Fluorescence fluctuation analysis of Arabidopsis thaliana somatic embryogenesis receptor-like kinase and brassinosteroid insensitive 1 receptor oligomerization
Fluorescence fluctuation analysis of Arabidopsis thaliana somatic embryogenesis receptor-like kinase and brassinosteroid insensitive 1 receptor oligomerization
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DOI：
10.1529/biophysj.107.112003
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发表时间：
2008-02-01
影响因子：
3.4
通讯作者：
Visser, Antonie J. W. G.
中科院分区：
文献类型：
--
作者：
Hink, Mark A.;Shah, Khalid;Visser, Antonie J. W. G.
受体激酶在信号的细胞感知中起关键作用。为了验证通过二聚化的受体活化模型，需要一个实验系统来确定活细胞内蛋白质的精确寡聚化状态。在这里，我们表明，光子计数直方图分析和双色荧光互相关光谱能够监测荧光标记的蛋白质在活的植物细胞中的单分子检测水平。在植物细胞中瞬时表达油菜素类固醇不敏感1（BRI1）受体和拟南芥体细胞胚胎发生受体样激酶1和3（AtSERK1和3）与增强的青色或黄色荧光蛋白的框内融合蛋白。虽然没有寡聚体结构被检测到AtSERK3，15%（AtSERK1）至20%（BRI1）的质膜中的标记蛋白质被发现是作为同源二聚体，而没有证据表明被发现为更高的寡聚体复合物。
Receptor kinases play a key role in the cellular perception of signals. To verify models for receptor activation through dimerization, an experimental system is required to determine the precise oligomerization status of proteins within living cells. Here we show that photon counting histogram analysis and dual-color fluorescence cross correlation spectroscopy are able to monitor fluorescently labeled proteins at the single-molecule detection level in living plant cells. In-frame fusion proteins of the brassinosteroid insensitive 1 (BRI1) receptor and the Arabidopsis thaliana somatic embryogenesis receptor- like kinases 1 and 3 (AtSERK1 and 3) to the enhanced cyan or yellow fluorescent protein were transiently expressed in plant cells. Although no oligomeric structures were detected for AtSERK3, 15% (AtSERK1) to 20% (BRI1) of the labeled proteins in the plasma membrane was found to be present as homodimers, whereas no evidence was found for higher oligomeric complexes.