Single Plane Illumination Microscopy (SPIM) revolutionized time lapse imaging of live cells and organisms due to its high speed and reduced photodamage. Quantitative mapping of molecular (co)mobility by fluorescence (cross-)correlation spectroscopy (F(C)CS) in a SPIM has been introduced to reveal molecular diffusion and binding. A complementary aspect of interactions is proximity, which can be studied by Förster resonance energy transfer (FRET). Here, we extend SPIM-FCCS by alternating laser excitation, which reduces false positive cross-correlation and facilitates comapping of FRET. Thus, different aspects of interacting systems can be studied simultaneously, and molecular subpopulations can be discriminated by multiparameter analysis. After demonstrating the benefits of the method on the AP-1 transcription factor, the dimerization and DNA binding behavior of retinoic acid receptor (RAR) and retinoid X receptor (RXR) is revealed, and an extension of the molecular switch model of the nuclear receptor action is proposed. Our data imply that RAR agonist enhances RAR-RXR heterodimerization, and chromatin binding/dimerization are positively correlated. We also propose a ligand induced conformational change bringing the N-termini of RAR and RXR closer together. The RXR agonist increased homodimerization of RXR suggesting that RXR may act as an autonomous transcription factor.

中文翻译：

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分子邻近性和协同性的同时定位揭示了激动剂增强的二聚作用和核受体的DNA结合。

单平面照明显微镜（SPIM）由于其高速度和减少的光损伤而彻底改变了活细胞和生物体的延时成像。通过在SPIM中通过荧光（互）相关光谱（F（C）CS）定量绘制分子（共）迁移率的图谱以揭示分子的扩散和结合。相互作用的一个补充方面是接近性，可以通过Förster共振能量转移（FRET）进行研究。在这里，我们通过交替的激光激发来扩展SPIM-FCCS，这减少了假正互相关并促进了FRET的包裹。因此，可以同时研究相互作用系统的不同方面，并可以通过多参数分析来区分分子亚群。在论证了该方法对AP-1转录因子的好处后，揭示了视黄酸受体（RAR）和类维生素A X受体（RXR）的二聚化和DNA结合行为，并提出了扩展核开关作用分子开关模型的方法。我们的数据暗示RAR激动剂可增强RAR-RXR异二聚化，并且染色质结合/二聚化呈正相关。我们还提出了配体诱导的构象变化，使RAR和RXR的N-末端更加靠近。RXR激动剂增加了RXR的同二聚化，提示RXR可以充当自主转录因子。与染色质结合/二聚化呈正相关。我们还提出了配体诱导的构象变化，使RAR和RXR的N-末端更加靠近。RXR激动剂增加了RXR的同二聚化，提示RXR可以充当自主转录因子。与染色质结合/二聚化呈正相关。我们还提出了配体诱导的构象变化，使RAR和RXR的N-末端更加靠近。RXR激动剂增加了RXR的同二聚化，提示RXR可以充当自主转录因子。