Brock Roland, Jovin Thomas M
Department of Molecular Biology, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.
Cytometry A. 2003 Mar;52(1):1-11. doi: 10.1002/cyto.a.10024.
The molecular analysis of intracellular signal transduction requires technologies that address quantitatively the activation of signaling proteins and formation of molecular complexes without disrupting cellular integrity.
Cells expressing the epidermal growth factor receptor (EGFR) in its endogenous form or fused to green fluorescent protein were incubated with 1-microm microspheres covalently functionalized with EGF. The disposition of the plasma membrane about the microspheres was analyzed by high-resolution confocal microscopy in combination with computational resolution enhancement and optimized fixation procedures. Receptor activation and translocation of signaling proteins to the microspheres was quantitated by image processing protocols for recovering the microsphere-associated fluorescence and the fluorescence in the local environment.
EGF-functionalized microspheres were internalized in an activation-dependent manner similar to that of the soluble growth factor. The correlation of receptor activation and recruitment of a signaling protein was analyzed quantitatively by isolating immunofluorescence signals from the microspheres and from their immediate environment.
The microsphere-based approach provides a quantitative analysis of cellular signal transduction with subcellular resolution under conditions maintaining cellular integrity. The analysis of signaling-induced (co)localization of proteins around a microsphere complements other technologies directly probing for molecular interactions such as fluorescence resonance energy transfer.
细胞内信号转导的分子分析需要能够在不破坏细胞完整性的情况下定量研究信号蛋白激活和分子复合物形成的技术。
将内源性表达表皮生长因子受体（EGFR）或与绿色荧光蛋白融合的细胞与用表皮生长因子共价功能化的1微米微球一起孵育。通过高分辨率共聚焦显微镜结合计算分辨率增强和优化的固定程序来分析微球周围质膜的分布情况。通过用于恢复微球相关荧光和局部环境中荧光的图像处理协议来定量受体激活和信号蛋白向微球的转位。
表皮生长因子功能化的微球以类似于可溶性生长因子的激活依赖方式被内化。通过从微球及其紧邻环境中分离免疫荧光信号，定量分析了受体激活与信号蛋白募集之间的相关性。
基于微球的方法在维持细胞完整性的条件下，以亚细胞分辨率对细胞信号转导进行定量分析。对微球周围信号诱导的蛋白质（共）定位分析补充了其他直接探测分子相互作用的技术，如荧光共振能量转移。
