Chang Wakam, Antoku Susumu, Gundersen Gregg G
Department of Pathology and Cell Biology, College of Physicians and Surgeons, Columbia University, P&S 15-420, 630 West 168th Street, New York, NY, 10032, USA.
Methods Mol Biol. 2016;1411:255-67. doi: 10.1007/978-1-4939-3530-7_17.
The rearward positioning of the nucleus is a characteristic feature of most migrating cells. Studies using wounded monolayers of fibroblasts and myoblasts have shown that this positioning is actively established before migration by the coupling of dorsal actin cables to the nuclear envelope through nesprin-2G and SUN2 linker of nucleoskeleton and cytoskeleton (LINC) complexes. During nuclear movement, these LINC complexes cluster along the actin cables to form adhesive structures known as transmembrane actin-associated nuclear (TAN) lines. Here we described experimental procedures for studying nuclear movement and TAN lines using wounded monolayers of fibroblasts and myoblasts, the acquisition of data using immunofluorescence microscopy and live-cell imaging, and methods for data analysis and quantification.
细胞核的向后定位是大多数迁移细胞的一个特征。使用成纤维细胞和成肌细胞损伤单层进行的研究表明，这种定位在迁移之前通过背侧肌动蛋白束通过nesprin-2G和核骨架与细胞骨架（LINC）复合体的SUN2连接蛋白与核膜耦合而被主动建立。在核移动过程中，这些LINC复合体沿着肌动蛋白束聚集，形成称为跨膜肌动蛋白相关核（TAN）线的粘附结构。在这里，我们描述了使用成纤维细胞和成肌细胞损伤单层研究核移动和TAN线的实验程序、使用免疫荧光显微镜和活细胞成像获取数据的方法，以及数据分析和定量的方法。
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