Zal Tomasz
Department of Immunology, University of Texas, MD Anderson Cancer Center, Unit 902, 7455 Fannin, Houston TX, USA.
Adv Exp Med Biol. 2008;640:183-97. doi: 10.1007/978-0-387-09789-3_14.
Ligand binding to cell membrane receptors sets off a series of protein interactions that convey the nuances ofligand identity to the cell interior. The information may be encoded in conformational changes, the interaction kinetics and, in the case of multichain immunoreceptors, by chain rearrangements. The signals may be modulated by dynamic compartmentalization of the cell membrane, cellular architecture, motility, and activation--all of which are difficult to reconstitute for studies of receptor signaling in vitro. In this chapter, we will discuss how protein interactions in general and receptor signaling in particular can be studied in living cells by different fluorescence imaging techniques. Particularly versatile are methods that exploit Förster resonance energy transfer (FRET), which is exquisitely sensitive to the nanometer-range proximity and orientation between fluorophores. Fluorescence correlation microscopy (FCM) can provide complementary information about the stoichiometry and diffusion kinetics of large complexes, while bimolecular fluorescence complementation (BiFC) and other complementation techniques can capture transient interactions. A continuing challenge is extracting from the imaging data the quantitative information that is necessary to verify different models of signal transduction.
配体与细胞膜受体的结合引发了一系列蛋白质相互作用，这些相互作用将配体特性的细微差别传递到细胞内部。信息可能编码在构象变化、相互作用动力学中，对于多链免疫受体而言，还可能通过链重排来编码。信号可能会受到细胞膜的动态分隔、细胞结构、运动性和激活的调节——所有这些在体外研究受体信号传导时都难以重建。在本章中，我们将讨论如何通过不同的荧光成像技术在活细胞中研究一般的蛋白质相互作用，特别是受体信号传导。利用福斯特共振能量转移（FRET）的方法特别通用，该方法对荧光团之间纳米级的距离和方向极为敏感。荧光相关显微镜（FCM）可以提供有关大复合物的化学计量和扩散动力学的补充信息，而双分子荧光互补（BiFC）和其他互补技术可以捕捉瞬时相互作用。一个持续的挑战是从成像数据中提取验证不同信号转导模型所需的定量信息。
