Max Planck Institute for Molecular Physiology, Department of Systemic Cell Biology, Dortmund, Germany.
Nat Methods. 2010 Jun;7(6):467-72. doi: 10.1038/nmeth.1458. Epub 2010 May 9.
Extracellular stimuli are transduced inside the cell by posttranslational modifications (PTMs), such as phosphorylation, of proteins in signaling networks. Insight into the structure of these networks requires quantification of PTM levels in individual cells. Fluorescence resonance energy transfer (FRET) measured by fluorescence lifetime imaging microscopy (FLIM) is a powerful tool to image PTM levels in situ. FLIM on cell arrays that express fluorescent protein fusions can quantify tyrosine phosphorylation patterns in large networks in individual cells. We identified tyrosine kinase substrates by imaging their phosphorylation levels after inhibition of protein tyrosine phosphatases. Analysis of the correlation between protein phosphorylation and expression levels at single cell resolution allowed us to identify positive feedback motifs. Using FLIM on cell arrays (CA-FLIM), we uncovered components that transduce signals from epidermal growth factor receptor.
细胞外刺激通过信号网络中蛋白质的翻译后修饰（PTMs），如磷酸化，在细胞内转导。要深入了解这些网络的结构，需要定量测定单个细胞中 PTM 的水平。通过荧光寿命成像显微镜（FLIM）测量的荧光共振能量转移（FRET）是原位成像 PTM 水平的有力工具。表达荧光蛋白融合物的细胞阵列上的 FLIM 可以定量测定单个细胞中大型网络中的酪氨酸磷酸化模式。我们通过抑制蛋白酪氨酸磷酸酶后成像其磷酸化水平来鉴定酪氨酸激酶底物。在单细胞分辨率下对蛋白质磷酸化与表达水平之间的相关性进行分析，使我们能够鉴定正反馈模体。我们使用细胞阵列上的 FLIM（CA-FLIM）揭示了从表皮生长因子受体传递信号的组件。
