Multiphoton-FLIM quantification of the EGFP-mRFP1 FRET pair for localization of membrane receptor-kinase interactions
Multiphoton-FLIM quantification of the EGFP-mRFP1 FRET pair for localization of membrane receptor-kinase interactions
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DOI：
10.1529/biophysj.104.050153
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发表时间：
2005-02-01
影响因子：
3.4
通讯作者：
Ng, T
中科院分区：
文献类型：
--
作者：
Peter, M;Ameer-Beg, SM;Ng, T
我们提出了一种改进的单体形式的红色荧光蛋白，mRFP 1，作为受体在生物荧光共振能量转移（FRET）实验中使用增强的绿色荧光蛋白作为供体。我们发现使用这种荧光团对定量测量FRET使用多光子荧光寿命成像显微镜（FLIM）的特别优势。该技术被用来证明一种新的受体激酶之间的相互作用的趋化因子受体（CXCR 4）和蛋白激酶C（PKC）α在癌细胞的活细胞和固定细胞实验。CXCR4-EGFP：PKCalpha-mRFP 1复合物被发现精确定位于细胞内囊泡和细胞突起时，通过多光子荧光-FLIM成像。使用mRFP 1标记的调控结构域或全长PKCalpha作为受体获得的FRET效率的比较显示，PKCalpha，在封闭（无活性）的形式，从与CXCR 4的细胞质部分相关联的限制。活细胞FLIM实验表明，该受体：激酶复合物的组装伴随着内吞过程。这被实验证据所证实，表明CXCR 4受体的再循环在用佛波酯刺激时增加，并且在通过双吲哚基马来酰亚胺抑制PKC时被阻断。EGFP-mRFP 1偶联应该是广泛适用的，特别是活细胞定量FRET测定。
We present an improved monomeric form of the red fluorescent protein, mRFP1, as the acceptor in biological fluorescence resonance energy transfer ( FRET) experiments using the enhanced green fluorescent protein as donor. We find particular advantage in using this fluorophore pair for quantitative measurements of FRET using multiphoton fluorescence lifetime imaging microscopy (FLIM). The technique was exploited to demonstrate a novel receptor-kinase interaction between the chemokine receptor (CXCR4) and protein kinase C (PKC) alpha in carcinoma cells for both live-and fixed-cell experiments. The CXCR4-EGFP: PKCalpha-mRFP1 complex was found to be localized precisely to intracellular vesicles and cell protrusions when imaged by multiphoton fluorescence-FLIM. A comparison of the FRET efficiencies obtained using mRFP1-tagged regulatory domain or full-length PKCalpha as the acceptor revealed that PKCalpha, in the closed ( inactive) form, is restrained from associating with the cytoplasmic portion of CXCR4. Live-cell FLIM experiments show that the assembly of this receptor: kinase complex is concomitant with the endocytosis process. This is confirmed by experimental evidence suggesting that the recycling of the CXCR4 receptor is increased on stimulation with phorbol ester and blocked on inhibition of PKC by bisindolylmaleimide. The EGFP-mRFP1 couple should be widely applicable, particularly to live-cell quantitative FRET assays.