Confocal Fluorescence Microscopy Study of Interaction Between Lens MIP26/AQP0 and Crystallins in Living Cells
Confocal Fluorescence Microscopy Study of Interaction Between Lens MIP26/AQP0 and Crystallins in Living Cells
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DOI：
10.1002/jcb.21598
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发表时间：
2008-05-01
影响因子：
4
通讯作者：
Liang, Jack J.
中科院分区：
文献类型：
--
作者：
Liu, Bing-Fen;Liang, Jack J.
MIP26/AQP0是主要的晶状体纤维膜蛋白，已被报道与许多其他晶状体成分相互作用，包括晶体蛋白、脂质和细胞骨架蛋白。关于晶体蛋白，许多先前的报道表明，MIP26/AQP0只与α-晶体蛋白或索恩特异的晶体锡相互作用。考虑到MIP26/AQP0在减少晶体中的光散射方面可能起到的重要作用，我们用共聚焦荧光共振能量转移(FRET)显微镜进一步研究了它与晶体蛋白的相互作用。具体地说，我们使用标记了绿色荧光蛋白(GFP)的MIP26作为供体，使用标记了红色荧光蛋白(RFP)的晶体蛋白(αA-、αB-、βB2-或伽马C-晶体蛋白)作为受体。将两种载体共转染入HeLa细胞。培养结束后，分别在GFP、RFP和FRET三个通道中获取激光散射显微图像。然后通过去除光谱渗漏的贡献得到净FRET图像。Net FRET的像素与GFP的像素进行归一化。结果表明，MIP26与所有晶状体蛋白之间存在可测量的相互作用，相互作用的程度从αA-和αB-晶状体蛋白递减到β-B2-和伽马C-晶状体蛋白。使用未标记的αA-晶体蛋白进行的竞争性相互作用研究表明，净FRET减少，表明MIP26与αA-晶体蛋白之间相互作用的特异性。我们的结论是，所有的晶体蛋白都与MIP26相互作用，其生理意义可能是缩小了膜和细胞质之间的折射率差。J.细胞。生物化学。104：51-58,2008。(C)2007威利-利斯公司。
MIP26/AQP0 is the major lens fiber membrane protein and has been reported to interact with many other lens components including crystallins, lipid, and cytoskeletal proteins. Regarding crystallins, many previous reports indicate that MIP26/AQP0 interacts with either only a.-crystallin or sorne specific -(-crystal tins. Considering the possibly important role of MIP26/AQP0 in the reduction of light scattering in the lenses, we have further investigated its interaction with crystallins using confocal fluorescence resonance energy transfer (FRET) microscopy. Specifically, we used MIP26 tagged with a green fluorescence protein (GFP) as a donor and a crystallin (alpha A-, alpha B-, beta B2-, or gamma C-crystallin) tagged with a red fluorescence protein (RFP) as an acceptor. The two plasmids were cotransfected to HeLa cells. After culture, laser scattering microscopy images were taken in each of the three channels: GFP, RFP, and FRET. The net FRET images were then obtained by removing the contribution of spectral bleed-through. The pixels of net FRET were normalized with those of GFP. The results show the presence of measurable interactions between MIP26 and all crystallins, with the extent of interactions decreasing from alpha A- and alpha B-crystallin to beta B2- and gamma C-crystallin. Competitive interaction study using untagged alpha A-crystallin shows decreased net FRET, indicating specificity of the interactions between MIP26 and alpha A-crystallin. We conclude that all crystallins interact with MIP26, the physiological significance of which may be a reduction in the difference of refractive index between membrane and cytoplasm. J. Cell. Biochem. 104: 51-58, 2008.(C) 2007 Wiley-Liss, Inc.