Three-color confocal Förster (or fluorescence) resonance energy transfer microscopy: Quantitative analysis of protein interactions in the nucleation of actin filaments in live cells.
Three-color confocal Förster (or fluorescence) resonance energy transfer microscopy: Quantitative analysis of protein interactions in the nucleation of actin filaments in live cells.
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DOI：
10.1002/cyto.a.22651
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发表时间：
2015-06
期刊：
影响因子：
3.7
通讯作者：
Bloom, George S.
中科院分区：
文献类型：
--
作者：
Wallrabe, Horst;Sun, Yuansheng;Fang, Xiaolan;Periasamy, Ammasi;Bloom, George S.
关键词：
使用活细胞3色FRET显微镜和相应的相同蛋白质的体外生物化学重建进行实验，以评估肌动蛋白丝成核。3色FRET数据的新应用被证明，扩展了传统的能量转移效率（E%）计算之外的分析。转染MDCK细胞以共表达Teal-N-WASP/Venus-IQGAP 1/mRFP 1-Rac 1、Teal-N-WASP/Venus-IQGAP 1/mRFP 1-Cdc 42、mCFP-Rac 1/Venus-IQGAP 1/mCherry-肌动蛋白或CFP-Cdc 42/Venus-IQGAP 1/mCherry-肌动蛋白，并使用单标记等效物进行光谱渗漏校正。使用确认的E%作为切入点，荧光水平和相关比率在细胞周边的离散累积水平上相关。CFP-Rac 1：Venus-IQGAP 1比率的上升与较低的整体肌动蛋白荧光相关，而CFP-Cdc 42：Venus-IQGAP 1比率在低比率下与增加的肌动蛋白荧光相关，并且在较高比率下是中性的。新的FRET分析还表明，mRFP 1-Cdc 42或mRFP 1-Rac 1水平的升高分别促进或抑制Teal-N-WASP与Venus-IQGAP 1的结合。这些3色FRET分析进一步支持我们的体外结果IQGAP 1，Rac 1和Cdc 42在肌动蛋白成核中的作用，以及Rac 1和Cdc 42对N-WASP与IQGAP 1的关联的差异影响。此外，这项工作强调了3色FRET作为同时评估单个活细胞中多种相互作用蛋白质的系统生物学策略的强大功能。
Experiments using live cell 3-color FRET microscopy and corresponding in vitro biochemical reconstitution of the same proteins were conducted to evaluate actin filament nucleation A novel application of 3-color FRET data is demonstrated, extending the analysis beyond the customary energy transfer efficiency (E%) calculations.. MDCK cells were transfected for co-expression of Teal-N-WASP/Venus-IQGAP1/mRFP1-Rac1, Teal-N-WASP/Venus-IQGAP1/mRFP1-Cdc42, mCFP-Rac1/Venus-IQGAP1/mCherry-actin or CFP-Cdc42/Venus-IQGAP1/mCherry-actin, and with single-label equivalents for spectral bleedthrough correction. Using confirmed E% as an entry point, fluorescence levels and related ratios were correlated at discrete accumulating levels at cell peripheries. Rising ratios of CFP-Rac1:Venus-IQGAP1 were correlated with lower overall actin fluorescence, whereas the CFP-Cdc42:Venus-IQGAP1 ratio correlated with increased actin fluorescence at low ratios and was neutral at higher ratios. The new FRET analyses also indicated that rising levels of mRFP1-Cdc42 or mRFP1-Rac1 respectively promoted or suppressed association of Teal-N-WASP with Venus-IQGAP1. These 3-color FRET assays further support our in vitro results about the role of IQGAP1, Rac1 and Cdc42 in actin nucleation, and the differential impact of Rac1 and Cdc42 on the association of N-WASP with IQGAP1. Moreover, this work emphasizes the power of 3-color FRET as a systems biology strategy for simultaneous evaluation of multiple interacting proteins in individual live cells.
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