Pernuš Agata, Langowski Jörg
Division Biophysics of Macromolecules, DKFZ, Heidelberg, Germany.
PLoS One. 2015 Apr 14;10(4):e0123070. doi: 10.1371/journal.pone.0123070. eCollection 2015.
We collected mobility and interaction maps of c-Fos-eGFP and c-Jun-mRFP1 transcription factors within living cell nuclei. c-Fos dimerizes with c-Jun to form the transcription activator protein-1 (AP-1) which binds to the specific recognition site. To monitor this process, we used fluorescence cross-correlation spectroscopy on a single plane illumination microscope (SPIM-FCCS), which provides diffusion coefficient and protein-protein interaction data in the whole image plane simultaneously, instead of just one point on conventional confocal FCS. We find a strong correlation between diffusional mobility and interaction: regions of strong interaction show slow mobility. Controls containing either an eGFP-mRFP dimer, separately expressing eGFP and mRPF, or c-Fos-eGFP and c-Jun-mRFP1 mutants lacking dimerization and DNA-binding domains, showed no such correlation. These results extend our earlier findings from confocal FCCS to include spatial information.
我们收集了活细胞核内c-Fos-eGFP和c-Jun-mRFP1转录因子的迁移率和相互作用图谱。c-Fos与c-Jun二聚化形成转录激活蛋白-1（AP-1），该蛋白与特定识别位点结合。为了监测这一过程，我们在单平面照明显微镜（SPIM-FCCS）上使用了荧光交叉相关光谱技术，该技术可同时在整个图像平面提供扩散系数和蛋白质-蛋白质相互作用数据，而不是像传统共聚焦FCS那样仅在一个点上获取数据。我们发现扩散迁移率与相互作用之间存在很强的相关性：强相互作用区域显示出缓慢的迁移率。含有eGFP-mRFP二聚体、分别表达eGFP和mRPF的对照，或缺乏二聚化和DNA结合结构域的c-Fos-eGFP和c-Jun-mRFP1突变体，均未显示出这种相关性。这些结果将我们早期共聚焦FCCS的研究结果扩展到了包括空间信息。
