院分区：
文献类型：
--
作者：
Huang H;Simsek MF;Jin W;Pralle A
膜结合细胞信号传导受膜超微结构调节，膜超微结构本身可能受信号传导影响。然而，实时测量完整细胞中膜蛋白与膜结构的相互作用带来了相当大的挑战。在本文中，我们提出了一种非破坏性的荧光方法，量化单细胞中的这些相互作用，并且能够连续监测同一细胞以观察微小的变化。这种方法结合了全内荧光显微镜和荧光相关光谱，同时测量不同大小区域中蛋白质的扩散和分子浓度。它能正确区分与膜栅栏相互作用的蛋白质和与胆固醇稳定结构域或脂筏相互作用的蛋白质。该方法检测膜超微结构或蛋白质二聚化倾向的小扰动。通过对单细胞的连续监测，我们展示了GPI锚定蛋白的二聚化如何增加其与结构域的关联。使用双色的方法，我们研究了一个GPI锚定蛋白的二聚化对同一细胞中表达的另一种GPI锚定蛋白的影响。细胞表面的扫描揭示了胆固醇稳定结构域和膜细胞骨架之间的相关性。
Membrane bound cell signaling is modulated by the membrane ultra-structure, which itself may be affected by signaling. However, measuring the interaction of membrane proteins with membrane structures in intact cells in real-time poses considerable challenges. In this paper we present a non-destructive fluorescence method that quantifies these interactions in single cells, and is able to monitor the same cell continuously to observe small changes. This approach combines total internal fluorescence microscopy with fluorescence correlation spectroscopy to measure the protein’s diffusion and molecular concentration in different sized areas simultaneously. It correctly differentiates proteins interacting with membrane fences from proteins interacting with cholesterol-stabilized domains, or lipid rafts. This method detects small perturbations of the membrane ultra-structure or of a protein’s tendency to dimerize. Through continuous monitoring of single cells, we demonstrate how dimerization of GPI-anchored proteins increases their association with the structural domains. Using a dual-color approach we study the effect of dimerization of one GPI-anchored protein on another type of GPI-anchored protein expressed in the same cell. Scans over the cell surface reveal a correlation between cholesterol stabilized domains and membrane cytoskeleton.
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