Homo-FRET Imaging as a Tool to Quantify Protein and Lipid Clustering
Homo-FRET Imaging as a Tool to Quantify Protein and Lipid Clustering
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Homo-FRET 成像作为量化蛋白质和脂质聚类的工具
DOI：
10.1016/j.bpj.2010.12.968
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发表时间：
2011
影响因子：
3.4
通讯作者：
G. Meer
中科院分区：
文献类型：
--
作者：
H. Gerritsen;A. Bader;E. Hofman;J. Voortman;P. Bergen;En Henegouwen;G. Meer
基于荧光各向异性的Homo-FRET方法有可能成为分子细胞生物学中有价值的工具。在这里，使用的是由于同质能量转移的荧光各向异性的快速变化。Homo-FRET测量可用于确定荧光团之间的距离，但也可用于量化簇的大小以及簇大小的分布。homo-FRET信号的解释是复杂的，因为荧光团的相互取向和每个簇的荧光团数量都以类似的方式影响荧光各向异性。荧光探针的性质是非常重要的，考虑到这些方面是至关重要的，正确的解释homo-FRET信号在蛋白质和脂质聚类研究。在这项研究中，集群的大小进行了量化，使用从控制二聚化和寡聚化实验获得的参考值。Homo-FRET应用于脂筏标志物GPI的聚类研究（见图）。和K-ras，以及EGF受体聚集在质膜。K-ras的结果与金标记K-ras的TEM研究进行了比较。
Fluorescence anisotropy based Homo-FRET methods have the potential to become valuable tools in molecular cell biology. Here, use is being made of the rapid change in fluorescenc anisotropy due to homo energy transfer. Homo-FRET measurements can be employed to determine the distance between fluorophores but also for quantifying the size of the clusters, as well as distributions of cluster sizes. The interpretation of homo-FRET signals is complicated by the fact that both the mutual orientations of the fluorophores and the number of fluorophores per cluster affect the fluorescence anisotropy in a similar way. The properties of the fluorescence probes are of great important and taking these aspects into account is crucial for the correct interpretation of homo-FRET signals in protein and lipid clustering studies. In this study cluster sizes are quantified using reference values obtained from controlled dimerization and oligomirization experiments. Homo-FRET is applied in a study on the clustering of lipid raft markers GPI (see Fig.) and K-ras, as well as for EGF receptor clustering in the plasma membrane. The results on K-ras are compared with an TEM study on gold labeled K-ras.