Dynamic assembly properties of nonmuscle myosin II isoforms revealed by combination of fluorescence correlation spectroscopy and fluorescence cross-correlation spectroscopy
Dynamic assembly properties of nonmuscle myosin II isoforms revealed by combination of fluorescence correlation spectroscopy and fluorescence cross-correlation spectroscopy
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DOI：
10.1093/jb/mvq134
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发表时间：
2011-03-01
影响因子：
2.7
通讯作者：
Takahashi, Masayuki
中科院分区：
文献类型：
--
作者：
Mitsuhashi, Mariko;Sakata, Hiroshi;Takahashi, Masayuki
肌球蛋白II分子通过其c端杆状区域组装成细丝，并负责多种细胞运动活动。非肌球蛋白II的三种亚型（IIA， IIB和IIC）在哺乳动物细胞中表达。然而，关于细丝的同工异构体组成，我们所知甚少。为了进一步了解肌球蛋白II亚型的组装特性，特别是在细丝中的亚型组成，我们进行了荧光相关光谱（FCS）和荧光互相关光谱（FCCS）的组合分析，这使我们能够同时获得相互作用和每个分子大小的信息。利用不同荧光探针标记的IIA和IIB的c端杆状片段（ARF296和BRF305），我们证明了ARF296和BRF305的混合物形成了异质组合，并且在每种异质异构体的预形成的同源组合之间以不依赖于异构体的方式发生了杆状片段的动态交换。我们还发现Mts1 （S100A4）特异性地将ARF296从异质组合中剥离，从而形成BRF305的同源组合。这些结果表明，IIA和IIB可以以不依赖于异构体的方式形成异构体，而Mts1等因子可以从异构体中去除一个异构体，从而形成由另一个异构体组成的同构丝。
Myosin II molecules assemble into filaments through their C-terminal rod region, and are responsible for several cellular motile activities. Three isoforms of nonmuscle myosin II (IIA, IIB and IIC) are expressed in mammalian cells. However, little is known regarding the isoform composition in filaments. To obtain new insight into the assembly properties of myosin II isoforms, especially regarding the isoform composition in filaments, we performed a combination analysis of fluorescence correlation spectroscopy (FCS) and fluorescence cross-correlation spectroscopy (FCCS), which enables us to acquire information on both the interaction and the size of each molecule simultaneously. Using C-terminal rod fragments of IIA and IIB (ARF296 and BRF305) labelled with different fluorescent probes, we demonstrated that hetero-assemblies were formed from a mixture of ARF296 and BRF305, and that dynamic exchange of rod fragments occurred between preformed homo-assemblies of each isoform in an isoform-independent manner. We also showed that Mts1 (S100A4) specifically stripped ARF296 away from the hetero-assemblies, and consequently, homo-assemblies of BRF305 were formed. These results suggest that IIA and IIB can form hetero-filaments in an isoform-independent manner, and that a factor like Mts1 can remove one isoform from the hetero-filament, resulting in a formation of homo-filaments consisting of another isoform.