Department of Mechanical Engineering, University of Michigan, Ann Arbor, MI 48019, USA.
Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, MN 55455, USA.
J Cell Sci. 2018 Oct 31;132(4):jcs219451. doi: 10.1242/jcs.219451.
The linker of nucleoskeleton and cytoskeleton (LINC) is a conserved nuclear envelope-spanning molecular bridge that is responsible for the mechanical integration of the nucleus with the cytoskeleton. LINC complexes are formed by a transluminal interaction between the outer and inner nuclear membrane KASH and SUN proteins, respectively. Despite recent structural insights, our mechanistic understanding of LINC complex assembly remains limited by the lack of an experimental system for its reconstitution and manipulation. Here, we describe artificial nuclear membranes (ANMs) as a synthetic biology platform based on mammalian cell-free expression for the rapid reconstitution of SUN proteins in supported lipid bilayers. We demonstrate that SUN1 and SUN2 are oriented in ANMs with solvent-exposed C-terminal KASH-binding SUN domains. We also find that SUN2 possesses a single transmembrane domain, while SUN1 possesses three. Finally, SUN protein-containing ANMs bind synthetic KASH peptides, thereby reconstituting the LINC complex core. This work represents the first reconstitution of KASH-binding SUN proteins in supported lipid bilayers using cell-free expression, which will be invaluable for testing proposed models of LINC complex assembly and its regulation.
核骨架和细胞骨架连接蛋白（LINC）是一种保守的核膜贯穿分子桥，负责将细胞核与细胞骨架进行力学整合。LINC 复合物由核膜内外腔 KASH 和 SUN 蛋白之间的穿核相互作用形成。尽管最近的结构研究取得了进展，但由于缺乏用于其重建和操作的实验系统，我们对 LINC 复合物组装的机制理解仍然有限。在这里，我们描述了人工核膜 (ANM) 作为一种基于哺乳动物无细胞表达的合成生物学平台，用于在支持的脂质双层中快速重建 SUN 蛋白。我们证明，SUN1 和 SUN2 在 ANM 中以溶剂暴露的 C 末端 KASH 结合 SUN 结构域定向。我们还发现 SUN2 具有单个跨膜结构域，而 SUN1 具有三个。最后，含有 SUN 蛋白的 ANM 结合合成 KASH 肽，从而重建 LINC 复合物核心。这项工作代表了首次使用无细胞表达在支持的脂质双层中重建 KASH 结合的 SUN 蛋白，这对于测试 LINC 复合物组装及其调控的拟议模型将是非常宝贵的。
