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Multi-color fluorescence fluctuation spectroscopy in living cells via spectral detection
eLife ( IF 7.7 ) Pub Date : 2021-09-08 , DOI: 10.7554/elife.69687
Valentin Dunsing 1 , Annett Petrich 1 , Salvatore Chiantia 1
Affiliation  

Signaling pathways in biological systems rely on specific interactions between multiple biomolecules. Fluorescence fluctuation spectroscopy provides a powerful toolbox to quantify such interactions directly in living cells. Cross-correlation analysis of spectrally separated fluctuations provides information about inter-molecular interactions but is usually limited to two fluorophore species. Here, we present scanning fluorescence spectral correlation spectroscopy (SFSCS), a versatile approach that can be implemented on commercial confocal microscopes, allowing the investigation of interactions between multiple protein species at the plasma membrane. We demonstrate that SFSCS enables cross-talk-free cross-correlation, diffusion and oligomerization analysis of up to four protein species labeled with strongly overlapping fluorophores. As an example, we investigate the interactions of influenza A virus (IAV) matrix protein 2 with two cellular host factors simultaneously. We furthermore apply raster spectral image correlation spectroscopy for the simultaneous analysis of up to four species and determine the stoichiometry of ternary IAV polymerase complexes in the cell nucleus.

中文翻译:

通过光谱检测实现活细胞中的多色荧光波动光谱

生物系统中的信号传导途径依赖于多个生物分子之间的特定相互作用。荧光波动光谱提供了一个强大的工具箱,可以直接量化活细胞中的这种相互作用。光谱分离波动的互相关分析提供了有关分子间相互作用的信息,但通常仅限于两种荧光团种类。在这里,我们提出了扫描荧光光谱相关光谱(SFSCS),这是一种可以在商用共聚焦显微镜上实施的多功能方法,可以研究质膜上多种蛋白质之间的相互作用。我们证明,SFSCS 能够对多达四种用强重叠荧光团标记的蛋白质种类进行无串扰的互相关、扩散和寡聚分析。例如,我们同时研究了甲型流感病毒 (IAV) 基质蛋白 2 与两种细胞宿主因子的相互作用。此外,我们还应用光栅光谱图像相关光谱法同时分析多达四个物种,并确定细胞核中三元 IAV 聚合酶复合物的化学计量。
更新日期:2021-09-08
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