Erdelyi Miklos, Simon Joseph, Barnard Eric A, Kaminski Clemens F
Department of Chemical Engineering and Biotechnology, University of Cambridge, Cambridge, United Kingdom; Analytical Science Division, National Physical Laboratory, Teddington, Middlesex, United Kingdom; Department of Optics and Quantum Electronics, University of Szeged, Szeged, Hungary.
Department of Pharmacology, University of Cambridge, Cambridge, United Kingdom.
PLoS One. 2014 Jun 19;9(6):e100526. doi: 10.1371/journal.pone.0100526. eCollection 2014.
Signaling within and between animal cells is controlled by the many receptor proteins in their membrane. They variously operate as trans-membrane monomers and homo- or hetero-dimers, and may assemble with ion-channels: analyses thereof are needed in studies of receptor actions in tissue physiology and pathology. Interactions between membrane proteins are detectable when pre-labeled with fluorophores, but a much fuller analysis is achievable via advanced optical techniques on living cells. In this context, the measurement of polarization anisotropy in the emitted fluorescence has been the least exploited. Here we demonstrate its methodology and particular advantages in the study of receptor protein assembly. Through excitation in both TIRF and EPI fluorescence illumination modes we are able to quantify and suppress contributions to the signal from extraneous intra-cellular fluorescence, and we show that the loss of fluorescence-polarization measured in membrane proteins reports on receptor protein assembly in real time. Receptor monomers and homo-dimers in the cell membrane can be analyzed quantitatively and for homo-dimers only a single fluorescent marker is needed, thus suppressing ambiguities that arise in alternative assays, which require multiple label moieties and which are thus subject to stoichiometric uncertainty.
动物细胞内及细胞间的信号传导由细胞膜中的多种受体蛋白控制。它们分别作为跨膜单体、同二聚体或异二聚体发挥作用，并且可能与离子通道组装在一起：在组织生理学和病理学中研究受体作用时需要对此进行分析。当用荧光团预先标记时，膜蛋白之间的相互作用是可检测的，但通过对活细胞采用先进的光学技术可以实现更全面的分析。在这种情况下，发射荧光中偏振各向异性的测量利用得最少。在此，我们展示了其在受体蛋白组装研究中的方法及独特优势。通过全内反射荧光（TIRF）和落射荧光（EPI）照明模式进行激发，我们能够量化并抑制细胞内无关荧光对信号的贡献，并且我们表明，膜蛋白中测得的荧光偏振损失实时反映了受体蛋白的组装情况。细胞膜中的受体单体和同二聚体可以进行定量分析，对于同二聚体仅需一个荧光标记，从而消除了在需要多个标记部分且存在化学计量不确定性的替代检测方法中出现的模糊性。
