Dynamics and molecular interactions of linker of nucleoskeleton and cytoskeleton (LINC) complex proteins
Dynamics and molecular interactions of linker of nucleoskeleton and cytoskeleton (LINC) complex proteins
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DOI：
10.1242/jcs.057075
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发表时间：
2009-11-15
影响因子：
4
通讯作者：
Worman, Howard J.
中科院分区：
文献类型：
--
作者：
Oestlund, Cecilia;Folker, Eric S.;Worman, Howard J.
核骨架和细胞骨架（LINC）复合物的连接子位于核膜中，形成层和细胞骨架元件之间的连接。Sun1， Sun2和nesprin2是LINC复合物的重要组成部分。我们在胚胎成纤维细胞中表达了这些蛋白与绿色荧光蛋白融合，并利用光漂白后的荧光恢复研究了它们的扩散流动性。我们发现它们在缺乏a型层粘连蛋白的小鼠的胚胎成纤维细胞中比在野生型小鼠的细胞中更具流动性。敲除Sun2也增加了短嵌合形式的nesprin2巨体（mini- nesprin2 - 2g）的迁移率，而缺乏emerin则不影响Sun1， Sun2或mini- nesprin2 - 2g的迁移率。荧光共振能量转移实验表明，Sun1比Sun2与层蛋白A的联系更紧密。在等离子体表面共振（Biacore）实验中，Sun1和Sun2对nesprin2 KASH结构域具有相似的亲和力。这种亲和性比先前报道的nesprin2和actin之间的亲和性高10倍。肌动蛋白结合域的缺失对mini- nespring - 2g迁移率没有影响。我们的数据支持一个模型，即a型层蛋白和Sun2将nesprin2锚定在外核膜上，而emerin、Sun1和actin对于这种锚定是必不可少的。
The linker of nucleoskeleton and cytoskeleton (LINC) complex is situated in the nuclear envelope and forms a connection between the lamina and cytoskeletal elements. Sun1, Sun2 and nesprin-2 are important components of the LINC complex. We expressed these proteins fused to green fluorescent protein in embryonic fibroblasts and studied their diffusional mobilities using fluorescence recovery after photobleaching. We show that they all are more mobile in embryonic fibroblasts from mice lacking A-type lamins than in cells from wild-type mice. Knockdown of Sun2 also increased the mobility of a short, chimeric form of nesprin-2 giant (mini-nesprin-2G), whereas the lack of emerin did not affect the mobility of Sun1, Sun2 or mini-nesprin-2G. Fluorescence resonance energy transfer experiments showed Sun1 to be more closely associated with lamin A than is Sun2. Sun1 and Sun2 had similar affinity for the nesprin-2 KASH domain in plasmon surface resonance (Biacore) experiments. This affinity was ten times higher than that previously reported between nesprin-2 and actin. Deletion of the actin-binding domain had no effect on mini-nesprin-2G mobility. Our data support a model in which A-type lamins and Sun2 anchor nesprin-2 in the outer nuclear membrane, whereas emerin, Sun1 and actin are dispensable for this anchoring.